Sage Journals: Discover world-class research

Abstract

BACKGROUND:

ALK receptor tyrosine kinase (ALK) aberrations have an established role in pathogenesis of many neoplasms, but their clinical significance in high grade serous ovarian carcinoma (HGSOC) is unclear.

OBJECTIVE:

To analyse the frequency of ALK overexpression, molecular abnormalities of ALK, and their impact on the progression-free survival (PFS) and overall survival (OS) in HGSOC.

METHODS:

Protein expression was examined by immunohistochemistry (IHC) using three different clones of anti-ALK antibody. The presence of translocations was analysed using fluorescent in situ hybridization. Next-generation sequencing was used for studying the copy number variation, as well as point mutation and translocations involving other commonly rearranged genes.

RESULTS:

ALK overexpression was demonstrated in up to 52% of tumours, whereas ALK copy gains in 8.2%, with no clear impact on survival. ALK point mutations were identified in 13 tumours (8.9%), with 3 belonging to the class IV showing significantly better OS. A trend suggesting better PFS was also noticed in these cases. Additionally, three gene fusions were found: ERBB2-GRB7, PRKCA-BRCA1 and SND1-BRAF, none of which has been previously described in HGSOC.

CONCLUSIONS:

HGSOC harbouring activating ALK mutations might be associated with a better survival, while ALK overexpression and ALK amplification does not impact the prognosis.

Keywords

BRCA1 BRAF ERBB2 fusion point mutations

1. Introduction

Every year, over 300,000 women worldwide are diagnosed with ovarian cancer [1], and the overall survival (OS) remains low despite the combination of surgical and systemic treatment, with most of the patients dying within 3 years of diagnosis [2]. Understanding the underlying molecular abnormalities seems to be crucial for selection of the most appropriate therapy and to explore the possibility of new therapeutic options.

ALK receptor tyrosine kinase (ALK, anaplastic lymphoma kinase) is a tyrosine kinase receptor with an established role in the pathogenesis of different malignant neoplasms, including non-small cell lung carcinoma (NSCLC) and neuroblastoma [3]. Depending on the specific tumour type, ALK overexpression as well as ALK translocations and point mutations were proven to have various impact on clinical behaviour. For example, in anaplastic large cell lymphoma (ALCL) the response to classic chemotherapy and overall prognosis is more favourable if a tumour harbours ALK rearrangements. This resulted in the separation of this entity into ALK-positive and ALK-negative ALCL in recent WHO classifications, despite otherwise similar morphology and phenotype of these tumours [4]. In other neoplasms, however, such as neuroblastoma or colorectal carcinoma, ALK amplifications, point mutations and translocations result in worse prognosis and response to treatment [5, 6], or lack of response to certain treatment modalities [7].

The ALK overexpression in ovarian cancer was first described in 2012 by Ren et al. [8]. In a cohort of 69 malignant ovarian neoplasms the phosphorylated form of ALK was found in 3 cases of serous ovarian carcinomas. Another report on the ALK significance in the ovarian cancer documented ALK overexpression identified immunohistochemically in 27% of serous ovarian carcinomas [9]. In the last year an additional report suggesting potential negative clinical implications of ALK overexpression was published, based on in vitro studies and immunohistochemical analysis [10].

The difference in incidence of ALK overexpression in previously published studies indicated the necessity to perform additional tests to establish the actual incidence of ALK aberrations in ovarian carcinoma. As differences in the frequency of various molecular aberrations of ALK may also depend on patients’ ethnicity, as shown in NSCLCs [11] and renal cell carcinoma [12], further analyses seem justified. Prior studies in ovarian carcinoma were based mainly on Asian population, potentially narrowing the genetic landscape of examined tumours, and stipulating the need to perform a study on European patients. The incidence of ALK-positivity in specific populations seems to be particularly relevant in the context of the promising results in the preclinical studies on the antitumor activity of the crizotinib combined with cisplatin in the ovarian cancer cell lines [13].

The main goal of this study was to determine the frequency of ALK overexpression and various ALK aberrations in high grade serous ovarian carcinomas in the Polish population, as well as to examine their role as a potential prognostic and predictive factor for standard chemotherapy. The secondary goal of the study was to establish the value of immunohistochemistry as a screening method in ovarian cancers with various molecular aberrations of ALK.

2. Materials and methods

2.1 Ethical committee approval

The study was approved by the Bioethical Committee of the Medical University of Gdansk (Poland).

2.2 Clinical data collection

A total of 150 high-grade serous ovarian carcinomas were selected for the study, 115 of which came from the archives of University Clinical Centre (UCC) of Gdansk (2009–2015), and 35 were obtained from the Military Institute of Medicine in Warsaw (MIM) (2004–2014). Clinicopathological data including date of birth, surgery date, clinical stage, applied therapy, biochemical response based on Ca125 levels and clinical PFS were obtained from hospital documentation. Selection of patients was based on the availability of the clinical data and the cancer tissue in the paraffin blocks. If applicable, date of death was obtained from the PESEL Registry of Polish Ministry of Digital Affairs.

2.3 Microarray preparations

Tissue microarrays (TMA) consisting of core sections (diameter: 2 mm) of primary tumors were prepared using the Manual Tissue Arrayer MTA-1 (Beecher Instruments, Inc., Sun Prairie, WI, USA) for the purpose of immunohistochemical staining and hybridization in situ. Non-neoplastic tissue fragments were used as a control and location markers in each microarray.

2.4 Immunohistochemistry

4 $\mu$ m-thick sections were taken from TMAs and stained with 3 clones of anti-ALK antibodies. For staining with ALK1 (Dako, Carpinteria, CA, USA) and 5A4 (NovocastraTM, LeicaBiosystems, Newcastle Upon Tyne, United Kingdom), ready-to-use primary antibodies autostainer Link 48 (Agilent Technologies, Santa Clara, CA, USA) was used. The pre-treatment was performed using heat-induced antigen retrieval in pH 6,1 buffer with subsequent 20-minute incubation with ALK1 antibody, and in pH 9,0 buffer with 45-minute incubation for 5A4. Antibody incubation was followed by the standard signal amplification with mouse LINKER at room temperature for 15 minutes. IHC for D5F3 (Ventana, Tucson, AZ, USA) was performed on Ventana Benchmark GX automated staining system (Ventana) with ultraView DAB detection kit (Ventana), using the protocol developed by Ventana. The H-score was evaluated in every case by multiplying the percentage of tumour that stain positively by the intensity (0 for negative, 1 for weak, 2 for moderate and 3 for strong staining; total H-score equaling 0–300). The score was assessed independently by two researchers (AG, KM) with consensus established in the case of discrepant scores.

2.5 Next-generation sequencing (NGS)

A custom-designed KAPA HyperChoice Enrichment Kit (Roche Holding, Basel, Switzerland) was used for the detection of single-nucleotide variants (SNVs), insertions, deletions, InDels and Copy Number Variation (CNV) of 21 exons of ALK. Automated target enrichment library preparation was performed by the i7 Robot (Beckman Coulter GmbH, Krefeld, Germany). Sequencing was done on the Illumina platform using NovaSeq 6000 (Illumina, San Diego, U.S.A.). Libraries were sequenced at 2 x 150 cycles and achieved a quality score of $\geqslant$ % of bases higher than Q30 at 2 x 150 bp. Variant analysis and interpretation was performed using CLC Genomics Workbench (QIAGEN, Venlo, Netherlands), and supplemented by the data available in public databases (COSMIC v96). Only mutant variants with an allelic frequency $>$ 5%, a forward-reverse balance $>$ 0.01 and an average quality $>=$ 24 were considered. Fusion and breakpoint detection was done using FACTERA with default parameters (Newman Lab) [14]. Copy gains were defined as copy number above 2. The mutation classification was based on the standards and guidelines of American College of Medical Genetics and Genomics [15].

Table 1
Pathoclinical data of all patients

Parameter		No.	Frequency
Stage	IA	1	0.7%
	IB	1	0.7%
	IC	6	4.4%
	II	2	1.5%
	II	3	2.2%
	II	0	0.0%
	IIIA	2	1.5%
	IIIB	5	3.7%
	IIIC	96	71.1%
	IV	19	14.1%
Age	$<$ 4	3	2.1%
	40–49	13	9.6%
	50–59	37	27.4%
	60–69	59	43.7%
	70–79	28	20.7%
	$>$ 8	5	3.7%
Progression	Ye	97	93.3%
	No	7	6.7%
PFS (months) (complete observations)	$<$ 1	67	69.1%
	12–24	12	12.4%
	24–36	4	4.1%
	36–60’	6	6.2%
	$>$ 60	8	8.2%
Survival	Yes	26	18.2%
	No	117	81.8%
OS (months) (fatal cases)	$<$ 1	19	16.1%
	12–24	27	22.9%
	24–36	21	17.8%
	36–60	25	21.2%
	$>$ 6	26	22.0%

No. – number of patients, PFS – progression-free survival (months). OS – overall survival (months).

Table 2

Results of immunohistochemical analysis

Name of antibody	Negative (hscore $<$ 10)	Positive
		Hscore 10–50	Hscore 50–10	Hscore $>$ 100	Total
ALK1	136 (100%)	0	0	0	0
D5F3	49 (48.0%)	30 (29.4%)	14 (13.7%)	9 (8.8%)	53 (52.0%)
5A4	94 (70.7%)	28 (21.1%)	9 (6.8%)	2 (1.5%)	39 (29.3%)

2.6 Fluorescent in situ hybridization (FISH)

FISH assay for ALK rearrangements was performed on TMAs by using dual-colour break apart ALK FISH probe set (Empire Genomics, Williamsville, NY, USA) according to the standard protocol. At least 100 cells were analysed. The cut-off for potential rearrangement of ALK was set at 10% of cells showing a split or isolated 3’ pattern, and in these cases the hybridization was to be repeated on the whole sections.

FISH assay for ALK amplification analysis was performed on 3 $\mu$ m sections cut from formalin-fixed, paraffin-embedded tissue blocks from tumours with copy gains in NGS. The procedure was carried out using the commercial probe set ZytoLight ${}^{\@setsize{\scriptsize}{8pt}{\viipt}{\@viipt}\textregistered}$ SPEC ALK/2q11 Dual Color Probe (ZytoVision) according to standard protocol. The slides were examined under a fluorescent microscope (Olympus BX43F) with an appropriate filter set. The criteria for copy number aberrations were based on those previously established in non-small cell lung carcinoma [16] with copy gains defined as a mean copy number between 3 and 5 in at least 10% of cells and amplification as the presence of at least 6 copies of ALK per cell.

2.7 Statistical analysis

PFS was defined as the time from the first diagnosis to the first documented progressive disease (PD) or censored at the last available follow-up. OS was calculated from the date of first diagnosis to the death of any cause or censored at the date of access to the PESEL Registry (consulted the 19.05.2022) for patients living at that time. For survival analysis, only patients treated with the standard chemotherapy regimen of paclitaxel and carboplatin, and with clinical follow-up available, were included.

Statistical analysis was performed using Statistica, version 13 (TIBCO Software Inc., San Francisco, CA, USA). Correlations were assessed using the Kendall $\tau$ method. Survival curves were generated using the Kaplan-Meier method, with significance evaluated using the Cox F test (significance level: $p$ -values $<$ 0.05). Univariate analysis using Cox proportional-hazards regression model was used.

Figure 1.

Immunohistochemical (IHC) and fluorescent in situ hybrydization (FISH) analysis. A and B – weak to moderate IHC staining with clone 5A4; C – moderate IHC staining with clone D5F3; D – increased number of ALK signals (green) both independently and in relations to centromer signal (red) in FISH, signifying ALK amplification.

Table 3

Correlations between immunohistochemistry, copy gain and pathoclinical data

Correlated parameter	Kendal tau	$p$
Progression & positive D5F3	$-$ 0.225	0.008
Progression & positive 5A4	0.002	0.977
Progression & copy gain	0.085	0.222
5 year PFS & positive D5F3	0.225	0.008
5 year PFS & positive 5A4	0.026	0.715
5 year PFS & copy gain	$-$ 0.060	0.383
Overall survival & positive D5F3	0.066	0.434
Overall survival & positive 5A4	0.062	0.390
Overall survival & copy gain	0.114	0.100
5 year OS & positive D5F3	0.044	0.603
5 year OS & positive 5A4	0.169	0.019
5 year OS & copy gain	$-$ 0.036	0.601
Stage & positive D5F3	$-$ 0.095	0.183
Stage & positive 5A4	$-$ 0.074	0.224
Stage & copy gain	$-$ 0.055	0.351
Age at admission & positive D5F3	0.021	0.757
Age at admission & positive 5A4	$-$ 0.069	0.237
Age at admission & copy gain	0.024	0.674
Positive 5A4 & positive D5F3	0.390	$<$ 0.001
Copy gain & positive D5F3	$-$ 0.054	0.427
Copy gain & positive 5A4	0.138	0.02

PFS – progression-free survival, OS – overall survival.

3. Results

3.1 Pathological re-evaluation and clinical data analysis

After pathological re-evaluation (A.G. and P.C.) 4 cases were removed from the study as representing entities other than HGSOC. The summary of the clinicopathological data for the remaining 146 cases is shown in Table 1. The median age at admission was 63 years (range 31–82). Data on disease progression was available in 104 cases, with the average time to progression of 12 months and the average time of observation in patients without progression of 91 months. In all these patients, first-line treatment included platinum derivatives-based chemotherapy. Six patients were removed from the survival analysis, four because of the first-line chemotherapy differing from the standard paclitaxel-carboplatin regimen (cisplatin, docetaxel, and capecitabine in one case, paclitaxel-cisplatin in another and cisplatin in monotherapy in two others), one due to missing information on treatment in the documentation and one due to the lack of data on overall survival in the PESEL database.

3.2 ALK overexpression

The immunohistochemical staining was completely negative with clone ALK1, which was excluded from the statistical analysis, and showed positivity ranging from 0 to 55% in two other clones, with most of the cases showing weak and focal cytoplasmic staining (Fig. 1a–c; Table 2), with only single cases presenting with concomitant membranous positivity. Statistical analysis showed a weak negative correlation between D5F3 positivity and disease progression – both during 5 years of observation (Kendall’s $\tau$ $=-$ 0.22; $p=$ 0.008) and the whole observation period (different for every patient; Kendall’s $\tau$ $=-$ 0.23; $p=$ 0.008), as well as a very weak positive correlation between 5A4 positivity and 5-year survival (Kendall’s $\tau$ $=$ 0.17; $p=$ 0.019). Staining positivity showed no clear association with age or stage (Table 3).

3.3 ALK rearrangements and amplification

FISH analysis with break-apart probe did not show any translocations including ALK locus. NGS results showed ALK copy gains in 11 cases, with CNV varying between 2.07 and 2.81. Statistical analysis showed weak correlation between the positive staining with 5A4 clone and the copy gain presence (Kendall’s $\tau$ $=$ 0.14; $p=$ 0.02), and no significant ( $p<$ 0.05) correlation with other clones or any clinical parameters (Table 3). In three of these patients, FISH analysis revealed an increased number of signals corresponding to ALK amplification (Fig. 1d).

3.4 ALK point mutations

NGS target coverage was at least 99% at 200x. ALK point mutations were found in 13 tumours (Table 4, patients 1–13), including three categorized as class IV variants, all of which came from stage IIIC HGSOC. The average PFS in these patients was longer than in other patients with progression (30 vs. 11 months). One of the patients harbouring the class IV mutation was still alive after 138 months of observation. The OS of the two other patients was also longer than the average (49 and 72 months vs. 38 months). Survival analysis confirmed that for patients with class IV ALK mutations OS was significantly longer than for the other patients from the similar stage group (Fig. 2a, Table 5). A similar trend was visible, albeit with no statistical significance, for OS in all patients regardless of the stage, as well as in PFS analysis (Fig. 2b–d, Table 5).

3.5 ALK point mutations

Fusion and breakpoint detection using FACTERA has shown rearrangements in three cases: ERBB2-GRB7 (break support 154/59), SND1-BRAF (break support 23/97 and 54/22) and PRKCA-BRCA1 (break support 33/62). None of these tumours showed any ALK aberrations by FISH or by NGS. Clinicopathological data on these patients are summarized in Table 4 (patients 14–16).

4. Discussion

This study is the first showing better prognosis in patients with potentially pathogenic ALK point mutations. Previous reports, based on in vitro, IHC and genetic profiling, suggested more aggressive behaviour of ALK-positive tumours [10], and worse overall prognosis and response to treatment in patients with ALK point mutations [17, 18]. This, however, was not confirmed in our research, which was based on methodology routinely used in tumour diagnostics.

It is worth noticing that in previously published studies the impact of ALK mutations on prognosis and treatment efficacy was analysed without discriminating between mutation classes. It also included mutations with little to no influence on pathogenesis [18]. It cannot be ruled out that most of those mutations appear as late and secondary events in advanced tumours with instable genome. If this is the case, the notion that they negatively impact the prognosis was already confirmed by the studies of ovarian tumours with high mutation burden [19]. In further studies, it seems appropriate to focus on the impact of aberrations with potentially pathogenic significance, excluding the tangential passenger mutations. Since ALK overexpression is an established positive prognostic factor in ALCL, both with and without ALK translocations [20], and might be associated with a better response to treatment in NSCLC [21], we cannot exclude potentially better prognosis and treatment response in HGSOC associated with pathogenetically relevant ALK-point mutations. Considering the genetic diversity of tumours in different populations, it cannot be completely ruled out that the obtained results may differ from the previous ones also due to the fact that the study was conducted on a different ethnic group.

Table 4

Summary of pathoclinical data of patients with molecular abberations

Age at admission

Stage

Observation-progression

Progression

ChTH

Overall observation

Fatal

D5F3 score

5A4 score

ALK1 score

Variant ALK (p.)

Variant ALK (c.)

Class

Cosmic

52 years

IIIC

57 months

Yes

P-C

138 months

p.(Val1265Ala)

c.3794T

>

No data

62 years

IIIC

25 months

Yes

P-C

72 months

Yes

–

p.(Gly903Glu)

c.2708G

>

Pathogenic, FATHMM 0.98

64 years

IIIC

8 months

Yes

P-C

49 months

Yes

p.(Gly882Ala)

c.2645G

>

No data

45 years

IIB

65 months

Yes

P-C

81 months

–

p.(Asn702Lys)

c.2106C

>

III

No data

67 years

1 month

Yes

P-C

13 months

Yes

p.(Pro1215Leu)

c.3644C

>

III

No data

62 years

IIIC

15 months

Yes

P-C

69 months

Yes

p.(Arg557Cys)

c.1669C

>

III

Pathogenic, FATHMM 0.93

44 years

–

p.(Arg311His)

c.932G

>

Neutral, FATHMM 0

65 years

IIIC

6 months

Yes

P-C

16 months

Yes

p.(Arg311His)

c.932G

>

Neutral, FATHMM 0

61 years

IIIC

6 months

Yes

P-C

19 months

Yes

p.(Arg557His)

c.1670G

>

No data

73 years

IIIC

15 months

Yes

P-C

32 months

Yes

110

p.(Tyr826

=

)

c.2478C

>

No data

55 years

IIIC

4 months

Yes

P-C

36 months

Yes

p.(Ser1053

=

)

c.3159C

>

Neutral, FATHMM 0.04

63 years

IIIC

105 months

P-C

105 months

p.(Leu1198

=

)

c.3594C

>

Pathogenic, FATHMM 0.83

74 years

IIIC

<

1 month

Yes

P-C

9 months

Yes

p. (Ala1200

=

)

c.3600G

>

No data

Age at admission

Stage

Observation-progression

Progression

ChTH

Overall observation

Fatal

D5F3 score

5A4 score

ALK1 score

Fusion

Break1

Break 2

65 years

IIIC

10 months

Yes

P-C

10 months

Yes

ERBB2:GRB7

chr17:37883152

chr17:37901703

54 years

16 months

Yes

P-C

32 months

Yes

PRKCA:BRCA1

chr17:64292876

chr17:41234430

79 years

1 month

Yes

P-C

2 months

Yes

SND1:BRAF

chr7:127567753

chr7:140489416

ChTH – chemotherapy, P-C – paclitaxel-carboplatin regimen; Patients 1–13 with ALK point mutations, Patients 14–16 with fusions involving genes other than ALK.

Table 5

Statistical analysis of ALK overproduction and ALK aberations

Factor	Parameter	Cox’s F test		Univariate Cox regression
		Cox’s F	$p$	Chi2	Risk ratio	$p$
ALK point mutations	PFS	1.321	0.165	0.771	0.760	0.380
	OS	1.371	0.15386	0.628	0.763	0.428
Class 4 ALK mutations	PFS	1.633	0.141	0.802	0.614	0.371
	PFS (stage IIIC)	2.085	0.059	1.939	0.476	0.164
	OS	2.301	0.061	1.807	0.432	0.179
	OS (stage IIIC)	2.661	0.036	2.375	0.386	0.123
Positive staining D5F3	PFS	1.271	0.170	0.623	0.818	0.430
	OS	1.010	0.483	0.006	0.980	0.940
Positive staining 5A4	PFS	1.042	0.446	0.029	1.043	0.865
	OS	1.314	0.130	1.733	0.714	0.188
ALK copy gain	PFS	1.002	0.457	0.004	0.977	0.950
	OS	1.197	0.290	0.034	0.925	0.853

PFS – progression-free survival, OS – overall survival.

Figure 2.

Kaplan-Meier curves of survival for patients with ALK class IV mutations vs other patients. A – Overall survival for stage IIIC HGSOC, B – Overall survival for all stages, C – Progression-free survival for stage IIIC HGSOC, D – Progression-free survival for all stages. OS – overall survival, PFS – progression-free survival.

Our analysis showed that three out of 146 examined tumours (2%) harboured novel ALK mutations classified as potentially pathogenic. All these patients were diagnosed in stage IIIC, but their OS was significantly better ( $p<$ 0.05) than the remaining patients in that stage. Two of the patients with tumours showing the mutation were treated in Gdansk. In the first patient, aged 52 at the time of diagnosis (2010), combined surgical treatment and chemotherapy resulted in remission which lasted 57 months. She was alive 138 months after the diagnosis. The second patient was operated at age 62. Pathological nodal mass was discovered 25 months after the surgery in the retroperitoneal space. The patient continued the therapy in a different treatment centre and died 72 months after the initial diagnosis. The tumour sample from the third patient was obtained from the archives of the Military Institute of Medicine. This patient had a relapse 8 months after the diagnosis and died 49 months after the initial surgery.

The weak correlation between the positive IHC and increased copy numbers in ALK 5A4 with complete lack thereof in two other anti-ALK clones and negative staining in all 3 clones in cases with FISH confirmed amplification indicates that overexpression of ALK and ALK amplification are independent events, neither of which had a strong impact on patients’ survival. This also decreases the value of IHC as a screening method for testing molecular aberrations. The weak correlation between positive IHC and the lack of progression as well as 5-year overall survival is worth mentioning, however, as it contradicts the previously mentioned data on the negative impact of ALK overexpression in ovarian carcinoma. It is further corroborated by the fact that in the univariate analysis, despite not showing statistical significance, all of the factors potentially resulting in the increased ALK activity (ALK copy gain, overexpression visible as IHC positivity, class III/IV point mutations) were associated with a lower risk of dying (RR $<$ 1.0), presenting a trend opposite to that described in the previously published data (Table 5).

In this study, we showed the presence of fusions involving other genes encoding protein kinases as demonstrated in three tumours having fusions of genes other than ALK (Table 4). In a 65-year-old female patient with stage IIIC HGSOC, who died 10 months after diagnosis, ERBB2-GRB7 fusion resulting from the deletion of the segment of 17q chromosome was discovered. Such an aberration has not been described so far in ovarian cancer. In colorectal cancer, it is associated with a lack of response to treatment with HER2 inhibitors, and a negative ERBB2 immunohistochemistry results even in cases with accompanying ERBB2 amplification in FISH [22]. It has also been reported in lobular breast carcinoma [23] and urothelial carcinoma [24].

Another fusion, PRKCA-BRCA1, occurred in a 54-year-old patient diagnosed with stage IV HGSOC with progression 16 months, and death 32 months after initial diagnosis. Such rearrangement has not been described in the literature so far. Both partner genes are known to be found in other fusions, including NF1, FAM134C, BECN1 and LSM12 for BRCA1 [25] and TANC2, SLC44A1, CDH8 and KIRREL for PRKCA [26], where their potentially pathogenic nature was indicated. Aberrant BRCA1 expression may affect the response to PARP inhibitors [27], which may indicate a potential clinical implication of such discovery in ovarian cancer.

The inversion resulting in SND1-BRAF was detected in an advanced (stage IV) tumour of a 79-year-old woman who progressed quickly and died within 2 months of surgery. It was previously reported in pancreatic acinar carcinoma [28] and NSCLC [29]. The research indicated, inter alia, the potential benefit of BRAF inhibitors and MEK inhibitors treatment in this group of patients. Our results expand the group of neoplasms with SND1-BRAF fusion, underlying the significance of further studies on potential efficacy of targeted therapeutics in those patients.

To summarize, the results of this study point out the potential role of ALK point mutations as an important factor for better response to chemotherapy and overall survival in a significant subset of locally advanced ovarian carcinomas. It also provides information on the frequency of ALK overexpression in the European population and expands the scope of tumours harbouring potentially targetable genetic fusions.

Author contributions

Conception: Adam Gorczynski, Wojciech Biernat, Piotr Czapiewski, Jörg Gabert, Karsten Neumann.

Interpretation or analysis of data: Adam Gorczynski, Kevin Miszewski, Piotr Czapiewski, Wojciech Biernat, Yann Gager, Sonja Koch, Jane Potschke, Dimitar Ugrinovski, Agata Pospieszynska, Bartosz Szymanowski, Szczepan Cierniak, Karsten Neuman, Katarina Mirkov.

Preparation of the manuscript: Adam Gorczynski, Piotr Czapiewski, Irene Kruecken.

Revision for important intellectual content: Yann Gager, Renata Duchnowska, Jörg Gabert, Wojciech Biernat.

Supervision: Piotr Czapiewski, Wojciech Biernat, Jörg Gabert, Renata Duchnowska, Dariusz Wydra.

Supplementary data

The supplementary files are available to download from http://dx.doi.org/10.3233/CBM-230117.

sj-xlsx-1-cbm-10.3233_CBM-230117.xlsx - Supplemental material

Supplemental material, sj-xlsx-1-cbm-10.3233_CBM-230117.xlsx

sj-xlsx-2-cbm-10.3233_CBM-230117.xlsx - Supplemental material

Supplemental material, sj-xlsx-2-cbm-10.3233_CBM-230117.xlsx

Footnotes

Acknowledgments

This project was financed from the PRELUDIUM 13 grant 2017/25/N/NZ4/00558 funded by National Science Centre, Poland.

The authors would like to thank dr. Magdalena Ratajska, dr. Victoria Tomkowicz, dr. Karolina Mollin, dr. Rafal Peksa, and dr. Michal Kunc for their invaluable input in designing and conducting the study.

References

Sung

Ferlay

Siegel

R.L.

Laversanne

Soerjomataram

Jemal

and Bray

, Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries, CA: A Cancer Journal for Clinicians 71 (2021), 209–249.

Arnold

Rutherford

Lam

Bray

Ervik

and Soerjomataram

, ICBP SURVMARK-2 online tool: International Cancer Survival Benchmarking Available from: http://gco.iarc.fr/survival/survmark, accessed [10/10/2022], 2019.

Gorczyński

Prełowska

Adam

Czapiewski

and Biernat

, ALK-positive cancer: Still a growing entity, Future Oncology (London, England) 10 (2014), 305–321.

Alaggio

Amador

Anagnostopoulos

Attygalle

A.D.

de O. Araujo

I.B.

Berti

Bhagat

Borges

A.M.

Boyer

Calaminici

Chadburn

Chan

J.K.C.

Cheuk

Chng

W.J.

Choi

J.K.

Chuang

S.S.

Coupland

S.E.

Czader

Dave

S.S.

de Jong

M.Q.

Elenitoba-Johnson

K.S.

Ferry

Geyer

Gratzinger

Guitart

Gujral

Harris

Harrison

C.J.

Hartmann

Hochhaus

Jansen

P.M.

Karube

Kempf

Khoury

Kimura

Klapper

Kovach

A.E.

Kumar

Lazar

A.J.

Lazzi

Leoncini

Leung

Leventaki

X.Q.

Lim

M.S.

Liu

W.P.

Louissaint

Marcogliese

Medeiros

L.J.

Michal

Miranda

R.N.

Mitteldorf

Montes-Moreno

Morice

Nardi

Naresh

K.N.

Natkunam

S.B.

Oschlies

Ott

Parrens

Pulitzer

Rajkumar

S.V.

Rawstron

A.C.

Rech

Rosenwald

Said

Sarkozy

Sayed

Saygin

Schuh

Sewell

Siebert

Sohani

A.R.

Tooze

Traverse-Glehen

Vega

Vergier

Wechalekar

A.D.

Wood

Xerri

and Xiao

, The 5th edition of the world health organization classification of haematolymphoid tumours: Lymphoid neoplasms, Leukemia 36 (2022), 1720–1748.

Bavi

Jehan

Prabhakaran

Al-Sanea

Al-Dayel

Al-Assiri

Al-Halouly

Sairafi

Uddin

and Al-Kuraya

K.S.

, ALK gene amplification is associated with poor prognosis in colorectal carcinoma, British Journal of Cancer 109 (2013), 2735–2743.

Bellini

Potschger

Bernard

Lapouble

Baulande

Ambros

P.F.

Auger

Beiske

Bernkopf

Betts

D.R.

Bhalshankar

Bown

Preter

K.D.

Clement

Combaret

Mora

J.F.D.

George

S.L.

Jimenez

Jeison

Marques

Martinsson

Mazzocco

Morini

Muhlethaler-Mottet

Noguera

Pierron

Rossing

Taschner-Mandl

Roy

N.V.

Vicha

Chesler

Balwierz

Castel

Elliott

Kogner

Laureys

Luksch

Malis

Popovic-Beck

Ash

Delattre

Valteau-Couanet

Tweddle

D.A.

Ladenstein

and Schleiermacher

, Frequency and prognostic impact of alk amplifications and mutations in the european neuroblastoma study group (siopen) high-risk neuroblastoma trial (hr-nbl1), Journal of Clinical Oncology 39 (2021), 3377–3390.

Pietrantonio

Maggi

Bartolomeo

M.D.

Facciorusso

M.G.

Perrone

Testi

Iacovelli

Miceli

Bossi

Leone

Milione

Pelosi

and Braud

F.D.

, Gain of ALK Gene Copy Number May Predict Lack of Benefit from Anti-EGFR Treatment in Patients with Advanced Colorectal Cancer and RAS-RAF-PI3KCA Wild-Type Status, PLOS ONE 9 (2014), e92147.

Ren

Tan

Z.P.

Zhu

Crosby

Haack

Ren

J.M.

Beausoleil

Moritz

Innocenti

Rush

Zhang

Zhou

X.M.

T.L.

Yang

Y.F.

and Comb

M.J.

, Identification of anaplastic lymphoma kinase as a potential therapeutic target in ovarian cancer, Cancer Research 72 (2012), 3312–3323.

Tang

Yang

Zhang

and Zhou

, Aberrant expression of anaplastic lymphoma kinase in ovarian carcinoma independent of gene rearrangement, International Journal of Gynecological Pathology 35 (2016), 337–347.

10.

Matsumoto

Oda

Hasegawa

Hashimura

Oguri

Inoue

Yokoi

Tochimoto

Nakagawa

Jiang

and Saegusa

, Anaplastic lymphoma kinase overexpression is associated with aggressive phenotypic characteristics of ovarian high-grade serous carcinoma, American Journal of Pathology 191 (2021), 1837–1850.

11.

Dearden

Stevens

Y.L.

and Blowers

, Mutation incidence and coincidence in non small-cell lung cancer: Meta-analyses by ethnicity and histology (mutMap), Annals of Oncology 24 (2013), 2371–2376.

12.

Gorczynski

Czapiewski

Korwat

Budynko

Prelowska

Okon

and Biernat

, ALK-rearranged renal cell carcinomas in Polish population, Pathology – Research and Practice 215 (2019), 152669.

13.

Huang

X.X.

Xie

F.F.

Hou

L.J.

Chen

X.X.

R.Y.

J.T.

Qiu

J.G.

Zhang

W.J.

Jiang

Q.W.

Yang

Zheng

D.W.

Chen

Huang

J.R.

Wang

Wei

M.N.

W.F.

Shi

and Yan

X.J.

, Crizotinib synergizes with cisplatin in preclinical models of ovarian cancer, American Journal of Translational Research 9 (2017), 1667.

14.

Newman

A.M.

Bratman

S.V.

Stehr

Lee

L.J.

Liu

C.L.

Diehn

and Alizadeh

A.A.

, FACTERA: A practical method for the discovery of genomic rearrangements at breakpoint resolution, Bioinformatics (Oxford, England) 30 (2014), 3390–3393.

15.

Richards

Aziz

Bale

Bick

Das

Gastier-Foster

Grody

W.W.

Hegde

Lyon

Spector

Voelkerding

and Rehm

H.L.

, Standards and guidelines for the interpretation of sequence variants: A joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology, Genetics in Medicine 17 (2015), 405–423.

16.

Salido

Pijuan

Martínez-Avilés

Galván

A.B.

Cañadas

Rovira

Zanui

Martínez

Longarón

Sole

Serrano

Bellosillo

Wynes

M.W.

Albanell

Hirsch

F.R.

and Arriola

, Increased ALK gene copy number and amplification are frequent in non-small cell lung cancer, Journal of Thoracic Oncology 6 (2011), 21–27.

17.

J.K.

Kim

Kang

Kim

S.W.

Song

Shim

S.H.

Choi

M.C.

J.H.

Song

J.Y.

Kim

Y.M.

Kim

J.H.

and Lee

J.W.

, Somatic genomic landscape of East Asian epithelial ovarian carcinoma and its clinical implications from prospective clinical sequencing: A Korean Gynecologic Oncology Group study (KGOG 3047), International Journal of Cancer 151 (2022), 1086–1097.

18.

Saifullah and Tsukahara

, Integrated analysis of the clinical consequence and associated gene expression of ALK in ALK-positive human cancers, Heliyon 8 (2022), e09878.

19.

Wang

Liu

Yang

Wang

Zhang

Peng

Wang

and Hong

, A novel tumor mutational burden-based risk model predicts prognosis and correlates with immune infiltration in ovarian cancer, Frontiers in Immunology 13 (2022).

20.

Chen

Zhou

Gao

Shi

Zhou

Xie

Liu

and Gong

, Extra copies of ALK gene locus is a recurrent genetic aberration and favorable prognostic factor in both ALK-positive and ALK-negative anaplastic large cell lymphomas, Leukemia Research 36 (2012), 1141–1146.

21.

Park

Kim

H.J.

Choi

C.M.

Lee

D.H.

Kim

S.W.

Lee

J.S.

Kim

W.S.

Choi

S.H.

Rho

J.K.

and Lee

J.C.

, Predictive factors for a long-term response duration in non-squamous cell lung cancer patients treated with pemetrexed, BMC Cancer 16 (2016), 1–8.

22.

Hechtman

J.F.

Zehir

Yaeger

Wang

Middha

Zheng

Hyman

D.M.

Solit

Arcila

M.E.

Borsu

Shia

Vakiani

Saltz

and Ladanyi

, Identification of targetable kinase alterations in patients with colorectal carcinoma that are preferentially associated with wild-type RAS/RAF, Molecular Cancer Research: MCR 14 (2016), 296.

23.

Bose

, A neu view of invasive lobular breast cancer, Clinical Cancer Research: An Official Journal of the American Association for Cancer Research 19 (2013), 3331–3333.

24.

Ross

J.S.

Wang

Gay

L.M.

Al-Rohil

R.N.

Nazeer

Sheehan

C.E.

Jennings

T.A.

Otto

G.A.

Donahue

Palmer

Ali

Nahas

Young

LaBrecque

Frampton

Erlich

Curran

J.A.

Brennan

Downing

S.R.

Yelensky

Lipson

Hawryluk

Miller

V.A.

and Stephens

P.J.

, A high frequency of activating extracellular domain ERBB2 (HER2) mutation in micropapillary urothelial carcinoma AC, Clinical Cancer Research 20 (2014), 68–75.

25.

Sun

Chao

Luo

and He

, Clinical characteristics and prognostic implications of BRCA-associated tumors in males: A pan-tumor survey, BMC Cancer 20 (2020), 1–16.

26.

Van

A.A.N.

Kunkel

M.T.

Baffi

T.R.

Lordén

Antal

C.E.

Banerjee

and Newton

A.C.

, Protein kinase C fusion proteins are paradoxically loss of function in cancer, Journal of Biological Chemistry 296 (2021), 100445.

27.

Brugge

P.T.

Kristel

Burg

E.V.D.

Boon

Maaker

M.D.

Lips

Mulder

Ruiter

J.D.

Moutinho

Gevensleben

Marangoni

Majewski

Jozwiak

Kloosterman

Roosmalen

M.V.

Duran

Hogervorst

Turner

Esteller

Cuppen

Wesseling

and Jonkers

, Mechanisms of Therapy Resistance in Patient-Derived Xenograft Models of BRCA1-Deficient Breast Cancer, JNCI: Journal of the National Cancer Institute 108 (2016).

28.

Chmielecki

Hutchinson

K.E.

Frampton

G.M.

Chalmers

Z.R.

Johnson

Shi

Elvin

Ali

S.M.

Ross

J.S.

Basturk

Balasubramanian

Lipson

Yelensky

Pao

Miller

V.A.

Klimstra

D.S.

and Stephens

P.J.

, Comprehensive genomic profiling of pancreatic acinar cell carcinomas identifies recurrent RAF fusions and frequent inactivation of DNA repair genes, Cancer Discovery 4 (2014), 1398–1405.

29.

Jang

J.S.

Lee

Liyanage

Yang

Guo

Asmann

Y.W.

P.W.

Erickson-Johnson

Sakai

Sun

Z.F.

Jeon

H.S.

Hwang

Bungum

A.O.

Edell

E.S.

Simon

V.A.

Kopp

K.J.

Eckloff

Oliveira

A.M.

Wieben

Aubry

M.C.

Wigle

Diasio

R.B.

Yang

and Jen

, Common Oncogene Mutations and Novel SND1-BRAF Transcript Fusion in Lung Adenocarcinoma from Never Smokers, Scientific Reports 5 (2015), 1–8.

Prognostic value of ALK overexpression and molecular abnormalities in high-grade serous ovarian carcinoma

Abstract

BACKGROUND:

OBJECTIVE:

METHODS:

RESULTS:

CONCLUSIONS:

Keywords

1. Introduction

2. Materials and methods

2.1 Ethical committee approval

2.2 Clinical data collection

2.3 Microarray preparations

2.4 Immunohistochemistry

2.5 Next-generation sequencing (NGS)

Table 1 Pathoclinical data of all patients

2.7 Statistical analysis

3.1 Pathological re-evaluation and clinical data analysis

3.2 ALK overexpression

3.3 ALK rearrangements and amplification

3.4 ALK point mutations

3.5 ALK point mutations

4. Discussion

Author contributions

Supplementary data

sj-xlsx-1-cbm-10.3233_CBM-230117.xlsx - Supplemental material

sj-xlsx-2-cbm-10.3233_CBM-230117.xlsx - Supplemental material

Footnotes

Acknowledgments

References

Table 1
Pathoclinical data of all patients