The relationship between free IGFBP3 and the esophageal cancer risk in a nested case-control study
Adachi Y, Nojima M, Mori M, Yamashita K, Yamano H, Endo T, Kato Y, Nakase H, Imai K, Sakata K and Tamakoshi A
Sapporo Medical University, Sapporo, Japan.
Background: Insulin-like growth factor-1 (IGF1) is a potent mitogen, whereas IGF-binding protein-3 (IGFBP3) binds and inhibits IGF1. Although high circulating IGF1 and low IGFBP3 are associated with increased risk of several cancers, there is no information in esophageal cancers.
Study aims: To assess the relationship between serum levels of IGF1 and IGFBP3 and the risk of esophageal carcinoma.
Materials and methods: We assessed the relationship between the serum levels of these molecules and the risk of esophageal cancer in a prospective, nested case-control study of participants from the Japan Collaborative Cohort Study. A baseline survey was conducted from 1988 to 1990. Of the 110,585 enrolled participants, 35% donated blood samples. Those who had been diagnosed with esophageal cancer were considered cases for nested case-control studies. A conditional logistic model was used to estimate odds ratios (ORs) for the incidence of esophageal cancer associated with serum IGF1 and IGFBP3 levels.
Results: In all, 31 cases and 86 controls were eligible for the present assessment. The molar ratio of IGF1/IGFBP3, which represents the free and active form of IGF1, was not correlated with the risk of esophageal carcinoma. A higher molar difference between IGFBP3 and IGF1, which estimates the free form of IGFBP3, was associated with a decreased risk of esophageal carcinoma (p = 0.0146), and people in the highest tertile has the lowest risk (OR = 0.107, 95% confidence interval (CI): 0.017-0.669). After adjustment for body mass index, tobacco use, and alcohol intake, the molar difference of IGFBP3−IGF1 was inversely correlated with the risk of esophageal carcinoma (p = 0.0150).
Conclusion: Thus, the free form of IGFBP3, which is estimated by this molar difference, may be inversely associated with esophageal cancer incidence.
High serum pentraxin-3 level predicts future hepatocellular carcinoma development in chronic hepatitis C patients with Child–Pugh class A liver cirrhosis
Zuwala-Jagiello J, Górka-Dynysiewicz J, Grzebyk E, Murawska-Cialowicz E and Pazgan-Simon M
Wroclaw Medical University, Wroclaw, Poland
Background: The soluble pattern recognition receptor long pentraxin-3 (PTX3), a modulator of tumor-associated inflammation, is known to be positively correlated with tumor grade and severity of malignancies. Increased PTX 3 production is implicated in the pathogenesis of hepatocellular carcinoma (HCC) in animal models. Although previous studies showed that HCC patients had higher serum PTX3 level at the time of diagnosis, it is unclear whether the glycoprotein contributes to the development of HCC or is just a reaction to cancer.
Study aims: To address this question, we performed a nested case-control study.
Materials and methods: Consecutive chronic hepatitis C (CHC) patients with Child–Pugh class A liver cirrhosis (cirrhotic patients; n = 43) were recruited from 2007 to 2009 and followed till 2016. Plasma level of PTX3, hsCRP, and pro-inflammatory cytokines was performed at baseline, the date of peak alanine aminotransferase (ALT) level, and the last visit. A total of 10 (35%) cirrhotic patients developed HCC at a median follow-up of 72 months (interquartile range: 68–76).
Results: Serum PTX3 was higher in patients with HCC than cirrhotic controls both during peak ALT and at the last visit (both p < 0.001). Cirrhotic patients with PTX3 above 5.1 ng/mL during peak ALT had increased risk of HCC (adjusted hazard ratio 1.5; 95% CI 1.1-2.1; p < 0.008). The sensitivity, specificity, and positive and negative predictive values of this cut-off to predict future HCC development were 67.9%, 75%, 73.1%, and 70%, respectively. Combination of PTX3 and alpha-fetoprotein (AFP) improved the sensitivity in diagnosing HCC or predicting future HCC development.
Conclusions: High serum PTX3 level predates the development of HCC in CHC patients with Child–Pugh class A liver cirrhosis and has moderate accuracy in predicting future cancer. This may assist clinicians in selecting high-risk patients for HCC surveillance program.
Integrated proteomic profiling technologies for novel gestational and cancer biomarker discovery
Terentiev A, Moldogazieva N and Mokhosoev I
N.I. Pirogov Russian National Research Medical University, Moscow, Russia
Background: In the past, the focus in biomarker discovery technologies was on the use of a single biochemical or immunochemical method such as immunoprecipitation analysis, radioimmunoassay (RIA), or enzyme-linked immunosorbent assay (ELISA). However, these tests have low specificity and sensitivity, especially in tumor patients.
Study aims: To review novel gestational and cancer biomarker discovery technologies based on integrated proteomic approaches.
Materials and methods: Literature search was performed to review the recent studies with emphasis on usage of high-throughput integrated proteomic profiling technologies that enhance novel biomarker and drug target discovery as well as protein structure/activity analysis.
Results: A large-scale quantitative proteomic profiling includes genetic, epigenetic, omics, systems biology, bioinformatics, and molecular imaging to be a useful tool in evaluating changes in gene expression patterns and comprehensive analysis for the identification, standardization, and validation of highly specific and sensitive biomarkers for cancer diagnosis, prognosis, and assessment of therapeutic strategies and discovery for novel anti-cancer drug targets. The co-expression and co-localization analysis provides information about coordinated and cooperative functioning of multiple proteins both in normal gestational development and in trophoblastic tumors. Clinical significance and screening performance improved when placental biomarkers were measured in combination with each other including pregnancy-specific beta1-glycoproteins (PSGs), human chorionic gonadotropin (hCG), human placental lactogen (hPL, the same is human chorionic somatomammotropin (hCS)), alpha-fetoprotein (AFP), glycodelin, transforming growth factor-β2 (TGF-β2), thrombospondin-1, pigment epithelium–derived factor (PEDF), galectin-1, and macrophage migration inhibitory factor.
Conclusion: They may be used as targets to design new drugs with ability to block cell signaling and/or to cause immunomodulatory, anti-inflammatory, proangiogenic, and anti-cancer effects.
Systems biology approach in discovery for novel hepatocellular carcinoma biomarkers
Terentiev A, Moldogazieva N and Mokhosoev I
N.I. Pirogov Russian National Research Medical University, Moscow, Russia
Background: Cancer is a multifactorial and complex disorder known as a process with non-linear dynamics. Hepatocellular carcinoma (HCC) is a heterogeneous disease with not fully elucidated pathogenesis and demonstrating high resistance to conventional chemotherapy.
Study aims: Review of cancer systems biology approaches as a logical extension and application of systems biology to the field of medical sciences. Systems biology and computational biology approaches along with various “omics” and structural biology technologies are presently being exploited to understand cancer complexity.
Materials and methods: Construction and analysis of protein–protein interaction (PPI) networks at the level of completely organism-specific or tissue-specific genomes, proteomes, and metabolomes or at the level of a network sub-circuits.
Results: Evaluability of network analysis in HCC was demonstrated based on the involvement of multiple signaling pathways in HCC progression and treatment. Activation of Ras/mitogen-activated protein kinase (MAPK), Akt/phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycin (mTOR), and Wnt-mediated signaling pathways has been shown to be risk factors for early recurrence and poor prognosis for the disease. Currently used HCC biomarkers, including alpha-fetoprotein (AFP), des-gamma-carboxyl prothrombin (DCP, also known as prothrombin induced by vitamin K absence or antagonist-II (PIVKA-II)), and Lens culinaris agglutinin-reactive fraction of AFP (AFP-L3), are considered to be useful for monitoring treatment responsiveness and tumor recurrence. Additionally, alpha-l-fucosidase, glypican-3 (GPC3), squamous cell carcinoma antigen-1 (SCCA-1), osteopontin (OPN), Golgi protein-73 (GOLPH2), carcinoembryonic antigen (CEA), vascular endothelial growth factor (VEGF), matrix metalloproteinases-2 and -9 (MMP-2 and MMP-9), cancer antigen 125 (CA125), prostate-specific antigen (PSA), and other secreted glycoproteins may be involved in synergistic functioning provided by the PPI network formation.
Conclusion: Network analysis provides very promising approach to reach this goal.
Biomarkers of oxidative protein degradation in cancer
Terentiev A, Mokhosoev I and Moldogazieva N
N.I. Pirogov Russian National Research Medical University, Moscow, Russia
Background: Cancer cells are often exposed to metabolic dysregulations, nutrient starvation, hypoxia, and oxidative stress. The vast majority of agents used cancer therapeutics (ionizing radiation, most chemotherapeutic agents, and some targeted drugs) work through either directly or indirectly generating reactive oxygen species (ROS) that cause protein degradation resulted in cancer cell damage.
Study aims: To study current state of protein oxidative degradation biomarker research.
Materials and methods: Literature search was performed to study recent data on the effects of ROS on protein structure and functioning with emphasis on a role of protein damage in cancer progression and discovery for novel drug targets.
Results: Oxidative damage of proteins can occur through different mechanisms that may include peptide bond cleavage and protein fragmentation, cross-linking, and chemical modification of virtually any amino acid side chain. This may have a wide range of functional consequences including altered gene expression and interactions with biological partners, decrease in enzymatic and binding activity, and modulation of protein turnover. Among the biomarkers of protein oxidative degradation are (1) the blood concentrations of reduced and oxidized forms of glutathione (GSH and GSSG, respectively) and S-glutathionylated proteins; (2) nitrated tyrosine (3-nitrotyrosine) and halogenated tyrosine (3-chlorotyrosine and 3-bromotyrosine) products; and (3) protein carbonyls generated by oxidation of several amino acids (K, R, P, and T) and by glycation/glycoxidation of ε-amino group of lysine. Successful therapeutic strategy could be the induction of cancer cell death using different unrelated oxidant compounds.
Conclusions: Biomarkers of oxidative stress correlate with chemotherapy response and upregulation of redox enzymes such as glutathione peroxidases and have been observed in the acquisition of chemotherapy resistance and genomic instability.
Unconventional evaluation of some promising A3B porphyrinic-type compounds as theragnostic agents
Socoteanu R, Boscencu R, Anastasescu M, Manda G and Vasiliu G
Institute of Physical Chemistry, Romanian Academy, Bucharest, Romania
Background: The potential and current use of porphyrins is well-known, especially in photodynamic treatment (PDT) of cancer field. The opportunity of using new related compounds is wide open due to their versatility and their capacity to act simultaneously as therapeutic agent and fluorescent marker.
Study aims: Evaluating how supplementary methods of studying the potential theragnostic agents could be linked with their biological activity.
Materials and methods: Compounds from unsymmetrical A3B-type porphyrinic structures with -(4acetoxy3-metoxyphenyl) in methinic bridges, -10, 15, and 20 meso-positions, are evaluated regarding one of the most important features, agglomeration capacity, by unconventional method—atomic force microscopy (AFM).
Results: The results corroborated with relevant biotests, as interaction with MCF-7 cell lines, and relevant view of the localization at cell level of the marker, conducts to interesting conclusions recommending the compounds and methodology for further development.
Conclusions: Coupling methods such as AFM and studies like interaction of markers with biological material could be an advantage in the process of understanding the behavior of the drug at the interface with cancer cell.
Increased risk of being diagnosed with subsequent primary malignancy in subjects with elevated levels of serological protein biomarker levels at diagnostic bowel endoscopy
Piper T, Johansen JS, Davis GJ, Dowell BI, Christensen IJ and Nielsen HJ
Department of Gastroenterology, Hvidovre Hospital, Hvidovre, Denmark
Background: Subjects with no colorectal cancer (CRC) at bowel endoscopy and no other type of cancer diagnosed at the time of the examination, but with increased serological protein biomarker levels have a subsequent increased risk of being diagnosed with malignant diseases.
Study aims: To evaluate whether increased serological protein biomarker levels in non-cancer subjects may aid in identifying subjects at increased risk of being diagnosed with subsequent primary malignancies.
Materials and methods: Plasma levels of TIMP-1, CEA, and CA19-9 and serum YKL-40 levels were determined in blood samples collected before diagnostic bowel endoscopy. Five ranked groups were made, and three of these groups were included in this study; groups included subjects with clean colorectal, or with diverticula, or with adenomas. A 7- to 10-year follow-up identified patients who subsequently developed a primary malignant disease; both intra- and extra- colonic malignant diseases were included. Reference intervals were constructed. Each biomarker level was defined as normal or elevated based on the 90th percentile from 400 age- and gender-adjusted subjects with no known comorbidity and with identified clean colorectal. Multivariate analyses were made and 5-year cumulated incidences were measured. Death was included as a competing risk.
Results: Primary malignancies were identified in 366 of 2635 patients; 38 of the cancers were of colorectal origin. Three groups were established—0: no increased biomarkers; 1: 1 of the 4 biomarkers increased; and 2: ≥2 biomarkers increased.
The cumulative 5-year incidences in the three subgroups were as follows: clean colon—0: 3.3%; 1: 5.8%; and 2: 7.8% (p = 0.002).
Diverticula: 0: 5.5%; 1: 10.1%; and 2: 18.8% (p < 0.0001).
Adenoma: 0: 6.9%; 1: 11.8%; and 2: 17.5% (p = 0.0009).
Conclusion: Increased protein biomarker levels at bowel endoscopy appear helpful to identify non-cancer subjects at increased risk of being diagnosed with subsequent primary malignancies.
Prognostic factor of measuring red blood cell distribution width and biochemical parameters in patients with advanced gallbladder cancer
Letelier P, Huilipang R, Saldías R, Martín AS, Araneda R, Andaur M and Guzmán N
Facultad de Ciencias de La Salud, Universidad Católica de Temuco, Temuco, Chile
Background: Gallbladder cancer (GBC) is a highly malignant and aggressive neoplasm. Currently, few biomarkers can support the prognosis. The red blood cell distribution width (RDW), a routine parameter in the blood count, has recently been proposed as a prognostic marker in solid tumors.
Study aims: The objective of the study was to evaluate the predictive value of RDW and biochemical parameters according to clinical–pathological condition and survival of patients with advanced GBC.
Materials and methods: This retrospective study included 35 patients diagnosed with GBC at the Hospital Hernán Henríquez Aravena from Temuco city in the period 2007–2010.
Results: RDW levels were classified into two groups: RDW <14.5% and RDW >14.5%. In this study, we found no statistically significant association with RDW level and clinical–pathological data nor with patient’s survival; however, it was observed that a high level of RDW was significantly associated with creatinine and sodium in serum. The Cox regression analysis revealed that the concentration of total bilirubin and alkaline phosphatase correlated with lower survival (p < 0.05), being an independent predictor factor (hazard ratio (HR) of 2.68 and 3.81, respectively).
Conclusions: Our study found no correlation between the RDW level and the prognosis of patients with advanced GBC, although the biochemical parameters (total bilirubin and alkaline phosphatase) proved to be useful as a prognostic factor, which could be related to the degree of hepatic and renal metastases of these patients. It is required to increase the sample size for the validation of these results.
Circulating microRNAs as biomarkers in biliary tract cancers
Letelier P, Riquelme I, Hernandez A, Guzmán N, Farías JG and Roa JC
Facultad de Ciencias de La Salud, Universidad Católica de Temuco, Temuco, Chile
Background: Biliary tract cancers (BTCs) are a group of highly aggressive malignant tumors with a poor prognosis. The current diagnosis is based mainly on imaging and intraoperative exploration due to brush cytology having a low sensitivity and the standard markers, such as carcinoembryonic antigen (CEA) and carbohydrate 19-9 (CA19-9), not having enough sensitivity nor specificity to be used in a differential diagnosis and early-stage detection. Thus, better non-invasive methods that can distinguish between normal and pathological tissue are needed. MicroRNAs (miRNAs) are small, single-stranded non-coding RNA molecules of ~20–22 nucleotides that regulate relevant physiological mechanisms and can be involved in carcinogenesis.
Study aims: This review highlights the progress in the analysis of circulating miRNAs in serum, plasma, and bile as potential diagnostic and prognostic markers of BTCs and aims to stimulate interest in the role of biomarkers in BTCs, which may open new avenues in the determination of BTCs pathogenesis.
Materials and methods: The electronic search was conducted in PubMed and Web of Science (up to 30 March 2016). We collected relevant articles to explore the association of biomarkers in all BTCs.
Results: Recent studies have demonstrated that miRNAs are detectable in multiple body fluids, showing great stability, either free or trapped in circulating micro vesicles, such as exosomes.
Conclusion: MiRNAs are ideal biomarkers that may be used in screening and prognosis in BTCs, aiding also in the clinical decisions at different stages of cancer treatment.
Application of PIVKA-II in hepatocellular carcinoma diagnosis in Chinese population: Preliminary data from Asian multicenter study
Zheng Y, Yang T, Soh A and Beshiri A
Medical Scientific Liaison Manager. Abbott Diagnostic Division, Abbott China
Background: Liver cancer is a health burden for Asian countries. Hepatitis is the most common risk factor for liver cancer while the infection rate of hepatitis virus is high in Asia. The most frequent liver cancer is hepatocellular carcinoma (HCC). Alpha-fetoprotein (AFP) is the most commonly used biomarker for HCC. However, the specificity of AFP is still low with around 65% in different reports. Thus, new biomarkers for detection of HCC are needed. Protein induced by vitamin K absence or antagonist-II (PIVKA-II) was first discovered in vitamin K deficiency patients and showed clinical potential in HCC patients. Although it has been used in clinical practice, most of the clinical experience has been from Japan. However, most of the HCC patients in Japan are hepatitis C virus (HCV) related, while other Asian countries are hepatitis B virus (HBV) related.
Study aims: To further support its clinical application, we initiated an Asian study on the application of PIVKA-II in HCC diagnosis and differential diagnosis including China, Singapore, Vietnam, and Thailand. Here, we report the preliminary results from China.
Materials and methods: In all, 333 cases of HCC and 164 cases of cirrhosis were recruited in China. Concentrations of PIVKA-II were detected by the ARCHITECT PIVKA-II assay according to manufacturer’s instructions.
Results: Receiver operating characteristic (ROC) analysis showed that the area under the curve (AUC) of AFP and PIVKA-II was 0.735 and 0.817, respectively (p < 0.05). We also analyzed the optimal cutoff of the biomarkers for HCC in Chinese population. The optimal cutoff for AFP to detect HCC was 30.5 ng/mL with the sensitivity of 63.5% and specificity of 76.1%. While the optimal cutoff for PIVKA-II to detect HCC was 93.0 mAU/mL, with the sensitivity of 70.2% and specificity of 82.1%.
Conclusion: The preliminary data support the clinical application of PIVKA-II in HCC diagnosis in China population. However, the detailed clinical diagnosis strategy is still under investigation.
Non-invasive detection of bladder cancer by the UBC Rapid test, ultrasonography, and cytology
Barak V1,2, Itzkovitch D1,2, Einarsson R1,2 and Pode D1,2
1Deptartments of Oncology and Urology, Hadassah University Medical Center, Jerusalem, Israel
2IDL Biotech AB, Bromma, Sweden
Background: At presentation, 75% of patients (pts) have superficial tumors confined to the mucosa (Ta) or the lamina propria (T1). After transurethral resection (TUR), 50% of patients will have recurrence within 2 years. Therefore, a close follow-up is mandatory. Cystoscopy is invasive, so there is a need for non-invasive methods.
Study aims: To assess sensitivity (Sens) and specificity (Spec) of three non-invasive methods to detect primary and recurrent bladder cancer: the UBC Rapid, ultrasonography (US), and cytology.
Materials and methods: A total of 89 patients following resection of bladder tumors and patients evaluated for hematuria provided urine for the UBC Rapid and cytopathological examination. Each patient had US and cystoscopy and biopsies, if needed. The sensitivity and specificity of the three non-invasive methods were compared to cystoscopy.
Results: No tumor in 54 patients and bladder tumor found in 35 patients. Overall sensitivity of the UBC Rapid test was 68.8% and specificity was 79.6%. Sensitivity of US was 68.8% and specificity was 98%. Sensitivity of cytology was 56.7% and specificity was 97.9%. The combination of all three methods had a sensitivity of 94.3 and a specificity of 71.1%. At least one of the tests was positive in 33/35 tumors. The combination of UBC Rapid and US had a sensitivity of 91.4% (95% CI = 76.9%–98.2%) and a specificity of 76%. The combination of UBC Rapid and Cytology had a sensitivity of 87.5% (95% CI = 71%–96.5%) and a specificity of 75%. The combination of US and Cytology had a sensitivity of 82.4% (95% CI = 65.5%–93.2%) and a specificity of 95.5%.
Conclusion: The combination of UBC Rapid, US, and cytology has very high sensitivity, can decrease the frequency of cystoscopies, and should be used as a routine non-invasive surveillance test.
Determination of a multivariable signature for prediction of overall survival for patients with colorectal cancer based on 14 protein biomarkers: A comparison of statistical methods
Christensen IJ, Rolff HC, Davis G, Gawell S, Martens F, Kos J, Hansen GH, Nielsen HJ and Ferm L
Hvidovre University Hospital, Hvidovre, Denmark
Background: The correct statistical method for the estimation of a predictive model for time to event data based on multiple biomarker measurements remains a challenge, in particular when relatively many markers are included.
Study aims: To assess suitable multivariable models for the prediction of time to death based on protein biomarkers in patients with colorectal cancer (CRC).
Materials and methods: A total of 540 CRC (245 rectal cancer patients) patients were accrued prospectively with clinical explanatory covariates and with preoperative blood samples drawn. Levels of protein biomarkers were determined using standardized and documented methods, and these were carcinoembryonic antigen (CEA), high-sensitivity C-reactive protein (hsCRP), Ferritin, CA19-9, Galectin 3, tissue inhibitor of metallo proteinase 1 (TIMP-1), CYFRA-21, Cathepsin S and X, Cystatin, collagen IV and three forms of the soluble urokinase plasminogen receptor (suPAR).
Results: These proteins levels were combined using multivariable statistical methods in order to develop a statistical model for the prediction of survival adjusted for clinical explanatory covariates (age, gender, tumor–node–metastasis (TNM) stage, location of tumor, and differentiation). The models tested were the Cox proportional hazards model with 10-fold cross-validation and the Adaptive Index Model (AIM).
Conclusion: The development of a signature for prediction of survival probabilities utilizing 14 protein biomarkers was feasible using both strategies. A reduced model was sufficient for accurate prediction of survival probabilities. These statistical methodologies yielded similar results.
Anti-tumor effect of anti-ErbB-2 trifunctional antibody
Kato Y, Hirata H, Tsujisaki M, Matsune T, Sasaki S, Hinoda Y, Nakase H and Imai K
Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan
Background: ErbB-2, a member of the epidermal growth factor (EGF) receptor tyrosine kinase family, is often overexpressed and/or amplified in breast cancer, gastric cancer, and other many malignancies.
Study aims: We established an anti-ErbB-2 mouse–human chimeric monoclonal antibody (MoAb) CH401, since ErbB-2 gene product is one of the best target molecules for cancer therapy. The MoAb CH401 was able to kill ErbB-2-positive cancer cells in vitro. We studied the mechanism of tumor suppression by MoAb CH401 and its therapeutic usefulness. Furthermore, we attempted to produce the anti-ErbB-2 and anti-CD3 trifunctional antibodies for seeking more powerful anti-tumor activity. In this study, this trifunctional antibody is examined to show the anti-tumor effect against Herceptin-resistant cancer cells in vitro and in vivo.
Material and methods: Western blot and flow cytometric and immunocytochemical analyses were performed to investigate the mechanism of tumor suppression by MoAb CH401 and its therapeutic usefulness.
Results: We found that MoAb CH401 induces apoptosis in ErbB-2 overexpressing cells by activating the Jun N-terminal kinase (JNK)/p38 pathway and downregulating the extracellular-signal-regulated kinase (ERK) pathway. In addition, we produced the anti-ErbB-2 and anti-CD3 trifunctional antibodies and tested its efficacy. We revealed its tumor suppression effect.
Conclusion: Our results suggest that using anti-ErbB-2 and anti-CD3 trifunctional antibodies is a promising approach to the treatment of erbB-2 expressing cancer cells. This trifunctional antibody was shown to have an effective potential for Herceptin-resistant cancer cells in vitro and in vivo.
Correlation between serum levels of total specific prostate antigen and Gleason score in patients with prostate adenocarcinoma
Andriolo A1, Mendes ME2 and Sumita NM2
1Escola Paulista De Medicina, Universidade Federal de São Paulo, São Paulo, Brazil
2Divisão de Laboratório Central, Hospital Das Clínicas, Faculdade de Medicina, Universidade de São Paulo, São Paulo, Brazil
Background: Prostate-specific antigen (PSA) is the most commonly used serum marker for the detection of prostate cancer. Its elevation in prostate cancer is due to a combination of factors such as changes in gland architecture, increased individual cell production, increased number of producing cells, increased local vascularization, and changes in capillary permeability.
The best known system of graduation of prostate adenocarcinoma is the Gleason score, which stratifies the cancer in five degrees, based on glandular patterns and degree of cell differentiation. Grade 1 tumors represent the more differentiated and grade 5 tumors, on the contrary, do not present glandular differentiation and the neoplastic cells infiltrate the stroma. As most tumors have more than one pattern, the lesions are graded according to the two predominant aspects, summed up, and resulting in the Gleason score.
Study aims: To evaluate whether there is a correlation between the serum of total PSA and the Gleason score performed in samples obtained by prostate biopsy.
Material and methods: We studied 150 patients with histological diagnosis of prostatic adenocarcinoma. Total PSA was measured in the serum by routine method according to all the recommendations of the supplier of the kit, and the samples obtained by biopsy were analyzed by two independent observers.
Results: The following table presents the serum levels of total PSA and Gleason score in patients with prostatic adenocarcinoma.
Conclusion: Our data show that there is a correlation between total circulating PSA concentration and Gleason grade, even by the analysis of the prostatic biopsy material.
Serum tumor markers panel by electrochemiluminescence: Methods evaluation in a clinical pathology service
Mendes ME, Barcos LC, Fragoso GVC, Batista VRM, Nakaema J and Sumita NM
Divisão de Laboratório Central, Hospital Das Clínicas, Faculdade de Medicina, Universidade de São Paulo, São Paulo, Brazil
Hypothesis: To know the application of electrochemiluminescence to a panel of serum tumor markers to be introduced in the laboratory routine of a large tertiary clinical laboratory.
Study aims: The aim of this study was to evaluate the analytical performance of five serum tumor markers assay by electrochemiluminescence method: carcinoembryonic antigen (CEA), Cyphra 21-1, CA125, CA15-3, and CA72-4.
Material and methods: These validations were part of the laboratory consolidation process in the new Core Laboratories automation site. The blood samples were obtained by peripheral venipuncture including pathological and normal values. The study included repeatability (within-run imprecision), total reproducibility (total within-lab imprecision), sensitivity, linearity, accuracy, robustness, carryover, sample stability, comparison between analytical systems, limit of detection (LOD), analytical measurement range (AMR), variation lot-to-lot, and specimen stability. The imprecision and LOD studies were based on CLSI EP5-A3 and CLSI EP17-A2 documents, respectively. The AMR was determined with serial dilution. The measurement was performed on Cobas 8000 e602 module (Roche Diagnostics GmbH, Germany) and compared to Cobas e411 (Roche Diagnostics GmbH, Germany) and analyzed by analysis of variance (ANOVA). Regarding the variation between lots, the goal for agreement of 10% was adopted. The specimen stability was defined too.
Results: The reproducibility was less than 2.5% for both methods, and the LOD for all parameters was low and the minimal lot-to-lot variability was documented. The clinical monitoring patterns correlated with established methods were higher than 90% of cases.
Conclusions: The electrochemiluminescence methods performed on the Cobas 8000 e602 module demonstrated acceptable imprecision, low LOD, a wide AMR, and an acceptable lot-to-lot stability. They were compared to established methods finding acceptable correlations and there were good diagnostic concordances. The system proved to be robust.
Assessment of circulating tumor cells in early-stage colon cancer
Abdallah EA, Silva VSE, Flores BCTCP, Alves VS, Braun AC, Urvanegia ACM and Chinen LTD
A.C.Camargo Cancer Center, São Paulo, Brazil
Background: Studies have shown that circulating tumor cells (CTCs) may be involved in the metastatization process. Therefore, its isolation constitutes a potential strategy for clinical follow-up as a non-invasive method.
Study aims: Our objective was to evaluate the role of circulating tumor cells and circulating tumor microemboli (CTM) in stages I–III colon cancer patients.
Material and methods: We collected 10 mL of blood from pre-surgery colon cancer and filtrated the sample in ISET® (Isolation by Size of Epithelial Tumor cells) device. This study started on July 2016. CTC counts were assessed and correlated with clinical and pathological data.
Results: We included 15 patients. However, one patient was identified as metastatic after surgery and therefore excluded of these analyses. From 14 patients analyzed, 8 (57.1%) were male, with a median age of 65 years (53–87 years). The most prevalent cancer sites were right colon (n = 6; 42.9%) and left colon (n = 4; 28.6%). CTCs’ detection rate was 85.7%, and the mean and median of CTCs were 5.1 and 2.6 mL, respectively (0–30.3 mL). We found CTM in 4/14 patients (28.5%). Histopathological analysis showed that no patient had blood vessel invasion, 5 (35.7%) had lymphatic invasion, and 4 (28.6%) had perineural invasion. Four patients (28.4%) were classified as stage I and 10 (71.4%) were classified as stages II and III. Although we present a limited sample, exact Fisher’s test showed an association between lymphatic invasion and CTC number above the median (p = 0.03), and the presence of CTM was most frequent in this group of patients, although without statistical significance (p = 0.09). CTM absence was associated with pathological T = 2 and 3 (p = 0.06), when compared to pathological T = 4.
Conclusion: Moreover, this is the first study to show the efficacy of ISET method to detect high rates of CTCs from stages I–III colon cancer patient. Our next steps are to evaluate some proteins in CTCs that could confer an invasive phenotype, resistance to adjuvant treatment, as well as verify the pattern of mutations in CTCs and CTMs.
Analysis of blood markers related to prognosis in locally advanced rectal cancer
Flores BCTCP, Silva VSE, Abdallah EA, Braun AC, Mello CAL, Urvanegia ACM, Alves VS and Chinen LTD
A.C.Camargo Cancer Center, São Paulo, Brazil
Background: Colorectal cancer is one of the principal causes of mortality nowadays, with rectal cancers comprising a third of cases. Rectal cancer represents worse prognosis, and in the locally advanced setting, it is treated differently when compared with colon cancer, with a therapy following three steps, consisting of neoadjuvant chemoradiation, surgery, and adjuvant chemotherapy. Studies have shown that circulating tumor cells (CTCs) may be involved in the metastatization process. Therefore, its isolation constitutes a potential strategy for clinical follow-up as a non-invasive method.
Study aims: Our objective is to evaluate the role of CTCs in stages I–III rectal cancer patients.
Material and methods: We do these analyses in 10 mL of blood before each new phase of the treatment (four moments) and process the sample on ISET® (Isolation by Size of Epithelial Tumor cells) device.
Results: From these 17 patients, CTCs’ detection rate was 100% in the baseline and 76.5% on the follow-up; 70.6% of the patients were classified as cT3 cN+ and clinical stage III. The mean of CTCs was 2.75 mL in patients with clinical stage N0 and 5.17 mL in patients with clinical stage N1 (p < 0.005). We already performed the next-generation sequencing, using Ion AmpliSeq™ Cancer Hotspot Panel v2, of the DNA derived from primary tumor, CTCs, and leucocytes of eight patients. As preliminary results, in average, 3 million mapped reads were generated, with 100% in 100× coverage. One patient (5.9%) presented NRAS mutation in the codon 61, in the primary tumor, unlike the genetic profile of CTCs, which did not present this mutation. However, the circulating tumor cells showed a mutation in the TYMS gene, seven mutations in the TYMS opposite strand gene, and two mutations of moderate impact on the gene TWIST2.
Conclusion: Although preliminary, our data suggest that CTCs can be used to define prognosis in patients with rectal cancer.
EGFR expression on CTCs and CTM as new tools in metastatic sarcomas
Braun AC, Mello CAL, Corassa MP, Alves VS, Abdallah EA, Díaz MTM, Urvanegia ACM, Flores BCTCP and Chinen LTD
A.C.Camargo Cancer Center, São Paulo, Brazil
Background: Sarcomas are rare malignances and presents high mortality rates as compared to other cancers. Half of patients will develop metastasis. The presence of isolated circulating tumor cells (CTCs) and circulating tumor microemboli (CTM) in the blood may be early markers of tumor invasion. Other factors also influence the process of metastases such as EGFR family receptors, involved in cell proliferation, migration, motility, and invasion of malignant cells.
Study aims: To isolate and quantify CTCs and CTMs, in addition to identify the expression of EGFR in these cells from patients with different types of metastatic sarcoma (leiomyosarcoma, synovial sarcoma, pleomorphic sarcoma, and liposarcoma).
Material and methods: Approximately 8 mL of blood was prospectively collected from patients with different types of metastatic sarcoma, before the beginning of chemotherapy. The samples were processed and filtered on ISET (Rarecells, France) system for the isolation and quantification of CTCs and CTMs. Later, immunocytochemistry (ICC) was performed. Anti-CD45 antibody was used to distinguish CTCs/CTMs from white blood cells, and EGFR antibody was used to search for its expression on CTCs/CTMs.
Results: We analyzed 18 patients with median age of 49 years (18–77 years). The median CTCs number was 2.0 CTCs/mL (0-11 CTCs/mL) and CTMs were found in 5 patients (27.7%). The positivity for EGFR protein in CTCs was observed in 83.33% of the patients (15 of 18) and in none CTM.
Conclusion: This is the first study to demonstrate, besides the CTC and CTM presence in sarcoma patients, the expression of EGFR protein in these cells. Our next step is to characterize these cells in a larger cohort of patients in an attempt to understand the role of EGFR in promoting tumor metastasis in sarcoma and relation of its expression with CTC migration.
Thyroid transcription factor 1 expression is associated with outcome of patients with non-squamous NSCLC treated with pemetrexed-based chemotherapy
Fiala O1, Pesek M2, Skrickova J3, Kolek V4, Salajka F5, Tomiskova M3, Satankova M3, Kultan J4, Kuliskova J4, Svaton M2, Hrnciarik M5, Hejduk K6, Chloupkova R6, Hornychova H7, Nova M7, Ryska A7 and Finek J1
1Department of Oncology and Radiotherapeutics, Medical School and Teaching Hospital Pilsen, Charles University, Prague, Czech Republic
2Department of Pneumology and Phthisiology, Medical School and Teaching Hospital Pilsen, Charles University, Prague, Czech Republic
3Department of Respiratory Diseases and TB, Medical School and Teaching Hospital Brno, Masaryk University, Brno, Czech Republic
4Department of Pneumology and Tuberculosis, Medical School and Teaching Hospital Olomouc, Palacky University Olomouc, Olomouc, Czech Republic
5Department of Pneumology, Medical School and Teaching Hospital Hradec Kralove, Charles University, Prague, Czech Republic
6Institute of Biostatistics and Analysis, Medical School and Teaching Hospital Brno, Masaryk University, Brno, Czech Republic
7The Fingerland Department of Pathology, Medical School and Teaching Hospital Hradec Kralove, Charles University, Prague, Czech Republic
Background: Pemetrexed is an antifolate cytostatic agent targeting several folate-dependent enzymatic pathways, which is widely used in the treatment of locally advanced or metastatic-stage non-small-cell lung cancer (NSCLC). Aside from the non-squamous histology, there is still no available molecular biomarker predicting treatment efficacy of pemetrexed-based chemotherapy.
Study aims: The aim of our retrospective study was to evaluate the association of thyroid transcription factor 1 (TTF-1) expression with outcome of a large cohort of patients with non-squamous NSCLC treated with pemetrexed.
Materials and methods: We retrospectively analyzed clinical data of 463 patients with advanced-stage NSCLC (IIIB or IV) treated with pemetrexed-based chemotherapy. TTF-1 expression was assessed using indirect immunohistochemical detection in formalin-fixed paraffin-embedded tumor tissue at the time of diagnosis.
Results: TTF-1 expression was detected in the tumor tissue from 76.0% of patients, and tumors from 24.0% of patients were TTF-1 negative. The median progression-free survival (PFS) and overall survival (OS) for patients with TTF-1-positive tumors were 4.8 and 11.8 compared to 2.8 and 8.3 months for those with TTF-1 negative tumors (p = 0.001 and p < 0.001). The multivariable Cox proportional hazards model revealed that TTF-1 expression was significantly associated with PFS (hazard ratio (HR) = 1.57, p < 0.001) and also with OS (HR = 1.73, p < 0.001).
Conclusion: The results of the conducted retrospective study suggest that the TTF-1 expression was independently associated with PFS and OS in patients with advanced-stage non-squamous NSCLC treated with pemetrexed-based chemotherapy.
Fibroblast growth factor 23 (FGF23) as a tumor marker of aggressiveness? A pilot study
Pestova M1, Topolcan O1, Kucera R1, Kinkorova J1, Simanek V1, Treska V2 and Fiala O3
1Department of Immunochemistry, University Hospital and Faculty of Medicine in Pilsen, Czech Republic
2Department of Surgery, University Hospital and Faculty of Medicine in Pilsen, Czech Republic
3Department of Oncology, University Hospital and Faculty of Medicine in Pilsen, Czech Republic
Background: The role of fibroblast growth factor 23 (FGF23) in the oncogenesis mechanism is currently not very clear. In the literature well-documented cases of tumor-induced osteomalacia which can be caused by high plasma levels of FGF23 due to genetic mutation in neoplastic tissue are available. The place of the effect of FGF23 are kidneys where the phosphate excretion increases in the proximal tubule. Osteomalacia is a logical consequence of the longer time of unresolved overproduction of FGF23. FGF23 is also an inhibitor of 1-alpha-hydroxylase, which catalyzes the conversion of vitamin D (calcidiol) into its active form (calcitriol). Some work deals with the relationship between FGF23 and vitamin D in the possible involvement of FGF23 in the oncogenesis process. Recent conclusions suggest that elevated levels of FGF23 may be involved in the development of colorectal neoplasia independent of vitamin D levels. The latest published information this year demonstrates the involvement of FGF23 in the angiogenesis process.
Study aims: To find out the utility of FGF23 in the early diagnosis and aggressiveness estimation of cancer.
Materials and methods: FGF23 was determined in 160 patients with the following types of cancer: colorectal cancer (40), breast cancer (20), liver metastasis (60), prostate cancer (20), and lung cancer (20). Control group consisted from 50 healthy persons. FGF23 was measured using chemiluminescent kit LIAISON FGF23 (DiaSorin, Italy).
Results: We proved significantly higher levels of FGF23 in patients with cancer compared to the healthy persons. Level of the FGF23 positively correlated with the stage of cancer. However, we were limited relatively small number of patients in the individual groups.
Conclusion: The pilot study shows that FGF23 is a promising biomarker, and importance for routine clinical practice will need to be tested in the larger multicentric study.
Can PHI better separate Gleason score 6 tumors and facilitate decision for right management for patients with prostate cancer?
Dolejsova O1, Fuchsova R2, Topolcˇan O2, Kucera R2, Hora M1, Svobodova H1, Hes O3, Eret V1 and Pecen L2
1Department of Urology, University Hospital and Faculty of Medicine in Pilsen, Czech Republic
2Department of Immunochemistry, University Hospital and Faculty of Medicine in Pilsen, Czech Republic
3Department of Pathology, University Hospital and Faculty of Medicine in Pilsen, Czech Republic
Background: Total level of prostate-specific antigen (tPSA) is used as a tumor marker in prostate cancer for a long time. It has limited specificity for detecting clinically significant prostate cancer. Its low specificity leads to an excessive number of prostate biopsies and resulted in an increase in detection of low-grade prostate cancer.
Study aims: The purpose of this study was to investigate the prostate health index (PHI) as a marker for tumor aggressiveness according to Gleason score (GS) in prostate biopsy and optimization for right indication for radical prostatectomy.
Materials and methods: Our cohort of 320 cases with preoperative measurements of total PSA, free PSA, -2proPSA, and calculated PHI was prospectively evaluated. All measurements were performed using chemiluminescent kits ACCESS (Beckman Coulter, USA). GS in biopsy and after radical prostatectomy was determined and compared to each other and with biochemical results. Specimens after radical prostatectomy were processed using whole-mount sections.
Results: The median PSA was 8.07 ng/mL. Median preoperative -2proPSA was 18.00 pg/mL (4.00–304.00) and PHI 57.32 (17.04–292.74). In 95 cases, definitive GS 6 appeared, median PHI is 43.24 ng/mL (17.04–103.79), -2proPSA 14.00 (4.00–44.0). We found statistically significant correlation between definitive GS6, PHI, and proPSA (p < 0.001) by comparison with definitive GS above 6.
Conclusion: PHI can better separate GS 6 (grade group 1) tumors and can facilitate decision for right management for patients with prostate cancer. PHI can be helpful marker for indication active surveillance and also can simplify urologist and patient’s decision for nerve sparing radical prostatectomy.
Biomarkers assess response to new therapies and provide prognosis in melanoma patients
Barak V, Leibovici V, Peretz T, Kalichman I, Lotem M and Merims S
Departments of Oncology and Dermatology, Hadassah–Hebrew University Medical Center, Jerusalem, Israel
Background: Malignant melanoma’s (MM) incidence is increasing lately, while mortality is strongly decreasing due to improved early detection, close monitoring of patients including biomarkers, and new therapies.
Aims: To evaluate a panel of biomarkers (S-100β, LDH, OPN, MIA, sIL-2R, and TK β2M) in MM patients to assess treatment response, especially to new immunotherapies (Anti-BRAF, IPI, and Anti-PD-1) and provide prognosis of those patients.
Materials and methods: We evaluated 247 MM patients and correlated markers to disease stage, metastases, and response to new immunotherapies and survival.
Results: Serum levels of S-100 were significantly higher in all patients before therapy, n = 89, (7.35 + 0.7) and decreased thereafter, n = 68 (1.4 + 0.3). Significantly higher levels were demonstrated in advanced disease including metastases (6.97 + 0.5) than in early disease (0.32 + 0.07) and no evidence of disease (NED) patients (0.16 + 0.04). Deceased MM patients had extremely high levels of S-100β (8.2 + 0.4), while significantly lower levels in alive patients (0.26 + 0.02) and controls (0.08 + 0.02) were found. Kinetic evaluations showed earlier response, a few months, to therapy than carcinoid tumor (CT) evaluations.
Conclusion: S-100β was the most useful biomarker for assessment of treatment response and prognosis, especially after using new immunological treatments in MM patients. Additional biomarkers, such as LDH, β2M, sIL-2R, and TK, may serve as a panel to improve recurrence detection and metastasis, affecting survival.
Investigation of BCR ABL fusion in a cohort of Brazilian patients with chronic myeloid leukemia symptoms
Agostinho LA1,2, Moreira TCG1,2, Samico RC1, Kussumi VM1 and Drummond M1
1Fundação Cristiano Varella – Hospital do Câncer de Muriaé, Muriaé city, Minas Gerais state, Brazil
2Centro Universitário Faminas (UNIFAMINAS), Muriaé city, Minas Gerais state, Brazil
Background: Chronic myeloid leukemia (CML) is a myeloproliferative disorder characterized by the presence of translocation between chromosomes 9 and 22 in at least 95% of cases. The p210 isoforms are the most frequent in CML, and their frequency varies in different populations. Individuals with CML bearing e1a2 isoform are rare, and this transcript encodes the p190 oncoprotein.
Study aims: The aim of this study was to determine the frequency of BCR-ABL gene fusions encoding the p210 (b3a2 and b2a2) and p190 (e1a2) transcripts in Brazilian patients with CML and acute lymphoblastic leukemia (ALL) symptoms at Cancer Hospital in Muriaé city, Minas Gerais state.
Materials and methods: The rearrangement between the BCR and ABL genes was detected by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) with TaqMan assay (Thermo Fisher Scientific).
Results: In 20 individuals investigated with suspected CML or ALL, 17 and 3, respectively, 3 had rare fusion p190, 13 had p210 fusion, and 4 did not have the fusion despite having a suspected clinical symptom. The e1a2 isoform was found in three patients: two with ALL and one with CML. The frequency of p190 fusion was observed in 15% of all sample (n = 20) and 7.1% in patients with CML symptoms (n = 14), and this frequency is higher than literature data (1%). The both patients affected by Acute Lymphoblastic Leukemia have HLA-DR and CD34 markers detected by immunophenotyping assay and, only one of them, has CD7 marker. The three patients bearing p190 isoform have at least a translocation between 9 and 22 chromosomes by karyotype. The p190 transcript considered rare was observed with higher frequency in our sample of patients with CML. The patients with CML and p190 transcript may be associated with reduced therapeutic response to tyrosine kinase inhibitors with higher molecular response in 10% of cases.
Conclusions: There is a controversy about the therapeutic response of patients with different transcripts being investigated, such as b3a2, b2a2, and e1a2; therefore, it is important to determine them in the diagnosis of leukemias.
Selection of reference genes for the normalization analysis of miRNA expression in liquid-based cervical cytology samples based on RT-qPCR
Causin RL, Souza KCB, Leal LF, Evangelista FM, Matsushita GM, Reis RM, Fregnani JHTG and Marques MMC
Barretos Cancer Hospital, Barretos, Brazil
Background: There are numerous and well-documented evidence in the literature showing that cervical cytology is limited as a single method for the screening of cervical cancer. In view of this, it would be extremely important to incorporate molecular tests that could improve accuracy for the detection of precursor lesions. In this scenario, the evaluation of the differential expression of microRNAs (miRNAs) has been very well pointed out in the literature, because these molecules regulate key processes for tumor development and progression. However, a few studies have been carried out in liquid cytology samples to detect the expression of miRNAs in cervical cancer precursor lesions. Furthermore, they used a small population or evaluated a few groups of miRNAs. In view of the above, there is a need for technical standardization for the analysis of miRNA expression in liquid-based cytology samples, because there is very little evidence in the literature that used liquid-based cytology samples for the analysis of miRNA expression.
Study aims: To select housekeeping for analysis of miRNA expression in liquid-based cervical cytology samples.
Materials and methods: Expression of U6, hsa-miR-16, and RNU44, 47, 48, and 49 was measured by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). Reference genes expression values were normalized to the reference using the comparative CT method (2-ΔCT). We used one common software, namely, NormFinder, to analyze expression stability of the six selected genes. In the NormFinder software, a stability value is also calculated for each gene by a model-based approach, taking into account its intra- and inter-group variations. NormFinder ranks candidate reference genes by calculating their stability values, with lower values indicating more stable genes.1
Results: The stability values calculated using NormFinder in six housekeepings were as follows: U6 = 0.787, miR-16 = 9.728, RNU-44 = 7.912, RNU-47 = 3.234, RNU-48 = 5.132, and RNU-49 = 1.716; the standard error: U6 = 2.127, miR-16 = 1.990, RNU-44 = 1.684, RNU-47 = 1.084, RNU-48 = 1.265, and RNU-49 = 1.226.
Conclusion: we conclude that the small nucleolar RNA transcript U6 was the best reference gene.
Distinct microRNA expression profile in hereditary breast tumors from BRCA1/2 mutation carriers
Pereira DP, Evangelista AF, Leal LF, Santana IVV, Machado LFA, Dufloth RM, Reis RM, Vieira RAC and Marques MMC
Barretos Cancer Hospital, Barretos, Brazil
Background: Many models based on clinical criteria have been developed to estimate the likelihood that an individual at high risk for hereditary breast and ovarian cancer predisposition syndrome has a germline mutation in BRCA1 or BRCA2. However, current prediction strategies have not been fully accurate. MicroRNAs (miRNAs) are small non-coding RNAs involved in gene expression regulation at post-transcriptional level and have been described as potential diagnostic biomarkers in several tumors, including breast cancer. Nonetheless, little has been reported about their role in hereditary breast cancer (HBC). Since miRNAs play important role in breast cancer onset and progression, our hypothesis is that there are specific differentially expressed miRNAs in HBC that can be used as biomarkers to increase the current clinical criteria to genetic test.
Study aim: To determine miRNA expression profiles for predicting BRCA1/2 mutations in HBC.
Material and methods: Differential expression profiling of miRNAs was performed using the NanoString technology and assessed on 21 HBC (BRCA1 and BRCA2), 8 sporadic BC, and 8 normal breast tissues (NBTs). We used fold changes values >2.0 to define differentially expressed miRNAs and t-test p value <0.05 to satisfy significance. Potential biomarkers were selected after ROC curve analysis considering area under the curve (AUC) > 0.90.
Results: Unsupervised hierarchical clustering analysis revealed miRNAs showing a statistically significant differential expression between HBC, sporadic BC, and NBT. Interestingly, we identify a specific molecular signature of breast tumors with mutation in BRCA1 versus in BRCA2.
Conclusion: MiRNA expression profiling revealed specifically deregulated miRNAs in HBC. Further analysis of these miRNAs in a large and independent cohort would provide a proper biomarker validation.
Early detection of luminal B breast cancer by circulating microRNAs using NanoString technology
Souza KCB, Evangelista AF, Leal LF, Causin RL, Vieira RAC, Neuber AC and Marques MMC
Barretos Cancer Hospital, Barretos, Brazil
Background: Breast cancer (BC) is the main cause of cancer death among women, and early diagnosis can improve the chances of cure. BC is a heterogeneous disease and clinical behaviors and therapies are different for each molecular subtype. Luminal B is a subtype characterized by expression of estrogen and progesterone receptors. Luminal B tumors comprise the majority of BCs and poor clinical outcome could be observed. The standard screening test in BC is Mammography that has certain limitations, such as false-negative results. Thus, an identification of minimally invasive biomarkers, such as serum and plasma, can be a promising tool that may contribute to early diagnosis. MicroRNAs (miRNAs), which are small molecules of RNA that regulate the expression of several genes, have been identified as potential biomarkers in cancer progression. Our hypothesis is that circulating miRNAs can be detected in blood and may be associated with the early diagnosis of luminal B BC.
Study aims: To identify miRNA expression profile in patients with onset-stage luminal B BC.
Materials and methods: In all, 16 cases were selected of luminal B BC stage I and II and 8 controls without risk for BC were included. We perform the RNA extractions from serum using miRNeasy kit (Qiagen) followed by molecular signatures of early-stage luminal B BC using NanoString technology. Statistical analyses were performed using the standard t-test method.
Results: Of the 800 miRNAs analyzed by this technology, 5 miRNAs had significant differential expression (p < 0.05, 1.5-fold): miR-520d, miR-151b, miR-455-5p, miR-513c-5p, and miR-3065-5p. Evaluating the area under ROC curve (>0.90), just miR-3065-5p shown potential as biomarker for early detection in luminal B BC.
Conclusion: We found five circulating miRNAs in serum from patients with BC in comparison with healthy women as potential biomarkers for early detection of luminal B BC, of which miR-3065-5p could be the most reliable.
Possible triage in fit screening for colorectal cancer
Petersen MM, Nielsen HJ, Christensen IJ and Ferm L
Department of Surgical Gastroenterology, Hvidovre Hospital, Hvidovre, Denmark
Background: Screening for colorectal cancer based on the fecal immunochemical testing (FIT) for occult blood concept has been implemented in a variety of European countries including Denmark. Although the compliance issue still exists, FIT screening has been a success with very high numbers of FIT-positive subjects. That has led to challenges with the colonoscopy capacity, which has prompted some countries to increase the present cutoff level of 100 ng/mL. Definitely increased cutoff levels miss subjects with non-detected neoplastic lesions; current research indicates that an increase in the FIT cutoff level to 200 ng/mL will miss 9% CRC and 30% high-risk and medium-risk adenoma patients. Present research with focus on blood-based biomarkers indicates that implementation of a triage test (FIT + blood tests = ±colonoscopy) may lead to reduction of colonoscopy demands and still may identify most of the patients missed by increased cutoff levels.
Study aims: To evaluate the feasibility to collect and determine biomarkers in blood samples within the timeframe from a positive FIT test result to the subsequent colonoscopy to select subjects in need for colonoscopy.
Materials and methods: Participants of the Danish CRC screening program with a positive FIT result between 100 and 400 ng/mL are invited to one of the five hospitals of The Capital Region of Denmark. A blood collection of 90 mL is performed and are handled and frozen at −80°C according to a validated standard operating procedure (SOP). On daily basis, some of the samples are transported to a central laboratory for determination of various biomarkers.
Results: Participants of the Danish CRC screening program with a positive FIT result between 100 and 400 ng/mL are invited to one of the five hospitals of The Capital Region of Denmark. A blood collection of 90 mL is performed and are handled and frozen at −80°C according to a validated SOP. On daily basis, some of the samples are transported to a central laboratory for determination of various biomarkers.
Conclusion: Implementation of Triage including FIT and blood-based biomarkers may be a possibility to improve the screen-associated colonoscopy capacity.
External quality assessment scheme (EQA) for isolation and analysis of circulating tumor DNA (ctDNA): Results and recommendations
Haselmann V1, Ahmad-Nejad P2, Geilenkeuser WJ3, Duda A1, Götz M1, Eichner R1, Patton S4 and Neumaier M1
1Institute for Clinical Chemistry, Medical Faculty Mannheim, University Hospital Mannheim, University of Heidelberg, Mannheim, Germany
2Institute for Medical Laboratory Diagnostics, Centre for Clinical and Translational Research (CCTR), HELIOS Hospital, Witten/Herdecke University, Wuppertal, Germany
3German Society for Clinical Chemistry and Laboratory Medicine (DGKL), Reference Institute for Bioanalytics, Bonn, Germany
4European Molecular Genetic Quality Network (EMQN), Manchester Centre for Genomic Medicine, St Mary’s Hospital, Manchester, UK
Background: Circulating tumor DNA is considered to have a high potential for future early diagnosis of relapse and therapy failure in the management of malignancies.
Study aims: This external quality assessment (EQA) scheme aimed to address issues of analytical quality in this new diagnostic area.
Material and methods: Each of the two EQA schemes consisted of three 2 mL ethylenediaminetetraacetic acid (EDTA)–plasma samples spiked with fragmented genomic DNA with a mutant allele frequency (MAF) ranging from 0% to 10% dedicated to the analysis of nine known sequence variations in KRAS codon 12/13 and of BRAF V600E. Laboratories reports are as follows: (1) time elapsed for processing, (2) storage temperatures, (3) method used for extraction and quantification, (4) genotyping methodologies, and (5) results.
Results: In the first EQA scheme, 42 laboratories from 10 European countries participated; 72.3% reported to isolate cfDNA manually, 62.5% used the entire plasma volume for cell-free DNA (cfDNA) extraction, and 38.5% used >10% of cfDNA isolated for downstream genotyping. Of the methods used for quantification, PicoGreen demonstrated the lowest CV (33.7%). For genotyping, of the 11 different methods used, the highest error rate was observed for Sanger sequencing and the lowest for highly sensitive approaches like digital PCR. In total, an error rate of 6.09% (12/197) was determined. Evaluation of the second EQA scheme revealed comparable results. Interestingly, we observed an increase in the error rate when decreasing the MAF, ranging from 0% for an MAF of 2% to 33% for an MAF of 0.1%.
Conclusion: This EQA scheme illustrates the current variability in multiple phases of cfDNA processing and analysis of ctDNA resulting in an overall error rate of 6.09%. The areas with the greatest variance and potential clinical impact included specimen volume, cfDNA quantification method, and preference of genotyping platform. Regarding quality assurance, there is an urgent need for harmonization of procedures and workflows in this new diagnostic field.
Phi and prostate cancer: Optimal management
Kucera R1, Topolcan O1, Dolejsova O1, Hora H2, Fuchsova R2 and Kinkorova J1
1Department of Immunochemistry, University Hospital and Faculty of Medicine in Pilsen, Czech Republic
2Department of Urology, University Hospital and Faculty of Medicine in Pilsen, Czech Republic
Background: Prostate cancer is the fifth leading cause of cancer death among men. More than one million cases are worldwide diagnosed every year, and the mortality is over 300,000 deaths per year. Prostate-specific antigen (PSA) is a serine protease composed of 240 amino acids in a single polypeptide chain. In serum, PSA is primarily bound to alpha1-antichymotrypsin (95% of total PSA), to a lesser extent to alpha2-macroglobulin; 10%–30% serum PSA occurs in the free form (fPSA). Since the 80s of the last century, total level of prostate-specific antigen (tPSA) is used as a tumor marker in the prostate cancer diagnostics. Most of the prostate tumors are diagnosed using biopsy based on the elevated levels of PSA. It is also useful to determine the part of PSA called proPSA. This part of PSA is produced more by tumor cells. Based on the levels of PSA, proPSA, and free PSA, the prostate health index PHI can be calculated. PHI value higher than 40 may indicate a high risk of prostate cancer.
Study aims: Demonstration of the usefulness of PHI in the management of prostate cancer patients.
Materials and methods: Cohort of 1865 patients was evaluated. In this cohort of patients the following was done: 2800 biopsies, 1848 MR, 150 PET/MR, 4900 samples with the results of PSA, fPSA, -2proPSA, fPSA/tPSA calculated results and PHI. Gleason score was established in all patients.
Results: When AUC sensitivity of each laboratory parameter was compared, PHI achieved the highest value (0.8118). PHI also achieved the best correlation with the Gleason score (G6–G9), which allows PHI to be a reliable marker of aggressiveness of the prostate cancer.
Conclusion: Determining PHI allows optimal diagnostic algorithm for prostate cancer, improvement of differential diagnosis of carcinoma versus prostate hypertrophy, reducing biopsies and imaging techniques, more accurate prognosis estimation, optimizing the type of surgery, optimizing the postoperative treatment, and optimizing the follow-up.
Serum HER-2 as a sensitive tumor marker in breast cancer patients
Barak V, Breuer S, Cohen D, Kalichman I, Peretz T and Uziely B
Immunology Laboratory for Tumor Diagnosis, Department of Oncology, Hadassah–Hebrew University Medical Center, Jerusalem, Israel
Background: The HER-2/neu oncogene is overexpressed in breast cancer (BC). The serum HER-2 assay was Food and Drug Administration (FDA) cleared in 2000 for monitoring metastatic breast cancer (MBC) patients. Its levels were found elevated in 25% of primary BC and 25%–75% of MBC patients.
Aims: To evaluate the sensitivity of the serum HER-2 Tumor Marker in Israeli BC patients, as to treatment response (hormonal or targeted therapy), prediction of recurrence or metastases, and prognosis of those patients.
Materials and methods: In all, 277 BC patients and 100 controls were evaluated for serum HER-2 levels, compared to established tumor markers: CA15-3, CA125, CEA, and also the Cytokeratin TPS. Their levels were correlated to disease activity, metastases number/location, treatment response, prognosis, and survival.
Results: Significantly higher levels of HER-2 (and also of other tumor markers) were recorded in MBC: 44.3 + 4.6, as opposed to low levels in no evidence of disease (NED): 11.7 + 0.4 and controls: 10.8+0.2, which were very similar. Increasing levels of HER-2 recorded during patient’ monitoring (6.2 up to 43.5) were correlated to recurrence or metastasis formation, shown only later (4.9–8.7 m) by computed tomography (CT) or magnetic resonance imaging (MRI). Decreases in HER-2 levels (48.7–11.3) were correlated to treatment response, both hormonal and targeted therapies and to a better clinical outcome and survival.
Conclusion: Serum HER-2 is a sensitive tumor marker for BC patients, indicating response to treatments and detecting earlier recurrence or metastases formation than CTs or MRI—a lead time which could enable treatment initiation or changes and might predict which patients will benefit from new treatments.
The diagnostic and prognostic values of sIL-2R as an immune biomarker in head and neck cancers
Barak V1, Meirovich A1, Leibovici V2, Kalichman I1, Peretz T1, Eliashar R3 and Gross M3
1Department of Oncology, Hadassah–Hebrew University Medical Center, Jerusalem, Israel
2Department of Dermatology, Hadassah–Hebrew University Medical Center, Jerusalem, Israel
3Department of Otolaryngology-Head and Neck Surgery, Hadassah–Hebrew University Medical Center, Jerusalem, Israel
Background: Head and neck cancer (HNC) patients are diagnosed with usually advanced disease and multimodality therapies are required, as well as prognostic biomarkers, to predict their response and assess survival.
Study aims: We aimed to evaluate the clinical significance of the immune biomarker sIL-2R in parallel to establish tumor markers in HNC patients to assess therapy response and prognosis.
Materials and methods: We evaluated 338 blood samples from 148 HNC patients from several subgroups: 84 larynx carcinoma pre- and 39 post therapy, 49 oral cavity pre and 29 post therapy, and 13 nasopharynx and 18 parotid and other salivary gland patients. The control group included 66 healthy subjects. Serum sIL-2R levels were evaluated by enzyme-linked immunosorbent assay (ELISA) and correlated to disease stage, lymph nodes, response to therapy, survival, and cancer differentiation.
Results: Significantly higher sIL-2R levels were recorded in all HNC patients, as opposed to controls, in advanced versus early-stage disease, which decreased following therapy. sIL-2R levels distinguished best, in comparison to other tumor markers, between HNC patients and controls. Survival was strongly associated to lower sIL-2R levels in patients entering the study.
Conclusion: sIL-2R is a sensitive immune biomarker for HNC patients. Its levels correlate to disease stage, assess response to therapy, and are predictive of recurrence or therapy response. We suggest therefore using sIL-2R as a reliable prognostic marker in HNC patients as single marker or in a combined panel of biomarkers.
Cost/efficiency of the tumor marker use in the differential diagnosis of paraneoplastic syndromes or in cancer of unknown origin
Molina R, Bosch X, Auge JM, Molina V, Gonzalez De Aledo JM, Filella X and López-Soto A
Hospital Clinic of Barcelona, Barcelona, Spain
Background: An important number of patients with cancer are diagnosed in advanced state. These patients are commonly present with non-specific symptoms and signs such as wasting syndrome, bone pain, and hemoptysis, among others, although some are finally diagnosed with benign disease. These patients are usually hospitalized, spending several days undergoing multiple diagnostic tests. While some patients are diagnosed with well-defined cancers, 2%–5% of all newly diagnosed patients with solid malignancies are classified as having cancer of unknown primary site (CUP).
Study aims: To study admitted patients with suspected cancer of unknown primary site.
Material and methods: We studied 7165 consecutive patients admitted to the Internal Medicine Department of our hospital with suspected cancer; 4601 patients had non-malignant processes and 2564 had malignant disease. Determinations were considered positive for suspected malignancy when serum levels were as follows: carcinoembryonic antigen (CEA) >15 ng/mL (>20 in patients with renal failure or liver disease), alpha-fetoprotein (AFP) >40 ng/mL (>80 in patients with liver diseases), CA19.9 >200 U/mL (>500 U/mL in patients with liver diseases or gamma-glutamyl transferase (GGT) <150 or effusions; >1000 in patients with jaundice or GGT >150), neuron-specific enolase (NSE) >45 ng/mL (renal failure >50 mg/mL; samples with hemolysis were excluded), PSA >30 ng/mL (excluding acute prostatitis), TAG-72 >80 U/mL, CYFRA 21–1 >7.5 ng/mL (>19 ng/mL in patients with renal failure; >11 ng/mL in patients with liver cirrhosis or jaundice), >3.5 ng/mL for SCC (excluding patients with renal failure or skin disorders), CA15.3 >100 U/mL, and CA125 >350 U/mL (>600 U/mL in patients with pleural effusion and >900 U/mL in those with ascites).
Results: There was a specificity of 97% in patients without malignancy, sensitivity of 65.3% in patients with malignancy (74.8% in epithelial tumors), and 81.3% in 417 CUP. Tumor markers were useful in the differential diagnosis between epithelial and non-epithelial tumors, between brain masses (metastases vs primary tumors), and between benign or malignant origin of different clinical situations such as wasting syndrome, effusions, liver or bone lesions, and effusions with a positive predictive value higher than 95%. It is interesting to indicate that some tumor markers such as S-100, ProGRP (excluding renal failure), or PSA are clearly indicating the presence of a specific cancer. Others such as AFP, HE4, B-HGC, or HER-2/neu clearly indicate a few options about tumor origin, easy to distinguish between them. Specificity in this situation is higher than 99%. This open new option about the use of them for diagnosis of cancer. Finally, the use of TM initially decreases significantly the cost, increasing the efficiency.
Conclusion: Tumor markers are useful as an aid in the evaluation of the risk of cancer of these patients with suspected cancer and may be useful to reduce the hospitalization time, morbidity, and the number of diagnostic tests required for diagnosis of different situations as lung, brain, liver masses, or bone images.
Tumor markers in lung cancer A new step in the diagnosis of lung cancer?
Molina R1,2, Marrades RM1,2, Augé JM1,2, Filella X1,2, Barco A1,2, Trape J1,2, Reguart N1,2, Molins L1,2 and Agusti A1,2
1Hospital Clinic of Barcelona, Barcelona, Spain
2Hospital Macarena De Sevilla Y Althaia, Spain
Background: Lung cancer (LC) is the most frequent and fatal human cancer. The diagnosis of LC might be relatively straightforward in some patients but cumbersome in others. The use of circulating biomarkers (tumor markers (TM)) in the clinical management of patients with suspected LC is not currently recommended because of the lack of specific TM for lung or because their individual sensitivity is low or suggesting that TM are not specific of any histological type. However, this idea is not totally true. Some TM used in lungs such as CEA, CA15.3, SCC, CYFRA 21-1, or CA19.9 are not lung specific. However, other such as neuron-specific enolase (NSE) and pro-gastrin-releasing peptide (ProGRP) are predominant in small-cell lung cancer (SCLC).
Study aims: To evaluate TM concentrations in patients with cancer of different sites.
Material and methods: A prospective evaluation of 2179 patients with cancer of different sites.
Results: We found that 99% of those with ProGRP serum levels higher than 150 pg/mL were SCLC or neuroendocrine tumors. Likewise, patients with suspicious signs of LC and ProGRP higher than 150 pg/L or NSE higher than 50 ng/mL had a 98% probability of SCLC. By contrast, those patients with abnormal levels of another TM such as CEA, CA15.3, SCC, CYFRA, or CA19.9 had a 99.1% probability of non-small-cell lung cancer (NSCLC). In summary, a simple blood test is able to suggest histology with a precision similar to the pathological evaluation. Individual TM sensitivity is lower than 65%. However, in an evaluation of 2681 patients with LC, sensitivity was 91.3% in NSCLC and 97.4% in SCLC. It is interesting to indicate that abnormal tumor marker serum levels were found in 75% of stage I and II patients. These results are complementary with image techniques, being the best efficiency obtained with the combined use of TM and CT scan.
Conclusion: TMs are useful tools in helping the diagnosis and histological diagnosis of LC. The potential of these TM in the diagnostic and screening settings deserves further research.
Tumor markers in ovarian cancer therapy monitoring: Comparison with clinical criteria
Molina R1, Rico M1,2, Mellado B1, Gonzalez De Aledo JM1, Fuste P1, Augé JM1 and Filella X1
1Hospital Clinic of Barcelona, Barcelona, Spain
2Puerta Del Mar University Hospital, Cadiz, Spain
Background: Worldwide, ovarian cancer is the leading cause of death from gynecological cancer. It is estimated that 225,500 new cases and 140,200 deaths occurred worldwide in 2010. Approximately 75% of ovarian cancer patients are detected at advanced stages (Stage IIb, III, or IV), when the 5-year survival rates are less than 20%. The vast majority of those patients have high-grade serous tumors. The high-grade serous ovarian cancers are often chemo-sensitive and respond well to initial chemotherapy, but tumor recurrence is frequent and resistance to further therapy develops in nearly all patients over time. Predictive and therapy monitoring factors are important to increase chemotherapy efficiency.
Study aims: The objective of this study is to determine the CA125 and HE4 utility in chemotherapy monitoring and as predictive factor in patients with advanced ovarian carcinoma.
Material and methods: CA125 and HE4 serum levels were determined prechemotherapy and in the pre-cycle evaluation in 110 patients with ovarian cancer (79 stage III and 31 stage IV), being the majority of them serous-papillary carcinoma (80.4%). Different chemotherapy courses have been use, being platinum based chemotherapy the most frequent (88.4%). Therapy response was improvement in the majority of patients (75%) the majority of them (70% with complete radiological response).
Results: Basal HE4 and CA15 were abnormal in 96% and 92% of patients treated. No relationship among pretreatment tumor marker (TM) levels and tumor response was found with a similar median concentration, time to progression or survival. By contrast when TM levels before chemotherapy and 3 weeks after finishing it, or normalization of these TM levels are related with a higher proportion of tumor response, time to progression (both p = 0.0001) and survival (both p = 0.009). Improvement was found in 92% of patients with CA125 normalization or with decreasing levels higher than 5 times in contrast to only 48% in those with higher values. Progression was found in 3% and in 39% of the patients in these groups. Similar results were found with HE4 with improvement in 92% of patients with decreasing levels in contrast to 43% in those with higher values. Similar results were obtained when basal TM levels and results before the third chemotherapy cycle (2 months) were compared.
Conclusions: HE4 and CA125 are predictive factors and parameters useful in therapy monitoring being able to suggest progression or lack of response earlier.
Could microRNA patterns be useful in colorectal cancer?
Pesta M1,2, Kucera R2, Kulda V3, Topolcan O2 and Vychytilova-Faltejskova P4
1Department of Biology, Faculty of Medicine and University Hospital in Pilsen, Charles University, Prague, Czech Republic
2Department of Immunochemistry, Faculty of Medicine in Pilsen, University Hospital in Pilsen, Charles University, Prague, Czech Republic
3Department of Medical Chemistry and Biochemistry, Faculty of Medicine in Pilsen, University Hospital in Pilsen, Charles University, Prague, Czech Republic
4Central European Institute of Technology, Masaryk University, Brno, Czech Republic
Background: Colorectal cancer (CRC) is the third most common malignancy worldwide. Despite improvements in diagnostics and therapy, more than 50% of patients will eventually die from their disease. MicroRNAs (miRNAs) are small non-coding RNA molecules participating in complex manner in the post-transcriptional regulation of gene expression including competing endogenous RNA (ceRNA) networks. The human genome encodes over 2500 miRNAs, which may target about 60% of mammalian genes.
Study aims: We systematically analyzed the expression of miRNAs in resected liver metastases and their corresponding primary tumors in order to identify those associated with CRC metastasis.
Materials and methods and Results: In total, 86 samples of primary tumors and 86 samples of corresponding liver metastases from patients with metastatic CRC were analyzed by gene expression profiling (752 miRNAs). It has revealed patterns of miRNA expression associated with CRC metastasis. Candidate miRNAs were chosen for subsequent validation by quantitative polymerase chain reaction (qPCR), which confirmed significantly (p < 0.05) reduced expression of miR-143, miR-10b, and miR-28-5p and increased expression of miR-122, miR-122*, and miR-885-5p in the tissue of liver metastases.
Conclusion: MiRNAs can generally be assessed with more precision and ease than messenger RNA (mRNA) of coding genes, and miRNA analysis is less demanding in terms of both quality and quantity of isolated RNA, features problematic in samples as formalin-fixed paraffin-embedded tissue (FFPE) or body fluids including blood plasma/serum. MiRNAs lack a direct executive function as enzymes or signal transduction molecules that are the targets of current anti-cancer therapy. For this reason, we believe that the most beneficial clinical use of miRNA pattern is to predict the disease prognosis that will help in decision on administering of the appropriate anti-tumor treatment. Tissue miRNAs or circulating cell-free miRNAs may become valuable prognostic markers for CRC.
Combination of RECAF with other markers improves cancer diagnosis
Moro R
Pacific Biosciences Research Centre, Richmond, BC, Canada
Background: Most cancer markers are unsuited for screening due to unacceptable high rates of false positives or low sensitivity.
Study aims: Herein, we show that combining carcinoembryonic antigen (CEA), cancer antigen 125 (CA125), or prostate-specific antigen (PSA) with RECAF (receptor for AFP) increases performance to levels consistent with screening requirements.
Material and methods: RECAF Assays: Competitive RIA and chemiluminescence on plates coated with anti-RECAF Mab. CEA, CA125, and PSA: Values were obtained from credited clinical laboratories. Cancer diagnosis was confirmed histologically.
Colorectal cancer (CEA + RECAF): Normal = 129, Cancers = 73.
Ovarian cancer (CA125 + RECAF): Normal = 105, Cancers = 80 (Stages I and II = 31, Stages III and IV = 49).
Prostate cancer (PSA + RECAF): Samples were blind. Normal = 24, Benign (BPH) = 118 and 2 sets of cancer samples: Set 1: 100 samples (Stage I = 22, Stage II = 30, Stage III = 20, and Stage IV = 28). Set 2 = 112 samples not staged.
Data analysis strategy: We determined the cutoff value for 100% specificity for each individual marker by ROC analysis. Samples above those cutoffs were considered as positive.
Results: Colon cancer: Cutoffs for 100% specificity: CEA = 8.3 ng/mL and RECAF = 5300 U. Sensitivity = 57.5% (area under the curve (AUC) = 0.907) and 72.6% (AUC = 0.818), respectively. Combining both markers yielded 91.8% sensitivity at 100% specificity.
Ovarian cancer: Cutoffs for 100% specificity: CA125 = 36 U/mL and RECAF = 7500 U. Sensitivities: Stages I and II = 76%, stages III and IV = 88%, and overall = 83%. At 97% specificity for RECAF (same CA125 cutoff), the sensitivity for stages I and II was 86%.
Prostate cancer: For samples in the PSA gray zone (4–10 ng/mL), best RECAF cutoff = 3900 U, resulting in eliminating ~70% of unnecessary biopsies.
Conclusion: The combination of RECAF and other markers resulted in sensitivity and specificity values consistent with the requirements necessary for cancer screening.
Will patients benefit from more precise determination of established markers in breast cancer stratification?
Faulstich K
BioNTech AG / BioNTech Diagnostics GmbH, Director Business Development and Project Management, Mainz, Germany
Background: Accurate determination of the predictive markers human epidermal growth factor receptor 2 (HER-2/ERBB2), estrogen receptor (ER/ESR1), progesterone receptor (PgR/PGR), and marker of proliferation Ki67 (MKI67) is indispensable for therapeutic decision making in early breast cancer.
Study aims: In a multicenter prospective study, we addressed inter- and intra-site reproducibility using the reverse transcriptase quantitative polymerase chain reaction (RT-qPCR)-based MammaTyper® test. Additionally, we show clinical validation data to address clinical utility of the test.
Material and methods: A total of 10 international pathology institutions participated in a precision study and determined mRNA expression levels of ERBB2, ESR1, PGR, and MKI67 in both centrally and locally extracted RNA from FFPE breast cancer specimens with the MammaTyper® test. Reproducibility was assessed by means of variance component analysis, Fleiss’ kappa statistics, and interclass correlation coefficients (ICCs). In an adjuvant clinical study, 719 retrospective samples from the prospectively designed FinHer trial were measured. Clinical outcome data for overall survival (OS) and distant disease-free survival (DDFS) were analyzed, Kaplan–Meyer analyses performed and concordance rates between MammaTyper® and immunohistochemistry (IHC) established.
Results: Intersite reproducibility showed excellent site agreement for single-marker quantitative assessments (ICC values: 0.980–0.998; standard deviation (SD): 0.21–0.44). Binary single-marker results (pos/neg) and subtype agreement were almost perfect (kappa values 0.90–1.00). Kaplan–Meyer analyses in adjuvant setting showed favorable prognosis of DDFS when determining MKI67 by MammaTyper® (HR = 0.42, p = 0.001) compared to IHC (HR = 0.56, p = 0.005). Patients classified as Luminal B-like (HER-2 negative) by MammaTyper® show benefit from Taxane-based chemotherapy (MammaTyper®: hazard ratio (HR) = 0.31, p = 0.012; IHC: HR = 0.63, p = 0.350). Prediction of complete pathological response in neoadjuvant setting was superior to IHC.
Conclusions: MammaTyper® has the potential to substantially improve the current standards of breast cancer diagnostics by providing a highly precise assessment of the established breast cancer markers.
Using fecal immunochemical test for hemoglobin in a quantitative way for the diagnosis of colorectal neoplasia
Augé JMM, Sandalinas S and Fernadez E; On Behalf of Procolon Group
Biochemistry and Molecular Genetics Department, Hospital Clinic of Barcelona, Barcelona, Spain
Background: Currently, fecal immunochemical test (FIT) for hemoglobin is the test of choice in colorectal cancer (CRC) screening programs. FIT has different advantages when compared with the guaiac fecal occult blood test (gFOBT). One of these advantages is the quantitative nature of the test that allows knowing the fecal hemoglobin (f-Hb) concentration of each sample. This property is used in CRC screening programs for adjusting the cutoff to the colonoscopy capacities and even to stratify the population in different risk groups.
Study aims: The aim of this study is to show different manners of using FIT in a quantitative way for the decision making in CRC screening programs and in symptomatic patients.
Material and methods: Different populations have been analyzed: CRC screening population that includes women and men without any CRC family history and without known colorectal disease, ranging from 50 to 69 years, that have been participated in Barcelona CRC screening program; however, a population of symptomatic and under surveillance patients with an history of gastrointestinal symptoms or with a previous diagnostic of CRC or presence of polyps has also been studied.
Results: Median fecal hemoglobin concentrations in participants with a positive FIT (≥20 µg Hb/g feces) in a CRC screening program are significantly higher in those with advanced colorectal neoplasia (ACRN (colorectal cancer (CRC) plus advanced adenoma (AA))) compared with participants with non-ACRN. Multivariate logistic regression analysis has identified sex, age, and fecal hemoglobin concentration as independent predictive factors for ACRN. Combining these factors, we are able to identify 16 risk categories associated with different probabilities of identifying ACRN. Risk for ACRN increased 12-fold among individuals in the highest category compared with the lowest category; positive predictive values ranged from 21% to 76%. In symptomatic and under surveillance patients, f-Hb concentrations from patients with ACRN are also significantly higher than from those with a normal colonoscopy examination. Sensitivity for ACRN ranges from 28.9% to 46.5% with a specificity ranging from 85.0% to 93.2%. The positive predictive values for detecting CRC and AA range from 11.6% to 20.6% and from 34.7% to 42.3%, respectively, and the negative predictive value for ACRN ranges from 90.2% to 88.4%.
Conclusion: Quantitative FIT is a useful tool in CRC screening programs and in the assessment of symptomatic patients, allowing a personalized evaluation in the risk stratification for ACRN and as a test for ruling out the presence of colorectal disease in symptomatic patients.
Utility of proGRP as a tumor marker in the medullary thyroid carcinoma
Parra-Robert M, Orois A, Augé JM, Halperin I, Filella X and Molina R
Department of Biochemistry and Molecular Genetics, Department of Endocrinology, Hospital Clinic of Barcelona, Barcelona, Spain
Background: Medullary thyroid carcinoma (MTC) is a neuroendocrine tumor that has its origin in parafollicular C-cells of the thyroid, where calcitonin (CT) is released. Currently, the accepted tumor markers (TMs) for the diagnosis and follow-up of MTC patients are CT and carcinoembryonic antigen (CEA). However, progastrin releasing peptide (proGRP) has been proposed as a TM useful in the MTC.
Study aims: Our aims were to investigate proGRP in thyroid tumors and its role in the assessment of advanced MTC as well as its utility in the differential diagnosis between MTC and non-MTC thyroid tumors.
Material and methods: Serum samples from 22 patients with MTC and 16 with non-MTC were collected. Patients were classified into advanced cancer or no evidence of disease (NED). ProGRP was performed by Architect (Abbott Diagnostics), CT by LIAISON (DiaSorin), and CEA by Cobas E601 (Roche Diagnostics).
Results: ProGRP median concentration in advanced MTC was significantly higher (1398.4 pg/mL) when compared with non-MTC, either in advanced disease (24.9 pg/mL) or NED (14.6 pg/mL). In non-MTC patients, proGRP median concentration was below its cutoff (50 pg/mL). Similar to CT. ProGRP was able to detect 88.9% of MTC patients, but with a slightly lower specificity of 76.9%. Using proGRP in combination with CT, the sensitivity increased to 100%.
Conclusion: The low prevalence of this malignancy strongly recommends further collaborative studies, mainly focused on monitoring proGRP during tyrosine kinase inhibitors treatment for early detection of resistance and assessing its usefulness to avoid the observed false-positive fluctuations that occur with CT and CEA.
Predictive role of KRAS mutation type in patients with metastatic colorectal cancer treated with bevacizumab
Fiala O1, Buchler T2, Mohelnikova-Duchonova B3, Melichar B3, Matejka M1, Kulhankova J1, Bortlicek Z4, Bartouskova M3, Liska V5, Topolcan O6, Kucera R6, Sedivcova M7 and Finek J1
1Department of Oncology and Radiotherapy, Medical School and Teaching Hospital in Pilsen, Charles University in Prague, Prague, Czech Republic
2Department of Oncology and First Faculty of Medicine, Charles University and Thomayer Hospital, Prague, Czech Republic
3Department of Oncology, Palacký University Medical School and Teaching Hospital, Olomouc, Czech Republic
4Institute of Biostatistics and Analyses, Faculty of Medicine, Masaryk University, Brno, Czech Republic
5Department of Surgery, Medical School and Teaching Hospital in Pilsen, Charles University in Prague, Prague, Czech Republic
6Department of Immunochemistry, Medical School and Teaching Hospital in Pilsen, Charles University in Prague, Prague, Czech Republic
7Molecular Pathology Laboratory, Bioptic Laboratory, Ltd., Pilsen, Czech Republic
Background: The KRAS mutations are found in 35%–45% of colorectal cancer (CRC) cases. Although the association between the RAS signaling and angiogenesis is well known, the negative predictive value of KRAS mutation has not been established in patients treated with bevacizumab.
Study aims: The aim of this study was to evaluate the association between specific KRAS mutation types and outcome of patients with metastatic CRC treated with bevacizumab. The study included 404 patients with metastatic colorectal cancer (mCRC) treated with bevacizumab.
Materials and methods: Clinical data obtained from the clinical registry CORECT were retrospectively analyzed.
Results: The shortest survival was observed in patients with tumors harboring G12V or G12A KRAS mutation (G12V/A). The median progression-free survival (PFS) and overall survival (OS) for patients with tumors harboring G12V/A KRAS mutation were 6.6 and 16.8 compared to 11.6 and 26.3 months, respectively, for patients with tumors harboring other KRAS mutation type (p < 0.001 and p < 0.001), while the survival of patients harboring other KRAS mutation types was comparable to those with tumors harboring wild-type KRAS gene. In the Cox multivariable analysis, KRAS G12V/A mutation type remains a significant factor predicting both PFS (hazard ratio (HR) = 2.18, p < 0.001) and OS (HR = 2.58, p < 0.001).
Conclusion: In conclusion, the results of this study indicate that there is a significant difference in biological behavior between tumors harboring G12V/A and other KRAS mutations. Moreover, comparison of survival of patients with tumors harboring G12V/A KRAS mutations with those harboring wild-type KRAS gene revealed that G12V/A KRAS mutations are prognostic biomarker for inferior PFS and OS in patients with mCRC treated with bevacizumab in univariate as well as multivariable analyses.
Importance of serum biomarkers for clinical response monitoring in small-cell lung carcinoma
Holdenrieder S
Institute of Laboratory Medicine, German Heart Center of the Technical University, Munich, Germany
Background: Progastrin-releasing peptide (ProGRP) is known to be a valuable serum biomarker for differential diagnosis of small-cell lung cancer (SCLC) and is widely used in follow-up of SCLC during and after therapies. However, systematic evaluations on its clinical significance for monitoring treatment response are rare. Existing studies on SCLC patients undergoing chemotherapies indicate a quick decrease of ProGRP to levels within the reference range until start of the second or third cycle even if pretherapeutic serum levels were quite high. In particular, patients with remission demonstrate a strong and sustained decrease to very low levels, while patients with insufficient response to therapy show early re-increases after an initial drop.
Methods: In a recent study performed by six lung cancer centers in Europe and China, ProGRP levels were prospectively measured before and during treatment of SCLC patients and correlated with imaging results. Percent changes of ProGRP from baseline were compared with macroscopic response to therapy.
Results: Majority of 215 patients with available CT result were either Caucasian (59.6%) or Asian (32.6%), presented with stage IIIB or IV disease (93.5%), and had efficient therapy response (stable disease or better; 86.5%). Decline of ProGRP levels strongly correlated with response to therapy (area under the curve (AUC): 81.2%). Different cutoff levels for ProGRP decline during treatment (–50%, –70%, or –90%), enabled the detection of progression with a sensitivity of 82.8%, 89.7%, and 96.6% and a specificity of 65.6%, 55.4%, and 39.8%, respectively.
Conclusion: This study impressively shows the clinical significance of ProGRP for monitoring the therapy response in SCLC patients. In addition, it reveals the challenges to appropriately interpret biomarker monitoring studies due to immanent difficulties such as (1) the questionable accuracy and clinical relevance of radiological staging (type of imaging, inter-observer variance, RECIST criteria, tumor size in computed tomography (CT) vs activity in positron emission tomography (PET)), (2) the type of progression (primary vs metastases, slow vs fast, different response of multiple lesions), and (3) the lead time of biomarkers that may lack a correlation with imaging at a given time point but anticipate later progression.
Diagnostic workflows for liquid biopsy approaches
Perakis S, Zhuo Q, Weber S, Ulz P, Geigl JB, Speicher MR and Heitzer E
Institute of Human Genetics, Medical University of Graz, Graz, Austria
Background: Tumor genomes are constantly changing, especially under the selective pressure of a given therapy. However, genomic alterations and the dynamics of tumor evolution in metastatic cancers remain incompletely characterized due to the challenge of repeated tumor sampling. The establishment of biomarkers from easily assessable biofluids like blood or plasma is beneficial compared to tissue biopsy as repeated sampling is easily achievable. In fact, circulating tumor DNA (ctDNA) was reported to represent a highly sensitive biomarker of metastatic cancer disease directly reflecting tumor burden and dynamics. Most studies have focused on point mutations although there is a clear need to move beyond these specific mutations and toward the full spectrum of genomic alterations, that is, aneuploidy, amplifications, deletions, and translocations, since these aberrations represent some of the most clinically useful genomic targets in cancer (e.g. ERBB2, AR, and KRAS amplifications).
Study aims: To define a diagnostic workflow for liquid biopsy approaches.
Material and methods: We use an untargeted approach called “plasma-Seq,” which is whole-genome sequencing of plasma DNA with a very shallow coverage of approximately 0.1×, for the establishment of genome-wide somatic copy number alterations (SCNA) as a monitoring tool for treatment response in metastatic cancer patients. In order to estimate the tumor content and to select samples that are suitable for plasma-Seq, we use our recently established mFAST-SeqS method as a pre-screening. Moreover, we apply several targeted approaches for the detection of driver mutations in cancer-associated genes.
Results: In addition to a clear correlation of changing ctDNA allele frequencies (AF) and treatment responses, we are able to observe the occurrence of resistance conferring SCNA before they become clinically obvious. Furthermore, novel therapy targets that have not been present in available tumor samples can be identified and the tumor evolution can be monitored.
Conclusion: Our data show that it is possible to non-invasively obtain molecular information on an individual patient’s cancer and to guide the choice of treatment or to understand why treatment has failed or is no longer working. Here, different workflows for ctDNA analyses will be presented and selected cases will demonstrate the potential clinical utility of ctDNA analyses.
Nucleosome positioning as cancer biomarker
Ulz P, Perakis S, Zhuo Q, Moser T, Speicher MR and Heitzer E
Institute of Human Genetics, Medical University of Graz, Graz, Austria
Background: The use of circulating tumor DNA (ctDNA) as a prognostic and/or predictive biomarker has been proven in numerous studies. To date, most studies focus on genetic alterations, that is, mutations or copy number alterations. However, recently evidence that cell-free DNA (cfDNA) reflects nucleosome footprints was reported. cfDNA/ctDNA fragments have been associated with the release of DNA from apoptotic cells after enzymatic processing since the distribution of their lengths has a mode near 166 bp in most analyses, a size which corresponds approximately to the DNA wrapped around a nucleosome (~147 bp) plus a linker fragment (~20 bp). This means cfDNA is actually released as nucleosomes into plasma and is partially protected from nuclease cleavage by packaging within nucleosomes.
Study aims: As nucleosomes are focal points of transcription control and are fundamental to DNA structure and gene regulation, we investigated whether plasma DNA is able to reflect expression-specific nucleosome occupancy at promoters. Furthermore, we assessed whether plasma DNA possesses the sensitivity and accuracy to predict whether genes are expressed or not, and furthermore, we determined whether blood samples from patients with cancer are informative for expressed cancer driver genes.
Materials and methods: Using whole-genome sequencing (WGS) datasets of plasma DNA, we identified two discrete regions at transcription start sites (TSS) where the nucleosome occupancy results in different read-depth coverage patterns in expressed and silent genes. By employing machine learning for gene classification, we found that the plasma DNA read depth patterns from healthy donors reflected the expression signature of hematopoietic cells. In cancer patients with metastatic disease, we were able to classify expressed cancer driver genes in regions with somatic copy number gains with high accuracy.
Conclusion: Our study suggests that read depth analyses of plasma DNA can reveal functional data such as the expression status of genes due to the nucleosome occupancy pattern at promoter regions and, furthermore, that even the expression status of cancer-related genes can be deduced from the blood of patients with cancer. Due to the increasing application of molecularly driven therapeutics, which rely on accurate and timely measurements of critical biomarkers, our results are of utmost significance, as they provide a new view on the genomes of the cells which release their DNA into the circulation. This significantly expands upon the currently existing options for cfDNA analysis.
Tumor markers as a diagnostic tool in patients with wasting syndrome
Trapé J1, Aligué J1, Arnau A1, San Jose A1,2, Ordeig J1, Ordeig R1, Franquesa F1, Bonet E M M1, Abril A1, González E1, Pruna M1, Sala M1 and Fígols C1
1Althaia—Xarxa Assistencial Universitària de Manresa, Manresa, Spain
2Vall d’Hebron Hospital, Barcelona, Spain
Background: Diagnosing patients with symptoms or signs of cancer is very difficult. Differential diagnosis with other pathologies may require a large number of tests. One of the signs of paraneoplastic syndrome is the isolated involuntary weight loss which is defined by a weight loss of more than 5% in 6 months with no other symptom and is characterized by anorexia, sarcopenia, cachexia, and dehydration.
Study aims: To study tumor marker expression in patients with wasting syndrome.
Materials and methods: In our institution, we studied 607 patients with isolated involuntary weight loss from primary care and emergency; from January 2007 to December 2013, the patients had a follow-up for 1 year.
Results: Of the patients studied, 24% were diagnosed of neoplasia, half were gastrointestinal (stomach, colon, liver, and pancreas), 43% of patients had non-neoplastic organic disease, and 30% had psychiatric disease. We found significant differences between patients who had cancer and benign diseases when using CEA, CYFRA 21-1, CA19-9, CA72-4, NSE, AFP, PSA, and CA15-3 but not for ß-2-microglobulin. We obtained a sensitivity of 66.7% at a specificity of 98.2% using following threshold values 15 µg/L, 7.8 µg/L, 200 KU/L, 80 KU/L, 45 µg/L, 40 µg/L, 30 µg/L, and 100 KU/L for CEA, CYFRA 21-1, CA19-9, CA72-4, NSE, AFP, PSA, and CA15-3, respectively. We established that the risk for malignancy in these patients was 3.3 95% confidence interval (CI: 1.6–6.6) when the more elevated tumor marker found between the upper reference limit value and cutoff, and the risk was 145 95% CI (57–369) when at least one marker exceeded the cutoff value. We detected 75% of epithelial tumors and 33% of non-epithelial tumors. In patients with a computed tomography (CT) scan suspicious of malignancy, all non-epithelial tumors were detected with the NSE, and the values of CEA, CYFRA21-1, CA19-9, CA72-4, AFP, PSA, or CA15-3 over the cut-off only identified epithelial tumors.
Conclusion: Tumor markers allowed to obtain relevant information in patients with involuntary weight loss by identifying those with a higher risk of cancer and also allowed to discriminate between patients with detectable mass by CT scan between epithelial and non-epithelial tumors.
New multifactorial approaches for simultaneous evaluation of pre-analytic factors on epigenetic nucleosome modification biomarkers
Uhlig S1, Hettwer K1, Frost K1, Schlierf A1, Trimpop N2, Kaiser V2, Semaan A2, Hartmann E2, Herzog M3, Micallef J3, Simon K1 and Holdenrieder S2,4
1QuoData—Quality and Statistics GmbH, Berlin, Germany
2University Hospital Bonn, Bonn, Germany
3Belgian VolitionRx, Namur, Belgium
4German Heart Centre, Munich, Germany
Background: Epigenetic nucleosome modifications such as DNA methylation, histone modifications, histone variants, and nucleosome adducts are promising new biomarkers for cancer diagnosis, prognosis, and monitoring that can be assessed by immunological tests.
Methods: The potential influence of preanalytical factors between blood drawing and centrifugation in the laboratory on the concentrations of two nucleosome biomarkers 5-methyl cytosine (5mc) and H4K20me3 was tested by a novel multifactorial setting in which each blood sample of 12 donors was handled in a different way. Thereby the factors donor type (cancer and healthy), sample matrix (serum and plasma), tube type (gel and kaolin), transport duration (1 and 6 h), transport temperature (4°C and 20°C), shaking during transport (yes and no), centrifugation duration (10 and 20 min), and cooling (4°C and off) were tested simultaneously.
Results: Specific sample collection and measurement settings enabled statistic testing of the influence of single factors, interactions thereof, estimation of the robustness of the results, as well as proband-specific factorial effects. Thereby, high stability of immunoassay results was shown for both markers with respect to all preanalytical factors in both patient groups. Differences were only seen with regard to the presence of disease.
Conclusion: This approach enables an elegant and time-, work-, and consumable-sparing procedure for simultaneous testing of the influence of preanalytical factors and the characterization of precision in the laboratory.
MR molecular imaging of cancer using targeted core/shell nanoparticles
Tomanek B1,2, Dash A3, Blasiak B2 and Van Veggel FCJM3,4
1Department of Oncology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada
2Institute of Nuclear Physics, Polish Academy of Sciences, Krakow, Poland
3Department of Chemistry, University of Victoria, Victoria, BC, Canada
4The Centre for Advanced Materials and Related Technology, University of Victoria, Victoria, BC, Canada
Background: While standard clinical magnetic resonance imaging (MRI) is a valuable tool for cancer detection, it often fails to detect cancer in its early stages or/and provides inconclusive results. Application of targeted nanoparticles to MRI could provide earlier and more accurate diagnosis. Contrast agents containing gadolinium are often used to increase MRI sensitivity, as they provide hyperintense T1-weighted MR image of tumor. However, superparamagnetic nanoparticles, such as iron oxide, reducing T2 and T2* and thus creating void signal, have also been employed to localize the disease. Accumulation of these contrast agents is based on differences in the vasculature of tumor and normal tissues, thus detection of tumor is limited and non-specific.
Study aims: To deal with limited contrast and lack of specificity of the current contrast agents, we developed new core/shell nanoparticles that reduce both T1 and T2 relaxation times and conjugated them with antibodies to render specificity using amine-functionalized coating.
Materials and methods: We investigated magnetic and biochemical properties of the contrast agents and their applications to glioma, breast, and prostate cancer detection. In vivo T1- and T2-weighted MR images were collected before and after injection of the targeted contrast agent using animal models. The agents consist of targeted NaDyF4/NaGdF4 nanoparticles, with the core and shell sizes controlled independently to provide maximum tumor contrast.
Results: This allows better tumor delineation and positive, tumor-specific contrast. A 9.4T MRI system was used for the experiments.
Conclusion: The results showed high efficacy of the new contrast agents, thus potential suitability for the early detection of cancerous tissues using molecular MRI.
Histone modification markers in colorectal cancer
Christensen IJ
Department of Gastroenterology, Hvidovre Hospital, Hvidovre, Denmark
Background: Circulating cell-free nucleosomes (ccfn) have shown promising association with colorectal cancer (CRC). However, the value in screening and early diagnosis of CRC needs further assessment.
Study aims: The aim was to evaluate serum levels of ccfn containing a variety of epigenetic signals including 5-methylcytosine DNA; histone modifications H3K9Me3, H3K9Ac, H3S10PO4, H3K36Me3, H4K20Me3, H4PanAc, and pH2AX; nucleosome variant H2AZ; and nucleosome adducts with HMGB1 and EZH2 as well as ccfn per se, in addition to develop and evaluate predictor models based on the above-mentioned ccfn and including serum levels of carcinoembryonic antigen (CEA) in early detection of CRC.
Material and methods: Blood samples were collected from 4105 individuals undergoing colonoscopy. Serum levels of ccfn and CEA were determined using enzyme-linked immunosorbent assay (ELISA) platforms.
Results: Individual assessment of levels of ccfn showed area under the receiver operating characteristic curve (AUCROC) = 0.525–0.576 in discrimination of individuals with CRC from individuals with non-malignant findings. Predictor models improved results (AUCROC = 0.736, sensitivity = 0.37 at specificity = 0.90). Further improvement was achieved in discrimination of individuals with CRC from individuals with clean colorectum (AUCROC = 0.846, sensitivity = 0.57 at specificity = 0.90).
Conclusion: The levels of ccfn among patients with CRC appeared to be stage-independent. In conclusion, the performance of the developed predictor models is very promising in early detection of CRC.
