Abstract
Invited lecture and oral presentation abstracts from the 44th Annual Congress of the International Society of Oncology and Biomarkers “Biomarkers in oncology: new horizons and challenges in the diagnosis and treatment of cancer.”
7–10 September 2017, Rio de Janeiro, Brazil.
An individual reference limit for “early” diagnosis of metastatic breast cancer during postoperative follow-up
This study is a clinical pilot study with the principal aim to investigate the accuracy of a panel of serum tumor markers (TMs) for the early diagnosis of relapses. We propose a systematic use of serum carcinoembryonic antigen–tissue polypeptide antigen–cancer antigen 15.3 (CEA-TPA-CA15.3) TM panel and criteria in order to make it an accurate tool for a postoperative breast cancer monitoring. A total of 211 disease-free breast cancer patients after mastectomy were intensively monitored with serial serum determination of CEA, CA15.3, and TPA. During a mean follow-up of 5 years, the sensitivity of the CEA-TPA-CA15.3 TM panel was 93%, the specificity was 97.6%, and the rate of false “warning signals” per year of follow-up was 9 per 100 patients. Our results show that the proposed tool is promising for a postoperative monitoring of breast cancer patients.
The importance of free light-chain dosage in multiple myeloma
Some hematological conditions may present with monoclonal gammopathies and could be indolent such as monoclonal gammopathy of undetermined significance or serious diseases such as multiple myeloma (MM). There are situations, such as non-secretory or oligosecretory MM, in which there is no correspondence between the tumor mass and the amount of immunoglobulin produced. Free light chains (FLCs) can be measured in serum through electrophoresis, which does not have sufficient sensitivity. Immunofixation in serum is more sensitive but not quantitative and therefore inappropriate for monitoring. The urinary protein electrophoresis is more sensitive than serum electrophoresis but urine concentration of FLCs is influenced by renal tubular absorption. Immunoassays specifically measure FLCs by detecting a “hidden” epitope on intact immunoglobulins and “visible” only on the FLCs. Renal tubules catabolize the light chains, reducing the amount excreted. Thus, the concentration of FLCs in the urine is determined by both tumor production and renal function. For this reason, serum FLC dosage is more sensitive than urine. Under normal conditions, about 500 mg of FLCs are produced per day, with a κ/λ ratio of about 2/1. As λ light chains (LCs) are dimeric, renal clearance is lower than that of κ chains, resulting in a serum κ/λ ratio of about 0.58 (range: 0.26–1.65). When FLCs are produced in excess, as in inflammation, immune disorders, renal failure, and plasma cell neoplasms, it results in serum accumulation. If the production is monoclonal, the ratio κ/λ changes and may be a useful parameter for the diagnosis and monitoring of this type of disease. Due to the short half-life of FLCs (2–6 h compared to 21 days for intact immunoglobulin G, e.g.), the assay may be useful for monitoring the efficacy of the therapy. In oligosecretory MM, in which the amount of secreted monoclonal protein is much lower than expected by the tumor burden, disease monitoring includes repeated testing for bone marrow and/or bone image plasma cell infiltration. In these cases, the evaluation of the FLCs and the relationship between them makes it possible to monitor the response to therapy. The measurement of serum FLCs has a greater correspondence with the tumor mass, especially when small amounts are present, as in residual disease. With this type of assay, reference ranges for the κ chain in serum are 3.3–19.4 mg, for λ chain 5.7–26.3 mg/L, κ/λ ratio without renal impairment 0.26–1.65, and κ/λ ratio with renal impairment 0.37–3.1.
Precision oncology for guiding and monitoring target therapy in lung tumors
Precision oncology is one of the most promising fields of cancer treatment. The ever-growing knowledge of the mutation repertoire that underlies the cancer development and progression has been fundamental for the definition of the altered gene pathways, which, in turn, has been the target of drug design. Epidermal growth factor receptor (EGFR) signaling pathway, a crucial pathway that regulates cell growth, survival, and differentiation, is recurrently altered in tumor cells and has been target for several and efficient tyrosine kinase inhibitors (TKIs). With the advances in genomics technologies, the state of the art in cancer treatment has changed and many clinical decisions have been made based on mutation genotype of specific genes. This new era of cancer treatment can be illustrated with lung adenocarcinoma (LA). A significant proportion of non–small-cell lung cancer (NSCLC), a subtype of LA, harbors specific activating mutations in kinase domain of EGFR, most of which predict sensitivity for TKI treatment. This discovery has dramatically changed the success in treating NSCLC patients. Taking advantage of this opportunity, I will show our experience in genomic testing for clinical decision for NSCLC patients. EGFR-activating mutation landscape of about 1000 cases as well as the sensitivity of different methodologies in detecting mutations will be presented. Despite the fact that the use of TKI in patients whose tumor harbor sensitivity mutations has showed considerable promise, the acquisition of resistance to TKI treatment has been an important issue. Monitoring this phenomenon in liquid biopsy to assess specific EGFR mutations in circulating tumor DNA (ctDNA) in plasma during treatment has substantially contributed to overcome this drawback. Some results about this approach will also be showed.
Emerging biomarkers for lung cancer immune therapy
The immune response is a very complex multifactorial “reaction.” Immunogenicity, cellular response available, and effective immune response are not consistent with tumor progression. The immune checkpoints present a number of negative regulatory immune checkpoints that negatively regulate immunologically primed and activated T cells and other immune cells at their end-effector site of activity, a physiological mechanism to avoid autoimmunity. Increased levels of PD-L1 protein expression (immunohistochemistry (IHC)) may inhibit immune response. As a result, high PD-L1 expression is assumed by tumors as a survival mechanism. Whether it is a poor prognostic factor in small-cell lung cancer (SCLC) remains to be determined. It is a therapeutic target in NSCLC and a predictive biomarker of anti-PD-1/PD-L1 therapy. The PD1–PD-L1 axis is a therapeutic target in NSCLC, so interruption of ligand–receptor binding can re-activate a primed but inhibited immune response. Biomarkers for immunotherapy are necessary because (1) precision medicine is a reality for advanced NSCLC, (2) only a minority of patients respond to and benefit from these treatments, (3) enrich the treatment population for benefit, (4) benefit is relative to standard of care, (5) the avoidance of harm, (6) there are toxicities from these drugs, and (7) is there a subgroup who fair worse on IO Rx? The cost of therapy is high. “Biomarkers” for PD1 axis inhibitors should take into account PD-L1 expression, mutational burden (or molecular surrogate), smoking history, tumor inflammation/immune response, the microbiome, general clinical status, and “trial of therapy.” “Immune contexture” varies intensely between tumors. The immune infiltration at the tumor site may be indicative of host response. Significant correlations were shown between the levels of immune cell infiltration in tumors and patient’s clinical outcome. Moreover, unbelievable progress comes from the discovery of mutation-encoded tumor neoantigens. In fact, as tumors grow, they acquire mutations that are able to influence the response of patients to immune checkpoint inhibitors (ICIs). It has been demonstrated that sensitivity to PD-1 and CTLA-4 blockade in patients with advanced NSCLC and melanoma was enhanced in tumors enriched for clonal neoantigens. CD8 and CD45RO densities were better predictor of recurrence and survival than TNM staging in colon cancer (Immunoscore, Paris, France). These correlations are not so clear in melanoma and NSCLC. Cancer immunotherapy may become a major treatment support in many cancers over the next decade. There are numerous immune cell types found in cancers and many components of an immune reaction to cancer. Thus, the tumor has many strategies to evade an immune response. It has been proposed that four different types of tumor microenvironment exist based on the presence or absence of tumor infiltrating lymphocytes and programmed death-ligand 1 (PD-L1) expression: type 1—adaptive immune resistance, type 2—immune ignorance, type 3—intrinsic induction, and type 4—alternative immune tolerance. Innate mechanisms driving PD-L1 expression may be stratified in genomic amplification events and constitutive oncogenic signaling such as that occurring in Hodgkin lymphoma, and biallelic loss of Lkb1+Pten leads to a mouse model of squamous cell carcinoma (SCC) with increased PD-L1, and JAK/STAT (Janus kinase/signal transducers and activators of transcription) pathway induces PD-L1. Adaptive mechanisms driving PD-L1 expression include the following: CD8+T-cell infiltrates release cytokines which activate tumor PD-L1 expression and PD-1 engagement and are found in LA, melanoma, Merkel cell carcinoma, NSCLC, breast cancer, and others. T-cell profiling was determined to predict “hot” immunophenotype and found that “Hot” tumors contain CD8+T cells expressing PD-1 and TIM3 inhibitory markers, PD-L1+tumor cells, and correlate with high mutational load, smoking history, and squamous morphology. “If the critical factor underlying the response to checkpoint blockade is the presence of leukocytes … then hybrid IHC/flow cytometry may improve upon PD-L1 IHC alone as a biomarker.” In addition, nonsynonymous mutations and smoking signature predict durable clinical benefit to pembrolizumab. In fact, nonsynonymous mutational burden from 300 gene panels correlates with clinical benefit but PD-L1 expression does not correlate with clinical benefit. An exploratory analysis was conducted in CheckMate 026 to test the hypothesis that patients with high tumor mutation burden (TMB) may derive enhanced benefit from nivolumab. For initial exploratory analysis, patients were divided into three subgroups based on TMB tertile distribution. Receiver operating characteristic (ROC) curves were generated and suggested that TMB has predictive power. Additional analyses to help further refine potential optimal cutpoints are ongoing. Recently, Food and Drug Administration (FDA) approves first cancer treatment for any solid tumor with a specific genetic feature with pembrolizumab. This agent is indicated for the treatment of patients with unresectable or metastatic solid tumors that have been identified as having a biomarker referred to as microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR). The efficacy results for patients (N = 149) with MSI-H/dMMR cancer showed objective response rate (ORR, 95% confidence interval (CI)) of 39.6% (31.7–47.9), complete response rate 7.4, partial response rate 32.2, response duration in months (range: 1.6–22.7), % >6 months 78%. All cancers are caused by somatic mutations. However, understanding of the biological processes generating these mutations is limited. The catalog of somatic mutations from a cancer genome bears the signatures of the mutational processes that have been operative. In one analysis of 4,938,362 mutations from 7042 cancers, the authors extracted more than 20 distinct mutational signatures. Some are present in many cancer types, notably a signature attributed to the APOBEC (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like) family of cytidine deaminases, whereas others are confined to a single class, such as BRCA and MMRd. Certain signatures are associated with age of the patient at cancer diagnosis, known mutagenic exposures or defects in DNA maintenance, such as tobacco, UV, POLE, and alkylator, but many are of cryptic origin. In addition to these genome-wide mutational signatures, hypermutation localized to small genomic regions, kataegis, is found in many cancer types. The results reveal the diversity of mutational processes underlying the development of cancer with potential implications for understanding of cancer etiology, prevention, and therapy.
Microbiome and cancer
Metagenomics, or the study of microorganisms from their genetic material in any given environment, is a rich source of potential biomarkers in oncology. The Laboratory of Medical Genomics at A.C. Camargo Cancer Center has been studying the complex interplay between microbes and cancer for a number of years. Several ongoing research projects will be discussed in this talk to illustrate the power of metagenomic profiling to uncover diagnostic and prognostic biomarkers that may contribute to disease detection and management. Oral cavity mucosa biofilm-associated bacteria may be involved with cancer development through the formation of carcinogenic substances in users of tobacco and alcohol. Dysbiosis in the normal tissue-associated microbiota composition may also contribute to carcinogenesis in rectal cancer. Changes in the diversity of microbial communities in the gastric tumor microenvironment, revealed by metagenomic analysis of gastric washes (GWs) obtained during endoscopy exam, have the potential to predict response to neoadjuvant chemotherapy and may help guide treatment decisions. Undesirable side effects of treatment in head and neck cancer, such as infection in the case of surgery and mucositis in the case of radiotherapy, may be circumvented with the identification of the tumor-associated microbial community changes that precede and accompany these events. Overall, our goal is to evaluate the impact of the microbiome in carcinogenesis and tumor progression and how this microbiome can be manipulated to improve treatment response and overall survival.
The whole population screening program for inherited cancers
We developed cancer risk assessment method, which predicts the risk of getting cancer, based on the clinical data provided by the patient and genomic data obtained by next-generation sequencing (NGS) of all the genes associated with familial cancers. Our goal is to identify all people with high risk of cancer in whole Polish population of 38 million. We just started the population screening program of the scale unheard of mainly due to high costs of genomic sequencing. To make the screening program possible, we developed a novel method of genomic sequencing and analysis, which decreased 20-fold the price of the multigene test, that is, below a hundred Euros. The method has been designed and tested by the interdisciplinary research team at the University of Warsaw and after validation immediately translated into clinical practice. It is estimated that there are at least a million women with high risk of getting breast cancer and the equal number of men with high risk of getting prostate cancer (PCa) in Poland. We target at finding them all with the aim of introducing them into personalized prophylactic program. The patient’s prophylactic plan is personalized based on his or her medical history and genetic mutations found in any of 70 cancer-related genes screened. During the presentation, we shall provide the preliminary results, including the prevalence and spectrum of gene mutations, based on first 3000 patients.
Personalized tumor markers
The cancer biomarker discovery pipeline is progressing slowly. The difficulties of finding novel and effective biomarkers for diagnosis and management of cancer patients are well known. We speculate that it is unlikely to discover new serological biomarkers characterized by high sensitivity and specificity. This projection is supported by recent findings that cancers are genetically highly heterogeneous. Here, we propose a new way of improving the landscape of cancer biomarker research. There are currently hundreds, if not thousands, of described biomarkers which perform at high specificity (>90%) but at relatively low sensitivity (<30%). We call these “rare TMs.” Borrowing from the principles of precision medicine, we advocate that among these low-sensitivity markers, some may be useful to specific patients. We suggest screening new patients for hundreds to thousands of cancer biomarkers to identify a few that are informative and then use them clinically. This is similar to what we currently do with genomics to identify personalized therapies. We further suggest this approach may explain as to why some biomarkers are elevated in only a small group of patients. It is likely that these differences in expression are linked to specific genomic alterations which could then be found with genomic sequencing.
Biomarkers in breast tumor tissue: role in determining prognosis and guiding treatment
Tissue biomarkers play an essential role in the management of patients with invasive breast cancer. For selecting patients likely to respond to endocrine therapy, both estrogen receptors (ERs) and progesterone receptors (PRs) should be measured on all newly diagnosed invasive breast cancers. On the other hand, for selecting likely response to all forms of anti-HER2 therapy (trastuzumab, pertuzumab, lapatinib, or ado-trastuzumab emtansine), determination of HER2 expression or gene copy number is mandatory. Where feasible, measurement of ER, PR, and HER2 should be performed on recurrent lesions and the primary invasive tumor. Although methodological problems exist in the determination of Ki67 because of its clearly established clinical value, wide availability, and low costs relative to the available multianalyte signatures, Ki67 may be used for determining prognosis, especially if values are low or high. In ER-positive, HER2-negative, lymph node–negative patients, multianalyte tests such as urokinase plasminogen activator (uPA)-PAI-1, Oncotype DX, MammaPrint, EndoPredict, Breast Cancer Index (BCI), and Prosigna (PAM50) may be used to predict outcome and aid adjunct therapy decision-making. Oncotype DX, MammaPrint, EndoPredict, and Prosigna may be similarly used in patients with 1–3 metastatic lymph nodes. All laboratories measuring biomarkers for patient management should use analytically and clinically validated assays, participate in external quality assurance programs, have established assay acceptance and rejection criteria, perform regular audits, and be accredited by an appropriate organization.
Genetics of endocrine tumors
The first genes that were found to be implicated in hereditary endocrine cancers were NF1 in 1990 and VHL and RET in 1993. Subsequently, other genes were also uncovered (MEN1, SDHB, SDHD, AIP) but each of these genes seemed to be causative of a unique spectrum of endocrine tumors, defining specific syndromes. Since then, more than 10 other genes have been discovered as causative of cancer endocrine syndromes. Of special relevance are (1) the steadily increasing number of genes implied in pheochromocytoma and paraganglioma syndromes, (2) the genes described to be implied in hyperparathyroidism, and (3) the genes causative of pituitary tumors. All these discoveries have led to the evidence that there is an important genetic–clinical overlap, expanding the endocrine cancer landscape. Here, we will present an update on the current knowledge of these genes and the spectra of these oncological endocrine diseases, altogether resulting in a more complex view than previous thought. We finally discuss how the NGS can help us to deal with these new discoveries.
Tumor markers in effusions
The effusions are a frequent reason for clinical consultations and about 5%–20% of all cases have a neoplastic origin. Between 24% and 50% of the patients with disseminated cancer develop pleural effusions, with about 100,000 new cases per year in the United States. These data clearly show the importance of having an efficient method for the differential diagnosis of patients with effusions. Cytology of the fluid is the mainly used technique, but unfortunately about 30% to 50% of malignant effusions display a negative cytology. This study presents the diagnostic possibilities of malignant effusions, using TMs. We describe the main problems in determination and interpretation of TMs in fluid effusions. In general, TM may be classified into two groups: those normally released by mesothelium, such as mesothelin, CA125, HE4, or CYFRA21-1, and those TMs not released by mesothelium, such as CEA, CA15-3, CA72-4, and CA19-9. Detection of high concentrations of TMs indicated the presence of neoplastic cells. However, important discrepancies have been reported in relation to the sensitivity, specificity, and cut-off; for example, CEA has described sensitivities between 27% and 82%, specificities between 77% and 100%, and cut-off from 3 to 275 ng/mL. We studied the main causes of these discrepancies and proposed procedures to avoid them. The simultaneous determinations of TM in effusions and serum have been suggested. When TM concentrations are higher in the effusion compared to the serum, it indicates local production and, indirectly, malignant effusion. Using these criteria in TM not released by mesothelium, the sensitivity is high, 70%–80%, with a high specificity (>98%) in both patients with negative or positive cytology.
Cancer classification and early cancer detection
The definitive diagnosis and classification of individual cancers underpins the care of individual cancer patients, as well as research into cancer causation, prevention, diagnosis, and treatment. Cancer classification has previously been based on consensus histopathological opinion with limited molecular input. Pathology is undergoing a rapid transformation due to the introduction of new technologies to practice. The understanding of cancer at a molecular level is now at a point where it needs to be integrated into its diagnosis. Digital pathology and image analysis are now also producing new insights, and providing quantitative justification of many existing diagnostic criteria, while challenging others. There is an urgent need to integrate these facets of diagnosis into cancer classification internationally. Early cancer detection may be followed by ablative or neoadjuvant treatment so that there may be little or no opportunity for histological diagnosis. This has implications for cancer epidemiology and research. The current (relatively) easy availability of tissue for research is also at risk. There is therefore a need to obtain diagnostic information from smaller and smaller samples, including liquid biopsy. This is a considerable challenge, and it is unclear to what extent it can be met. The use of genetic information from cell-free DNA (cfDNA) is one potential answer, while the isolation of circulating tumor cells (CTCs) represents another. However, few assays can match the diagnostic yield of histopadiamanthology. Correlation of these and other assays with histopathological diagnosis is important for validation of tests for early cancer detection.
A multiparametric approach including prostate health index to improve upon existing prostate cancer screening and biopsy recommendations
Screening for PCa is controversial because of concerns about over-diagnosis and overtreatment of non–life-threatening tumors. Therefore, an approach to screening that includes a detailed family history with genetic testing of risk single-nucleotide polymorphisms and high-penetrance genetic variants should be considered to determine the initiation and frequency of prostate-specific antigen (PSA) testing. After an elevated serum PSA level has been confirmed, additional testing should be considered before recommending a prostate biopsy. These additional tests should include prostate health index (PHI), a test that can help identify which men are most at risk of harboring PCa and aggressive disease. In addition, multiparametric magnetic resonance imaging (MRI) can be performed to improve upon the accuracy of tumor detection. The path forward involves this multiparametric risk assessment based on clinical information, genetic biomarkers, serum biomarkers, and imaging techniques. This multiparametric approach provides personalized screening that will overcome many of the current controversies surrounding PCa screening.
Personalized treatment for cancer: how biomarkers are showing the way
Personalized treatment can be defined as using the biological characteristics of a patient’s disease in order to administer the most effective therapy at the optimum dose. To provide personalized treatment for cancer, four main types of biomarkers are necessary. These include prognostic biomarkers to identify who should or should not receive adjuvant treatment, predictive biomarkers to identify the most appropriate therapy, toxicity biomarkers to identify potential severe toxicity, and monitoring biomarkers to assess real-time response to treatment. Validated prognostic biomarkers include uPA, Oncotype DX, and MammaPrint for lymph node–negative breast cancer, MSI for stage II colon cancer and alpha-fetoprotein (AFP), human chorionic gonadotropin (HCG), and lactate dehydrogenase (LDH) for advanced germ cell tumors. Everyday used predictive biomarkers include ER and HER2 for endocrine and anti-HER2 therapy, respectively, in breast cancer, mutation status of BRAF for anti-BRAF therapy in melanoma, KRAS/NRAS mutation status for anti-EGFR antibodies in colorectal cancer, and EGFR mutational status for anti-EGFR TKIs in NSCLC. Newly emerging predictive biomarkers for immunotherapy include MSI and PD-L1 levels. For upfront identification of patients at high risk of suffering from severe therapy–related toxicity in colorectal cancer, specific variants of dihydropyrimidine dehydrogenase (DPD) may be measured for predicting toxicity from fluoropyrimidines and uridine diphosphate glucuronosyltransferase*28 (UGT1A1*28) for predicting toxicity from irinotecan. Therapy-monitoring biomarkers include CEA for colorectal cancer, PSA for PCa, CA125 for ovarian cancer, and CA15-3, CEA, TPA, and tissue polypeptide-specific antigen (TPS) for breast cancer. The use of prognostic, predictive, toxicity, and monitoring biomarkers can thus help match each patient to the most effective and least toxic therapy and as a result avoid useless toxicity and unnecessary costs.
Prostate-specific antigen in screening for prostate cancer: more good than harm or more harm than good?
PSA is the most widely used cancer biomarker, worldwide. Indeed, it is the only serum biomarker widely used in cancer screening. Since the introduction of PSA screening in the late 1980s and early 1990s, the number of men diagnosed with PCa has greatly increased in several Western countries. Indeed, in many countries, PCa has become the most common non-skin cancer diagnosed in men, surpassing lung cancer. It is widely believed that this increase in PCa diagnosis directly relates to the increased use of PSA screening. As a result of this screening, most men who are diagnosed with PCa now present with localized and asymptomatic disease. While the incidence of PCa has increased since the introduction of PSA screening, mortality from the disease has decreased. Indeed, by 2008, mortality rates in the United States had decreased by almost 40%, relative to the levels in the early 1990s. At least part of this decrease has been speculated to be due to early detection due to PSA screening. Despite this apparent benefit of PSA screening for PCa, the practice is highly controversial, with mixed views from expert panels as to whether or not it should be performed. As a screening test for PCa, PSA lacks sensitivity and specificity for early disease. Furthermore, screening may lead to unnecessary biopsies, over-detection, and overtreatment. Although a large European randomized clinical trial showed that PSA screening resulted in an approximately 20% reduction in death from PCa, it is unclear whether the benefits of screening outweigh the potential harms associated with over-diagnosis and overtreatment. Although published guidelines differ in their recommendation for PSA screening, almost all state that prior to PSA testing, men should be informed of the risks and benefits of the process. Most guidelines also state that men with a life expectancy of less than 10 years should not be screened. New markers such as −2proPSA, PHI, and PCA3 may complement PSA in the detection of early PCa.
MicroRNAs in the tumor microenvironment
MicroRNAs (miRs), short, non-coding RNA molecules, are key post-transcriptional regulators of gene expression and, as such, control diverse biological functions and are implicated in the pathogenesis of cancer. Over the past decades, it has been well-documented that tumor progression does not rely solely on the intrinsic features of cancer cells, but it is strongly related to tumor microenvironment. Accumulating evidence shows that miRs significantly modulate tumor microenvironment, by influencing tumor cells, by affecting contribution of cell populations, and by playing an important role in immunosuppression, inflammatory reactions, and drug resistance. Some miRs, such as miR-155, miR-125b, and miR-21, are implicated in numerous mechanisms and are thought to significantly contribute to creating tumor-promoting microenvironment. We have shown significant changes in miR expression in gynecological cancers and central nervous system lymphomas, exemplifying the potential of miRNAs as tumor biomarkers. Manipulating the levels of miRs in the tumor microenvironment may provide new strategies to enhance anticancer therapies.
Protein electrophoresis and immunofixation in the diagnosis and follow-up of multiple myeloma
Protein electrophoresis and immunofixation are still crucial for MM management. A brief review and update will be presented about the fundamentals and interpretation of these tests in the diagnosis and follow-up of MM.
Tumor markers in gastrointestinal cancer
TMs are substances produced by cells tumors or by other cells in response to the tumor. They can be found in the blood, urine, stools, or saliva and need to have a high sensibility and accuracy. A lot of TMs had been studied for screening, diagnosis, and follow-up or to evaluate response during treatment. Most TMs are proteins; however, recent studies are trying to find genes or lipids that can help to diagnose and follow cancer patients. The markers commonly adopted on clinical use in gastrointestinal cancer are AFP, CEA, CA19-9, and CA72-4 and are measured in the serum. AFP and abdomen ultrasound are used on hepatocarcinoma screening in patients with chronic hepatitis. However, elevated levels of AFP >400 ng/mL are observed in only 20% of the patients and almost half of them had serum levels <20 ng/mL. Computed tomography (CT) and MRI are recommended for patients with rising levels of AFP. In the absence of mass in the liver, AFP and liver image must be repeated after 3 months. CEA, CA19-9, and CA72-4 are not used for cancer screening because they do not have the necessary specificity or sensitivity but is commonly used after cancer diagnosis. Colorectal cancer patients with higher levels of CEA or CA19-9 had worst prognosis. Higher levels of CEA during the follow-up are commonly associated with recurrence or disease progression. For colorectal cancer surveillance, CEA is measured every 3 months for 2 years and then every 6 months for more 3 years in patients with stage III or II with higher risks. Increased levels of CEA during surveillance detected more recurrences with possibility of curative treatment. CA19-9 is a sialylated Lewis A blood group protein used mainly in pancreatic and biliary cancers but raised levels may also be detected in other gastrointestinal cancers. Increased levels of this TM may indicate cancer recurrence or progression that needs to be confirmed by CT scans. In symptomatic patients with cancer of the pancreas, CA19-9 had 80% of sensitivity and 80%–90% of specificity. During chemotherapy treatment, the decrease in the levels of the TM in advanced pancreatic cancer is associated with an increase in survival. Although these markers had not a high sensitivity or specificity on diagnosis, the periodic determination of these markers has a high importance in the follow-up.
The fraction of cancer attributable to ways of life, infections, occupation, and environmental agents in Brazil in 2020
This lecture will present the estimates of the attributable fractions of 25 types of cancers in Brazil calculated to 2020 resulting from exposure of a wide range of modifiable risk factors potentially related to the environment and ways of life. These estimates were based on the prevalence of exposure to selected risk factors among adults obtained from population-based surveys conducted from 2000 to 2008 and on data drawn from meta-analyses or large, high-quality studies. The comparison of our results with the findings of other similar studies conducted in different countries as well as the need to improve strategies for primary prevention of the main risk factors and the promotion of a healthy diet and physical activity in the country will be discussed.
Prostate-specific antigen in the early diagnosis of prostate cancer: state of the art
PSA remains the most used biomarker in the detection of PCa. However, because of its low specificity, biopsy is positive only in around 25% of patients with PSA from 2 to 10 µg/L. Furthermore, the detection of non-significant PCa increased in relation to the extensive use of PSA in PCa screening. Active surveillance is now accepted as a valuable strategy in the management of non-aggressive PCa, decreasing the negative effects of over-diagnosis and overtreatment. The criteria for the selection of patients for active surveillance include PSA serum levels, biopsy Gleason score, and the percentage of biopsy material involved by the tumor. However, these criteria misclassify some patients. Novel tools are necessary in order to improve patient selection for active surveillance. New biomarkers have been proposed in recent years with the aim of increasing specificity and better distinguishing aggressive from non-aggressive PCa. PHI (PSA, fPSA, and p2PSA) and the 4Kscore (PSA, fPSA, iPSA, and hK2) are blood-based tests related to the aggressiveness of the tumor, which provide the risk of suffering PCa, avoiding negative biopsies. Furthermore, the use of urine has emerged as a non-invasive way to identify novel biomarkers, including PCA3 and TMPRSS2:ERG fusion gene. Available results showed the value of PCA3 score to decide the repetition of biopsy in patients with a previous negative result, although its relationship with the aggressiveness of the tumor is controversial. More recently, several authors reported the usefulness of exosomal and non-exosomal urinary miRNAs in the detection and prognosis of PCa. According to our experience, miR-21, miR-141, and miR-214 were found significantly deregulated in intermediate-/high-risk PCa versus low-risk/healthy subjects in urinary pellets. Significant differences between both groups were found in urinary exosomes for miR-21, miR-375, and let-7c. To summarize, current results suggest that emerging biomarkers may be useful as part of a multivariable approach for early detection of PCa. Although several of these new biomarkers have been recommended by different guidelines, large prospective and comparative studies are necessary to establish their value in PCa detection and prognosis.
The role of exosomes in tumor biology and tumor biomarker
The cellular secretion of vesicles in particular exosomes represents a remarkable system for short- and long-range cell-to-cell communication. Exosomes are extracellular vesicles defined as homogeneous membranous vesicles lined by a lipid bilayer, sized between 30 and 100 nm, and containing specific sets of proteins, lipids, and nucleic acids, in particular, miRs and non-coding RNAs. Tumor development and metastatic progression are supported by exosomes, which are released from the tumor cells to the blood or lymphatic system. Indeed, these extracellular vesicles also represent important circulating biomarkers. One of the most important problems in oncology is the resistance to treatment. We demonstrated that Ku70 and Ku80, two proteins of DNA repair, and Rab5C, a protein of the endocytic pathway, are highly expressed in the tumor tissue of patients with rectum cancer resistant to neoadjuvant radiotherapy. These proteins are also abundantly expressed in rectum tumor–derived cell lines resistant to ionizing irradiation, and functional assays demonstrated their direct involvement with the resistant phenotype. Moreover, exosomes secreted by the latter cells contain high levels of Ku70 and Ku80, and remarkably they can transfer the resistant phenotype to non-resistant cells, thus indicating that Ku70 and Ku80 in circulating exosomes of patients under neoadjuvant radiotherapy for rectum cancer should be explored as biomarkers for therapy response. The identification of biomarkers for resistance to 5-Fluorouracil neoadjuvant therapy in circulating exosomes from gastric cancer is also been explored. Another interesting opportunity is to evaluate the importance of proteins responsible for exosome biogenesis and secretion as prognostic biomarkers. We explored the levels of Rab7 and Rab27b, which, respectively, decrease and increase exosome secretion in tissue samples from 223 cases of head and neck squamous cell carcinoma (HNSCC) and correlated it with clinical data. Patients with HNSCC presenting higher levels of Rab7 or lower levels of Rab27b show a better prognosis with lower cancer recurrence or cancer-related death during 5 years of follow-up than those expressing lower levels of Rab7 or higher levels of Rab27, thus showing the importance of RAb7 and Rab27b expression as prognostic biomarkers for HNSCC. Therefore, the exosome protein cargo and cellular proteins associated with exosomes biogenesis and secretion should be explored as cancer biomarkers.
Immunotherapy in lung cancer
The incorporation of immunotherapy in the armamentarium for NSCLC treatment has been proven as a new and successful strategy to improve the outcomes of NSCLC patients diagnosed in advanced stages. ICIs directed to modulate the PD1/PDL-1 synapse have been used as single agents in either first-line or further line therapies. Pembrolizumab, an anti-PD1 antibody, has been proven to improve overall survival rates in comparison with platinum-based chemotherapy as a first-line treatment in metastatic NSCLC patients whose tumors present a tumor proportional score (TPS, a score of the PDL-1 expression, using clone 22C3) ≥50% in neoplastic cells. As second-line treatment, nivolumab (anti-PD1), pembrolizumab (in tumors with TPS ≥1%), and atezolizumab (anti-PDL1) were shown to improve overall survival in comparison with chemotherapy (docetaxel). Other outcomes, such as response rates, progression-free survival, and quality of life, are also in favor of the ICIs. The best predictive marker of benefit from these ICIs remains to be defined: each of the above-mentioned antibodies were developed in parallel with respective companion diagnostic tests, and despite similar analytical performance of PD-L1 expression for three assays, interchanging assays and cut-offs can lead to “misclassification” of PD-L1 status for some patients. Patient selection, diagnosis, and treatment of immune-related toxicities; the value of combining ICIs with other therapies (chemotherapy, radiation therapy, and anti-CTLA4 agents); and cost-effectiveness currently remain as challenges regarding the incorporation of ICIs in the daily practice.
Circulating tumor cells as a tool for personalized medicine
Metastases are the cause of 90% of cancer deaths. It is known that the spread of tumor cells can occur through the bloodstream. Studies have shown that the presence of CTCs is a worse prognostic factor and are also related to the greater tumor aggressiveness in several neoplasms. Studying these cells, then, one can have a view of the biology of the disease individually. Since 2004, many groups have been trying to develop technologies for CTC detection. Most methods rely on biological characteristics of tumor cells, such as epithelial surface markers. Studies have shown that the detection of CTCs by physical characteristics, such as cell size, may be more sensitive and specific. In this class, basic concepts on CTCs, as well as studies carried out by the main international groups, will be approached. We will show some results of our group evaluating markers of response to chemotherapy treatment in CTCs and their correlation with clinical evolution.
Gastric washes: an alternative approach for liquid biopsy-based biomarker identification in gastric adenocarcinoma patients
Gastric cancer, the third leading cause of cancer-related deaths worldwide, is a heterogeneous disease with high phenotypic and molecular diversity. Traditional histopathological classification methods are based on biopsies that represent a fraction of the tumor, collected at a given time point, and fail to properly represent global tumor aspects and its molecular diversity. Using next-generation deep sequencing techniques, we are now able to identify mutations in body fluids and track tumor-derived DNA in order to perform a close patient follow-up during disease progression. Now, the non-invasive tests available for the early detection of cancerous lesions in the gastric mucosa are scarce and non-specific. The presence of tumor DNA is not restricted to blood samples and can be found in other body fluids, including urine and saliva. For gastric cancer patients, our group has recently identified that the liquid derived from the GW obtained during patient routine endoscopy can be a valuable source of tumor-derived DNA. This fluid can be representative of different parts of the gastric mucosa, and not only of a specific site, a current limitation of tissue biopsies. Sequencing of the TP53 gene in tumor biopsies, plasma (PL) and GW from gastric cancer patients allowed the identification of mutations found exclusively in the GW and that were absent from the corresponding paired biopsies, suggesting tumor heterogeneity not captured in the tissue samples obtained by endoscopy. When both GW and PL are considered together, we can achieve a detection level higher than those reported so far for liquid biopsies in gastric cancer studies. Such data would allow tracking of tumor mutational dynamics at diagnosis and during tumor progression and cancer treatment in a personalized fashion.
The challenges of using circulating tumor DNA as tumor biomarkers
The detection and characterization of ctDNA in plasma brought new possibilities and high expectations in the precision oncology field. The genetic information captured from the ctDNA analysis can be used in several stages of the cancer treatment, such as in early diagnosis, as a predictive or prognostic marker, or to monitor treatment effectiveness and resistance emergence, with minimal risk for the patient. In addition, this method permits assessing the tumor variability and heterogeneity, once ctDNA are expelled from cells from different regions of the tumor and metastases. In the clinics, currently, the use of ctDNA mutation analysis is focused mainly in molecular profiling and monitoring of diseases with known frequently mutated driver genes, such as lung and colon adenocarcinomas. However, recent improvements in molecular methods and discoveries about the biology of tumor cfDNA are expanding the variety of applications ctDNA, both in research and in clinical settings. In A.C. Camargo Cancer Center, our group is presently performing exploratory studies in diverse disease contexts to demonstrate the translational potential of ctDNA. Briefly, I will summarize our recent results using different approaches of ctDNA analysis, such as NGS-targeted panels, digital polymerase chain reaction (PCR), detection of specific genomic breakpoints of fusion genes, and genome-wide profiling of rare tumors, highlighting our key findings and challenges. Finally, I will discuss emerging genomics technologies and concepts that may further propel the ctDNA research field and its implementation in routine clinical practice.
Liquid profiling—new perspectives for prediction and monitoring of therapy response in cancer patients
The concept of “companion diagnostics” in the treatment of cancer patients with “targeted therapies” implies the pretherapeutic therapy stratification on the basis of tissue mutation status such as EGFR mutations for TKI therapies in lung cancer and absence of K-Ras mutations for cetuximab therapy in colorectal cancer. However, therapy response rates and duration are still limited due to primary or secondary resistances. Recently, genetic heterogeneity including spatial and temporal variability within a tumor or between primary tumor, lymph node, and distant metastases was uncovered explaining the difficulties of one-time molecular examinations in tissue biopsy material. Therefore, the need for continuous monitoring of the overall mutation status in the patient body as stratification tool for therapy modification became evident. Nowadays, sensitive blood-based diagnostics identifying mutations in CTCs and in circulating cell-free plasma DNA (cfDNA) are available. They could overcome the genetic heterogeneity as circulating cfDNA reflects the cancerous DNA changes in the whole body. As this concept of “liquid profiling” is only minimally invasive, it can be used to complement tissue biopsy for patient stratification and for the serial monitoring of successfully treated and newly occurring resistant cell clones at an individual level. Digital PCR technologies such as BEAMing (beads, emulsion, amplification, and magnetics) allow the sensitive quantification of specific mutations and have recently shown great potential for therapy monitoring, early recurrence detection, and resistance monitoring in colorectal and lung cancer. In a single-center study, we demonstrated the highly sensitive detection and quantification of K-Ras mutations in plasma DNA of 32 patients with pancreatic cancer receiving chemotherapy. Most remarkably, K-Ras mutation status in plasma was highly predictive for response to chemotherapy and for prognosis of progression-free and overall survival, while K-Ras tissue status was not. During the course of therapy, the amount of mutated plasma DNA correlated with therapy response and tumor recurrence. These findings underline the high clinical relevance of this “liquid profiling” approach for the individualized guidance of cancer patients.
The role of host’s genetic polymorphism in the determination of gastric disease severity
Helicobacter pylori causes one of the most common chronic infections in humans, being present in the gastric mucosa of more than 50% of the world’s population. The infection is predominantly acquired in childhood and persists for life unless treated. In most persons, the natural history of the infection is without complications, but peptic ulcer disease, distal gastric carcinoma, or mucosa-associated lymphoid gastric lymphoma may occur in a percentage of the infected individuals. The presence of the infection is not enough to explain why only a small percentage of the infected individuals will develop diseases that are more serious. Thus, an influence of host factors, environmental, and bacterial virulence has been investigated. The role of the host is not completely known. Genetic factors, age, and sex can determine the type of immune response to the infection, which can define the disease outcomes. In relation to genetic factors, polymorphisms on genes that encode cytokines have been target of studies. Polymorphisms in the gene that codifies interleukin (IL)-1β and the IL-1β antagonist receptor, for example, were associated with gastric cancer increased risk in Caucasian populations. However, these results were not observed in Asian countries. In Brazil, only polymorphisms in IL-1RN antagonist receptor gene were associated with increased gastric cancer risk. These results suggest an existence of ethnic and region differences. Other genes associated with gastric diseases have been studied. Although the found results are controversial, they might be important in the understanding of the pathogenesis of gastric cancer.
Cancer immunotherapy: breakthrough of “déjà vu all over again”?
From the application of Coley’s toxin in the early 1900s to the present clinical trials using immune checkpoint regulatory inhibitors, the history of cancer immunotherapy has consisted of extremely high levels of enthusiasm after anecdotal case reports of enormous success, followed by decreasing levels of enthusiasm as the results of controlled clinical trials are available. In this presentation, this pattern will be documented for various immunotherapeutic approaches over the years, the sole exception being vaccination against cancer-causing viruses, which have already prevented thousands of cancers. We can only hope that the present high level of enthusiasm for the use of immune stimulation by removal of blocks to cancer immunity will be more productive than the incremental improvements using previous immunotherapies.
Aflatoxin, stem cells, and cholangiocarcinogenesis in glutathionine-S-transferase A3 knockout mice
Aflatoxin (AFB1), a product of moldy grain (Aspergillus flavus), is a highly potent hepatocarcinogen for most species, including humans, rats, fish, and ducks. In areas of the world with endemic hepatitis B virus (HBV) infection and consumption of moldy grain, there was a very high incidence of hepatocellular carcinoma. In contrast, wild-type adult mice are completely resistant to AFB1. However, newborn mice and partially hepatectomized (PH) mice are sensitive during active hepatocyte proliferation. This AFB1 sensitivity is due to lack of a major detoxification mechanism, glutathione-S-transferase (GST) conjugation of the activated hydroxylated form of AFB1. There are at 21 GST subunits in the mouse and 17 in humans. GSTA3 is the major, and most likely only, subunit responsible for AFB1 detoxification. We engineered an mGSTA3 knockout (KO) mouse. Both male and female mGSTA3 KO mice show high binding of activated AFB1-hydroxylated moieties to DNA and acute liver injury to high doses of AFB1. There is no compensatory increase in other GST isoenzymes and our data indicate that mGSTA3 is the only subunit of GST that is active in AFB1 detoxification. During chronic AFB1 exposure of mGSTA3 KO mice, there are toxic hepatocellular findings including microvesicular fatty change, nuclear inclusions, and megahepatocytes. There is marked oval cell proliferation, much greater than that seen in any other model of hepatocarcinogenesis in mice. Mouse oval cells have been cultured and cell lines established that express markers of hepatic stem cells. The ability of these cell lines to initiate tumors or repair liver injury is under investigation. After long-term AFB1 exposure (0.5 mg/kg AFB1 for weekly for 12 weeks followed by 6 to 12 months without treatment), KO mice have multiple cholangiomas and cholangiocarcinomas (CCAs), but not hepatocellular carcinomas. Control wild-type mice show no changes. AFB1 contains a furan structure and furan is known to induce CCAs in rats. In conclusion, AFB1 given to mGSTA3 KO mice induced liver injury, massive oval (stem) cell proliferation, and CCAs showing that oval cells may give rise to not only hepatocellular carcinomas (in rats) but also CCAs. This is consistent with the hypothesis that oval cells are bi-polar liver stem cells.
Stem cell origin of cancer and a pre-clinical breast cancer stem cell model based on MMTV-PyMT mice for testing nanoparticle-directed therapy
A review of the role of stem cells in various theories of carcinogenesis (field, chemical, infectious, mutation, epigenetic) reveals that regardless of the cause, cancer arises from tissue stem cells. The stem cell model of cancer predicts that cancer treatment failure can be overcome if treatment is directed to the self-renewing cancer stem cell (CSC). Most therapeutic approaches are directed to the proliferating cells of the tumor (transit-amplifying cells, TACs) and not the CSCs. To advance therapy to CSCs, we need models of cancer in which the both the proliferating TACs and the non-proliferating CSCs can be identified, targeted, and treated. Cells comprising the microenvironment (stem cell niche) of CSCs also play a defining role in optimal tumor initiation (TI) potential and are a third target for treatment. The MMTV-PyMT transgenic mouse model of spontaneous breast cancer is used to identify CSCs, TACs, and tumor-supporting mesenchymal cells (cancer-associated fibroblasts, CAFs). Female mouse mammary cancer (FFMC) cell lines consisting of two major populations were generated from primary breast cancers: CD24+CD29+CD44lowCD49f+Sca-1low tumor initiation cells (TICs) and CD24-CD44+CAFs. CD24+CD49f+419II cells at 1 × 103 consistently initiated mammary gland tumors upon transplantation (TI), whereas the CD24− CAFs failed to initiate such tumors at 1 × 107 cells. Co-transplantation of CD44+ CAFs and CD49f TICs resulted in a significant increase in TI to the level of a single CSC. Knockdown of Bmi-1, a polycomb group protein and inhibitor of transcription of differentiation genes, by short hairpin RNA (shRNA) or small molecular inhibitor PTC 209, prevented 419II cells from initiating tumors. TI was restored when Bmi-1 was reintroduced via an expression vector or expression of the Bmi-1 inhibitor PTEN was suppressed. Preliminary data show localization of anti–CD49f-coated particles to growing breast cancers transplanted to syngeneic FVB/N mice. We now propose to use anti–CD49f-coated nanoparticles containing PTC 209 to inhibit Bmi-1 in CSCs, anti-CD24+ nanoparticles containing docetaxel to target therapy to the TACs, and anti-CD44+ particles to inhibit CAFs.