Molecular Analysis of Biliary Tract Cancers with the Custom 3' RACE-Based NGS Panel.

The technique 3' rapid amplification of cDNA ends (3' RACE) allows for detection of translocations with unknown gene partners located at the 3' end of the chimeric transcript. We composed a 3' RACE-based RNA sequencing panel for the analysis of FGFR1-4 gene rearrangements, detection of activating mutations located within FGFR1-4, IDH1/2, ERBB2 (HER2), KRAS, NRAS, BRAF, and PIK3CA genes, and measurement of the expression of ERBB2, PD-L1, and FGFR1-4 transcripts. This NGS panel was utilized for the molecular profiling of 168 biliary tract carcinomas (BTCs), including 83 intrahepatic cholangiocarcinomas (iCCAs), 44 extrahepatic cholangiocarcinomas (eCCAs), and 41 gallbladder adenocarcinomas (GBAs). The NGS failure rate was 3/168 (1.8%). iCCAs, but not other categories of BTCs, were characterized by frequent FGFR2 alterations (17/82, 20.7%) and IDH1/2 mutations (23/82, 28%). Other potentially druggable events included ERBB2 amplifications or mutations (7/165, 4.2% of all successfully analyzed BTCs) and BRAF p.V600E mutations (3/165, 1.8%). In addition to NGS, we analyzed microsatellite instability (MSI) using the standard five markers and revealed this event in 3/158 (1.9%) BTCs. There were no instances of ALK, ROS1, RET, and NTRK1-3 gene rearrangements or MET exon 14 skipping mutations. Parallel analysis of 47 iCCA samples with the Illumina TruSight Tumor 170 kit confirmed good performance of our NGS panel. In conclusion, targeted RNA sequencing coupled with the 3' RACE technology is an efficient tool for the molecular diagnostics of BTCs.

ALK, ROS1, RET and NTRK1-3 Gene Fusions in Colorectal and Non-Colorectal Microsatellite-Unstable Cancers.

This study aimed to conduct a comprehensive analysis of actionable gene rearrangements in tumors with microsatellite instability (MSI). The detection of translocations involved tests for 5'/3'-end expression imbalance, variant-specific PCR and RNA-based next generation sequencing (NGS). Gene fusions were detected in 58/471 (12.3%) colorectal carcinomas (CRCs), 4/69 (5.8%) gastric cancers (GCs) and 3/65 (4.6%) endometrial cancers (ECs) (ALK: 8; RET: 12; NTRK1: 24; NTRK2: 2; NTRK3: 19), while none of these alterations were observed in five cervical carcinomas (CCs), four pancreatic cancers (PanCs), three cholangiocarcinomas (ChCs) and two ovarian cancers (OCs). The highest frequency of gene rearrangements was seen in KRAS/NRAS/BRAF wild-type colorectal carcinomas (53/204 (26%)). Surprisingly, as many as 5/267 (1.9%) KRAS/NRAS/BRAF-mutated CRCs also carried tyrosine kinase fusions. Droplet digital PCR (ddPCR) analysis of the fraction of KRAS/NRAS/BRAF mutated gene copies in kinase-rearranged tumors indicated that there was simultaneous co-occurrence of two activating events in cancer cells, but not genetic mosaicism. CRC patients aged above 50 years had a strikingly higher frequency of translocations as compared to younger subjects (56/365 (15.3%) vs. 2/106 (1.9%), p = 0.002), and this difference was particularly pronounced for tumors with normal KRAS/NRAS/BRAF status (52/150 (34.7%) vs. 1/54 (1.9%), p = 0.001). There were no instances of MSI in 56 non-colorectal tumors carrying ALK, ROS1, RET or NTRK1 rearrangements. An analysis of tyrosine kinase gene translocations is particularly feasible in KRAS/NRAS/BRAF wild-type microsatellite-unstable CRCs, although other categories of tumors with MSI also demonstrate moderate occurrence of these events.

Cost-Efficient Detection of NTRK1/2/3 Gene Fusions: Single-Center Analysis of 8075 Tumor Samples.

The majority of NTRK1, NTRK2, and NTRK3 rearrangements result in increased expression of the kinase portion of the involved gene due to its fusion to an actively transcribed gene partner. Consequently, the analysis of 5'/3'-end expression imbalances is potentially capable of detecting the entire spectrum of NTRK gene fusions. Archival tumor specimens obtained from 8075 patients were subjected to manual dissection of tumor cells, DNA/RNA isolation, and cDNA synthesis. The 5'/3'-end expression imbalances in NTRK genes were analyzed by real-time PCR. Further identification of gene rearrangements was performed by variant-specific PCR for 44 common NTRK fusions, and, whenever necessary, by RNA-based next-generation sequencing (NGS). cDNA of sufficient quality was obtained in 7424/8075 (91.9%) tumors. NTRK rearrangements were detected in 7/6436 (0.1%) lung carcinomas, 11/137 (8.0%) pediatric tumors, and 13/851 (1.5%) adult non-lung malignancies. The highest incidence of NTRK translocations was observed in pediatric sarcomas (7/39, 17.9%). Increased frequency of NTRK fusions was seen in microsatellite-unstable colorectal tumors (6/48, 12.5%), salivary gland carcinomas (5/93, 5.4%), and sarcomas (7/143, 4.9%). None of the 1293 lung carcinomas with driver alterations in EGFR/ALK/ROS1/RET/MET oncogenes had NTRK 5'/3'-end expression imbalances. Variant-specific PCR was performed for 744 tumors with a normal 5'/3'-end expression ratio: there were no rearrangements in 172 EGFR/ALK/ROS1/RET/MET-negative lung cancers and 125 pediatric tumors, while NTRK3 fusions were detected in 2/447 (0.5%) non-lung adult malignancies. In conclusion, this study describes a diagnostic pipeline that can be used as a cost-efficient alternative to conventional methods of NTRK1-3 analysis.

Rapid and Cost-Efficient Detection of RET Rearrangements in a Large Consecutive Series of Lung Carcinomas.

RET-kinase-activating gene rearrangements occur in approximately 1-2% of non-small-cell lung carcinomas (NSCLCs). Their reliable detection requires next-generation sequencing (NGS), while conventional methods, such as immunohistochemistry (IHC), fluorescence in situ hybridization (FISH) or variant-specific PCR, have significant limitations. We developed an assay that compares the level of RNA transcripts corresponding to 5'- and 3'-end portions of the RET gene; this test relies on the fact that RET translocations result in the upregulation of the kinase domain of the gene and, therefore, the 5'/3'-end expression imbalance. The present study included 16,106 consecutive NSCLC patients, 14,449 (89.7%) of whom passed cDNA quality control. The 5'/3'-end unbalanced RET expression was observed in 184 (1.3%) tumors, 169 of which had a sufficient amount of material for the identification of translocation variants. Variant-specific PCR revealed RET rearrangements in 155/169 (91.7%) tumors. RNA quality was sufficient for RNA-based NGS in 10 cases, 8 of which carried exceptionally rare or novel (HOOK1::RET and ZC3H7A::RET) RET translocations. We also applied variant-specific PCR for eight common RET rearrangements in 4680 tumors, which emerged negative upon the 5'/3'-end unbalanced expression test; 33 (0.7%) of these NSCLCs showed RET fusion. While the combination of the analysis of 5'/3'-end RET expression imbalance and variant-specific PCR allowed identification of RET translocations in approximately 2% of consecutive NSCLCs, this estimate approached 120/2361 (5.1%) in EGFR/KRAS/ALK/ROS1/BRAF/MET-negative carcinomas. RET-rearranged tumors obtained from females, but not males, had a decreased level of expression of thymidylate synthase (p < 0.00001), which is a known predictive marker of the efficacy of pemetrexed. The results of our study provide a viable alternative for RET testing in facilities that do not have access to NGS due to cost or technical limitations.

KRAS, NRAS, BRAF, HER2 and MSI Status in a Large Consecutive Series of Colorectal Carcinomas.

This study aimed to analyze clinical and regional factors influencing the distribution of actionable genetic alterations in a large consecutive series of colorectal carcinomas (CRCs). KRAS, NRAS and BRAF mutations, HER2 amplification and overexpression, and microsatellite instability (MSI) were tested in 8355 CRC samples. KRAS mutations were detected in 4137/8355 (49.5%) CRCs, with 3913 belonging to 10 common substitutions affecting codons 12/13/61/146, 174 being represented by 21 rare hot-spot variants, and 35 located outside the "hot" codons. KRAS Q61K substitution, which leads to the aberrant splicing of the gene, was accompanied by the second function-rescuing mutation in all 19 tumors analyzed. NRAS mutations were detected in 389/8355 (4.7%) CRCs (379 hot-spot and 10 non-hot-spot substitutions). BRAF mutations were identified in 556/8355 (6.7%) CRCs (codon 600: 510; codons 594-596: 38; codons 597-602: 8). The frequency of HER2 activation and MSI was 99/8008 (1.2%) and 432/8355 (5.2%), respectively. Some of the above events demonstrated differences in distribution according to patients' age and gender. In contrast to other genetic alterations, BRAF mutation frequencies were subject to geographic variation, with a relatively low incidence in areas with an apparently warmer climate (83/1726 (4.8%) in Southern Russia and North Caucasus vs. 473/6629 (7.1%) in other regions of Russia, p = 0.0007). The simultaneous presence of two drug targets, BRAF mutation and MSI, was observed in 117/8355 cases (1.4%). Combined alterations of two driver genes were detected in 28/8355 (0.3%) tumors (KRAS/NRAS: 8; KRAS/BRAF: 4; KRAS/HER2: 12; NRAS/HER2: 4). This study demonstrates that a substantial portion of RAS alterations is represented by atypical mutations, KRAS Q61K substitution is always accompanied by the second gene-rescuing mutation, BRAF mutation frequency is a subject to geographical variations, and a small fraction of CRCs has simultaneous alterations in more than one driver gene.

Comprehensive evaluation of the test for 5'-/3'-end mRNA unbalanced expression as a screening tool for ALK and ROS1 fusions in lung cancer.

BACKGROUND: Despite the progress in the development of next-generation sequencing (NGS), diagnostic PCR assays remain to be utilized in clinical routine due to their simplicity and low cost. Tests for 5'-/3'-end mRNA unbalanced expression can be used for variant-independent detection of translocations, however, many technical aspects of this methodology require additional investigations. METHODS: Known ALK/ROS1 fusions and 5'-/3'-end unbalanced expression were analyzed in 2009 EGFR mutation-negative non-small cell lung cancer (NSCLC) samples with RT-PCR tests, which were optimized for the use with FFPE-derived RNA. RESULTS: Variant-specific PCR tests for 4 common ALK and 15 common ROS1 translocations detected 115 (5.7%) and 44 (2.2%) rearrangements, respectively. Virtually all samples with common ALK fusions demonstrated some level of 5'/3' mRNA ends unbalanced expression, and 8 additional NSCLCs with rare ALK fusions were further identified by PCR or NGS among 48 cases selected based on ALK expression measurements. Interestingly, NSCLCs with unbalanced 5'-/3'-end ALK expression but without identified ALK translocations had elevated frequency of RAS mutations (21/40, 53%) suggesting the role of RAS activation in the alternative splicing of ALK gene. In contrast to ALK, only a minority of ROS1 translocation-positive cases demonstrated unbalanced gene expression, with both 5'- and 3'-end mRNA expression being elevated in most of the samples with translocations. Surprisingly, high ROS1 expression level was also found to be characteristic for NSCLCs with activating mutations in other tyrosine kinases such as EGFR, ALK, or MET. CONCLUSIONS: Comprehensive ALK analysis can be performed by the test for 5'-/3'-end unbalanced expression with minimal risk of missing an ALK rearrangement. In contrast, the use of the test for 5'-/3'-end unbalanced expression for the detection of ROS1 fusions is complicated; hence, the utilization of variant-specific PCR assays for ROS1 testing is preferable.

Efficacy of lorlatinib in lung carcinomas carrying distinct ALK translocation variants: The results of a single-center study.

BACKGROUND: Lorlatinib is a novel potent ALK inhibitor, with only a few studies reporting the results of its clinical use. METHODS: This study describes the outcomes of lorlatinib treatment for 35 non-small cell lung cancer patients with ALK rearrangements, who had 2 (n = 5), 1 (n = 26) or none (n = 4) prior tyrosine kinase inhibitors and received lorlatinib mainly within the compassionate use program. RESULTS: Objective tumor response (OR) and disease control (DC) were registered in 15/35 (43%) and 33/35 (94%) patients, respectively; brain metastases were particularly responsive to the treatment (OR: 22/27 (81%); DC: 27/27 (100%)). Median progression free survival (PFS) was estimated to be 21.8 months, and median overall survival (OS) approached to 70.1 months. Only 4 out of 35 patients experienced no adverse effects; two of them were the only subjects who had no clinical benefit from lorlatinib. PFS and OS in the no-adverse-events lorlatinib users were strikingly lower as compared to the remaining patients (1.1 months vs. 23.7 months and 10.5 months vs. not reached, respectively; p < 0.0001 for both comparisons). ALK translocation variants were known for 28 patients; there was no statistical difference between patients with V.1 and V.3 rearrangements with regard to the OS or PFS. CONCLUSION: Use of lorlatinib results in excellent disease outcomes, however caution must be taken for patients experiencing no adverse effects from this drug.

Revisiting multiple erroneous genetic testing results and clinical misinterpretations in a patient with Li-Fraumeni syndrome: lessons for translational medicine.

BACKGROUND: Many cancer patients undergo sophisticated laboratory testing, which requires proper interpretation and interaction between different specialists. CASE PRESENTATION: We describe a patient with an extensive family history of cancer, who was diagnosed with bilateral breast cancer and two lung cancer lumps by the age of 40 years. She submitted a lung cancer specimen to a genetic profiling service, which reported the presence of the EGFR mutation (a combination of G719S and L833V substitutions) and the TP53 с.322_327del (p.G108_F109del) mutation in the tumor tissue. Possible therapeutic options were discussed at a medical conference, where one of the discussants raised a concern that the identified TP53 mutation may not necessarily be somatic, but reflect the germ-line status of the gene. Review of clinical records and follow-up dialog with the patient revealed, that she previously provided her blood for DNA analysis in two laboratories. The first laboratory utilized a custom NGS assay and did not detect the TP53 mutation, instead pointed to a potential pathogenic significance of the MSH6 c.2633 T > C (p.V878A) allele. The second laboratory revealed the TP53 с.322_327del (p.G108_F109del) allele but stated in the written report that it has an unknown pathogenic significance. To resolve the possible uncertainty regarding the role of the TP53 с.322_327del (p.G108_F109del) variant, we suggested that the patient invite her second cousin for genetic testing, as she was affected by neuroblastoma at the age of 3 years. This analysis revealed the presence of the same TP53 variant. CONCLUSION: We provide point-by-point discussion, reviewing multiple laboratory mistakes and clinical misinterpretations occurred with this patient. This case report exemplifies the need to involve rigorous clinical expertise in the daily practice of medical laboratory facilities.

Gene rearrangements in consecutive series of pediatric inflammatory myofibroblastic tumors.

BACKGROUND: Inflammatory myofibroblastic tumors (IMTs) are exceptionally rare neoplasms, which are often driven by rearranged tyrosine kinases. METHODS: This study considered 33 consecutive patients with IMT (median age, 6.6; age range, 0.6-15.8 years). RNA and cDNA were successfully obtained in 29 cases. The molecular analysis included sequential tests for 5'/3'-end unbalanced gene expression, variant-specific PCR, and next-generation sequencing (NGS). RESULTS: 5'/3'-end unbalanced ALK expression was revealed in 15/29 (52%) IMTs. Strikingly, all these tumors demonstrated high amount of ALK protein detected by immunohistochemistry. Variant-specific PCR was capable of identifying the type of ALK rearrangement in 11/15 IMTs with 5'/3'-end unbalanced ALK expression. The remaining four tumors were analyzed by NGS; two known and two novel (CLTC-ins6del84-ALK and EEF1G-ALK) ALK rearrangements were detected. Five IMTs demonstrated 5'/3'-end unbalanced ROS1 expression, and all these tumors carried TFG-ROS1 fusion. Nine tumors, which were negative for 5'/3'-end unbalanced ALK/ROS1 expression, were subjected to further analysis. Variant-specific PCR revealed two additional tumors with gene rearrangements (TFG-ROS1 and ETV6-NTRK3). The remaining seven IMTs were tested by NGS; single instances of TFG-ROS1 and novel SRF-PDGFRb translocations were detected. CONCLUSIONS: Twenty-four of 29 IMTs (83%) were shown to have druggable rearrangements involving tyrosine kinases, 20 of these 24 gene fusions were detectable by simple and inexpensive PCR assay, which is based on the detection 5'/3'-end unbalanced gene expression.

Comparative analysis of expression of mutant and wild-type alleles is essential for reliable PCR-based detection of MET exon 14 skipping.

MET exon 14 skipping (exon 14Δ) mutations are associated with tumor sensitivity to a number of tyrosine kinase inhibitors, however clinical testing for MET gene status remains complicated. We developed a simple allele-specific PCR cDNA-based test, which allowed for the identification of MET exon 14Δ allele in 35 (2.5%) out of 1415 EGFR mutation-negative lung carcinomas (LCs). MET exon 14Δ was significantly associated with elderly age and non-smoking status of the patients. A total of 34 (97%) out of 35 tumors carrying MET exon 14Δ showed preferential expression of the mutated allele; this imbalance was attributed to the down-regulation of the expression of the wild-type gene copy. Sanger sequencing confirmed the presence of genomic exon 14 splice site mutations in 24/35 (68.6%) cases, which showed MET exon 14 skipping by PCR. In addition to LCs described above, some carcinomas demonstrated low-abundance MET exon 14Δ-specific signal. Low-level expression of MET exon 14Δ allele may potentially compromise the results of allele-specific PCR-based tests, therefore comparison of the level of expression of mutated and normal alleles is essential for the reliability of MET gene testing.

Variability in lung cancer response to ALK inhibitors cannot be explained by the diversity of ALK fusion variants.

Multiple laboratory evidences indicate that distinct variants of ALK translocations differ in their biochemical properties and responsiveness to ALK tyrosine kinase inhibitors (TKIs). These data are supported by some clinical studies, which showed improved responses to crizotinib in non-small cell lung cancer (NSCLC) patients carrying particular variants of ALK translocation. We retrospectively considered 64 Russian patients with ALK-rearranged NSCLC, who were treated by crizotinib (n = 23), ceritinib (n = 39) or alectinib (n = 2). ALK fusion variants were genotyped by PCR. Median progression-free survival (PFS) approached to 18 and 21 months in subjects with "short" (v.3a/b, v.5a/b) vs. "long" (TAPE-domain containing) fusion variants (p = 0.783), respectively; similar data were obtained while comparing EML4/ALK variant 1 vs. other ALK translocations (19 and 21 months, respectively; p = 0.604). Objective response rates were also strikingly similar in the above groups ("short": 88%, "long": 77%, p = 0.479; variant 1: 76%, other translocations: 81%, p = 0.753). Furthermore, ALK variants did not influence the disease outcomes when patients treated by crizotinib and ceritinib were analyzed separately. Overall, PFS on ALK TKI did not depend on whether the drug was administered upfront or after chemotherapy. Ceritinib produced significantly longer PFS than crizotinib (p = 0.022). In conclusion, this study revealed that distinct ALK translocation variants render similar clinical responsiveness to ALK inhibitors.

EGFR T790M Mutation in TKI-Naïve Clinical Samples: Frequency, Tissue Mosaicism, Predictive Value and Awareness on Artifacts.

BACKGROUND: This study evaluated the distribution of epidermal growth factor receptor (EGFR) T790M mutations in treatment-naïve tumor and normal samples obtained from cancer patients. METHODS: We utilized allele-specific PCR (AS-PCR), digital droplet PCR (ddPCR) and next generation sequencing (NGS) to detect EGFR T790M allele in several collections of tumor and normal human tissues. RESULTS: AS-PCR analysis of treatment-naïve tumor samples revealed somatic T790M mutation in 3/394 (1%) non-small cell lung carcinomas (NSCLC) carrying the tyrosine kinase inhibitor (TKI)-sensitizing EGFR mutation, but in none of 334 NSCLC lacking EGFR exon 19 deletions (ex19del) or L858R substitutions and in none of 235 non-lung tumors. Use of highly sensitive and quantitative assays, such as ddPCR and NGS, produced a high number of T790M-specific signals even in presumably T790M-negative DNA specimens. This background noise was evidently higher in degraded DNA isolated from formalin-fixed paraffin-embedded tissues as compared to high molecular weight DNA. A combination of AS-PCR, ddPCR and NGS revealed mosaic EGFR T790M allele in 2/68 (3%) NSCLC treated with the first-generation TKI. Both these tumors produced evident and durable response to gefitinib. CONCLUSION: Detection of mosaic EGFR T790M mutation in treatment-naïve samples may be compromised by yet unresolved technical issues and may have limited clinical value.

Rapid selection of BRCA1-proficient tumor cells during neoadjuvant therapy for ovarian cancer in BRCA1 mutation carriers.

Ovarian carcinomas (OC) often demonstrate rapid tumor shrinkage upon neoadjuvant chemotherapy (NACT). However, complete pathologic responses are very rare and the mechanisms underlying the emergence of residual tumor disease remain elusive. We hypothesized that the change of somatic BRCA1 status may contribute to this process. The loss-of-heterozygosity (LOH) at the BRCA1 locus was determined for 23 paired tumor samples obtained from BRCA1 germ-line mutation carriers before and after NACT. We observed a somatic loss of the wild-type BRCA1 allele in 74% (17/23) of OCs before NACT. However, a retention of the wild-type BRCA1 copy resulting in a reversion of LOH status was detected in 65% (11/17) of those patients after NACT. Furthermore, we tested 3 of these reversion samples for LOH at intragenic BRCA1 single nucleotide polymorphisms (SNPs) and confirmed a complete restoration of the SNP heterozygosity in all instances. The neoadjuvant chemotherapy for BRCA1-associated OC is accompanied by a rapid expansion of pre-existing BRCA1-proficient tumor clones suggesting that continuation of the same therapy after NACT and surgery may not be justified even in patients initially experiencing a rapid tumor regression.

Novel ALK fusion partners in lung cancer.

Detection of ALK rearrangements in patients with non-small cell lung cancer (NSCLC) presents a significant technical challenge due to the existence of multiple translocation partners and break-points. To improve the performance of PCR-based tests, we utilized the combination of 2 assays, i.e. the variant-specific PCR for the 5 most common ALK rearrangements and the test for unbalanced 5'/3'-end ALK expression. Overall, convincing evidence for the presence of ALK translocation was obtained for 34/400 (8.5%) cases, including 14 EML4ex13/ALKex20, 12 EML4ex6/ALKex20, 3 EML4ex18/ALKex20, 2 EML4ex20/ALKex20 variants and 3 tumors with novel translocation partners. 386 (96.5%) out of 400 EGFR mutation-negative NSCLCs were concordant for both tests, being either positive (n = 26) or negative (n = 360) for ALK translocation; 49 of these samples (6 ALK+, 43 ALK-) were further evaluated by FISH, and there were no instances of disagreement. Among the 14 (3.5%) "discordant" tumors, 5 demonstrated ALK translocation by the first but not by the second PCR assay, and 9 had unbalanced ALK expression in the absence of known ALK fusion variants. 5 samples from the latter group were subjected to FISH, and the presence of translocation was confirmed in 2 cases. Next generation sequencing analysis of these 2 samples identified novel translocation partners, DCTN1 and SQSTM1; furthermore, the DCTN1/ALK fusion was also found in another NSCLC sample with unbalanced 5'/3'-end ALK expression, indicating a recurrent nature of this translocation. We conclude that the combination of 2 different PCR tests is a viable approach for the diagnostics of ALK rearrangements. Systematic typing of ALK fusions is likely to reveal new NSCLC-specific ALK partners.

Candidate gene analysis of BRCA1/2 mutation-negative high-risk Russian breast cancer patients.

Twenty one DNA repair genes were analyzed in a group of 95 BC patients, who displayed clinical features of hereditary disease predisposition but turned out to be negative for mutations in BRCA1 and BRCA2 entire coding region as well as for founder disease-predisposing alleles in CHEK2, NBN/NBS1 and ATM genes. Full-length sequencing of CHEK2 and NBN/NBS1 failed to identify non-founder mutations. The analysis of TP53 revealed a woman carrying the R282W allele; further testing of additional 108 BC patients characterized by a very young age at onset (35 years or earlier) detected one more carrier of the TP53 germ-line defect. In addition, this study confirmed non-random occurrence of PALB2 truncating mutations in Russian hereditary BC patients. None of the studied cases carried germ-line defects in recently discovered hereditary BC genes, BRIP1, FANCC, MRE11A and RAD51C. The analysis of genes with yet unproven BC-predisposing significance (BARD1, BRD7, CHEK1, DDB2, ERCC1, EXO1, FANCG, PARP1, PARP2, RAD51, RNF8, WRN) identified single women carrying a protein-truncating allele, WRN R1406X. DNA sequencing of another set of 95 hereditary BC cases failed to reveal additional WRN heterozygous genotypes. Since WRN is functionally similar to the known BC-predisposing gene, BLM, it deserves to be analyzed in future hereditary BC studies. Furthermore, this investigation revealed a number of rare missense germ-line variants, which are classified as probably protein-damaging by online in silico tools and therefore may require further consideration.