Pathways and targeting avenues of BRAF in non-small cell lung cancer.

INTRODUCTION: BRAF is a serine-threonine kinase implicated in the regulation of MAPK signaling cascade. BRAF mutation-driven activation occurs in approximately 2-4% of treatment-naive non-small cell carcinomas (NSCLCs). BRAF upregulation is also often observed in tumors with acquired resistance to receptor tyrosine kinase inhibitors (TKIs). AREAS COVERED: This review describes the spectrum of BRAF mutations and their functional roles, discusses treatment options available for BRAF p.V600 and non-V600 mutated NSCLCs, and identifies some gaps in the current knowledge. EXPERT OPINION: Administration of combined BRAF/MEK inhibitors usually produces significant, although often a short-term, benefit to NSCLC patients with BRAF V600 (class 1) mutations. There are no established treatments for BRAF class 2 (L597, K601, G464, G469A/V/R/S, fusions, etc.) and class 3 (D594, G596, G466, etc.) mutants, which account for up to two-thirds of BRAF-driven NSCLCs. Many important issues related to the use of immune therapy for the management of BRAF-mutated NSCLC deserve further investigation. The rare occurrence of BRAF mutations in NSCLC is compensated by high overall incidence of lung cancer disease; therefore, clinical studies on BRAF-associated NSCLC are feasible.

Preparation of Duplex Sequencing Libraries for Archival Paraffin-Embedded Tissue Samples Using Single-Strand-Specific Nuclease P1.

DNA from formalin-fixed paraffin-embedded (FFPE) tissues, which are frequently utilized in cancer research, is significantly affected by chemical degradation. It was suggested that approaches that are based on duplex sequencing can significantly improve the accuracy of mutation detection in FFPE-derived DNA. However, the original duplex sequencing method cannot be utilized for the analysis of formalin-fixed paraffin-embedded (FFPE) tissues, as FFPE DNA contains an excessive number of damaged bases, and these lesions are converted to false double-strand nucleotide substitutions during polymerase-driven DNA end repair process. To resolve this drawback, we replaced DNA polymerase by a single strand-specific nuclease P1. Nuclease P1 was shown to efficiently remove RNA from DNA preparations, to fragment the FFPE-derived DNA and to remove 5'/3'-overhangs. To assess the performance of duplex sequencing-based methods in FFPE-derived DNA, we constructed the Bottleneck Sequencing System (BotSeqS) libraries from five colorectal carcinomas (CRCs) using either DNA polymerase or nuclease P1. As expected, the number of identified mutations was approximately an order of magnitude higher in libraries prepared with DNA polymerase vs. nuclease P1 (626 ± 167/Mb vs. 75 ± 37/Mb, paired t-test p-value 0.003). Furthermore, the use of nuclease P1 but not polymerase-driven DNA end repair allowed a reliable discrimination between CRC tumors with and without hypermutator phenotypes. The utility of newly developed modification was validated in the collection of 17 CRCs and 5 adjacent normal tissues. Nuclease P1 can be recommended for the use in duplex sequencing library preparation from FFPE-derived DNA.

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.

Frequency and molecular characteristics of PALB2-associated cancers in Russian patients.

PALB2 is а high-penetrance gene for hereditary breast cancer (BC). Our study aimed to investigate the spectrum of PALB2 mutations in Russian cancer patients. PALB2 sequencing revealed pathogenic variants in 3/190 (1.6%) young-onset and/or familial and/or bilateral BC cases but none in 96 ovarian cancer (OC) or 172 pancreatic cancer patients. Subsequently, seven recurrent PALB2 pathogenic alleles were selected from this and previous Slavic studies and tested in an extended patient series. PALB2 pathogenic variants were detected in 5/585 (0.9%) "high-risk" BC, 10/1508 (0.7%) consecutive BC and 5/1802 (0.3%) OC cases. Haplotyping suggested that subjects with Slavic alleles c.509-510delGA (n = 10) and c.172-175delTTGT (n = 4) as well as carriers of Finnish c.1592delT mutation (n = 4) originated from a single founder each, while PALB2 p.R414X allele (n = 4) had at least two independent founders. Somatic loss of heterozygosity (LOH) was revealed in 5/10 chemonaive BCs and in 0/2 BC samples obtained after neoadjuvant therapy. Multigene sequencing identified somatic PALB2 inactivating point mutation in one out of two tumors without PALB2 LOH but in none of four BCs with PALB2 LOH. Genomic instability, as determined by NGS, was observed in four out of five tumors with biallelic PALB2 inactivation but not in the BC sample with the preserved wild-type PALB2 allele. PALB2 germ-line mutations contribute to a small fraction of cancer cases in Russia. The majority although not all PALB2-driven BCs have somatic inactivation of the remaining PALB2 allele and therefore potential sensitivity to platinum compounds and PARP inhibitors.

Systemic investigations into the molecular features of bilateral breast cancer for diagnostic purposes.

Introduction: Many breast cancer (BC) patients develop the disease bilaterally. The emergence of two tumors in the same host is unlikely to be a random co-incidence: bilateral BC (biBC) patients are enriched by women who are susceptible to this disease due to genetic or non-genetic factors.Areas covered: Data on molecular pathogenesis and translational aspects of biBC research are summarized.Expert opinion: Studies on concordant and discordant molecular events occurring in paired tumors resemble twin studies, as they help to reveal core components of BC pathogenesis and to analyze interactions between host factors and tumor phenotype. Mutation profiling of biBC pairs suggested that most biBCs are clonally independent malignancies, although some instances of presumably contralateral metastatic spread were shown as well. Many biBCs, especially synchronous ones, demonstrate the similarity of essential tumor characteristics, which can be explained by sharing of genetic background, hormonal milieu, metabolic environment, and external exposures. biBC is strongly associated with BC-predisposing germline mutations; therefore, clinical management of biBC patients must include comprehensive genetic testing. Some contralateral metachronous BCs demonstrate high-level microsatellite instability (MSI-H). MSI-H is sometimes observed in radiation- and chemotherapy-induced tumors; therefore, it is possible that some second BCs are causally related to the therapy applied for the first cancer. MSI-H tumors are responsive to immune checkpoint blockade; hence, MSI-H analysis is advisable for biBC molecular testing. Systematic cataloging of biBC molecular portraits is likely to provide valuable information on fundamental aspects of cancer pathogenesis.

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.

First-Line Cetuximab Monotherapy in KRAS/NRAS/BRAF Mutation-Negative Colorectal Cancer Patients.

BACKGROUND: Colorectal carcinomas (CRCs) are sensitive to treatment by anti-epidermal growth factor receptor (EGFR) antibodies only if they do not carry activating mutations in down-stream EGFR targets (KRAS/NRAS/BRAF). Most clinical trials for chemo-naive CRC patients involved combination of targeted agents and chemotherapy, while single-agent cetuximab or panitumumab studies included either heavily pretreated patients or subjects who were not selected on the basis of molecular tests. We hypothesized that anti-EGFR therapy would have significant efficacy in chemo-naive patients with KRAS/NRAS/BRAF mutation-negative CRC. METHODS: Nineteen patients were prospectively included in the study. RESULTS: Two (11%) patients experienced partial response (PR) and 11 (58%) subjects showed stable disease (SD). Median time to progression approached 6.1 months (range 1.6-15.0 months). Cetuximab efficacy did not correlate with RNA expression of EGFR and insulin-like growth factor 2 (IGF2). Only one tumor carried PIK3CA mutation, and this CRC responded to cetuximab. Exome analysis of patients with progressive disease (PD) revealed 1 CRC with high-level microsatellite instability and 1 instance of HER2 oncogene amplification; 3 of 4 remaining patients with PD had allergic reactions to cetuximab, while none of the subjects with PR or SD had this complication. Comparison with 19 retrospective KRAS/NRAS/BRAF mutation-negative patients receiving first-line fluoropyrimidines revealed no advantages or disadvantages of cetuximab therapy. CONCLUSIONS: Cetuximab demonstrates only modest efficacy when given as a first-line monotherapy to KRAS/NRAS/BRAF mutation-negative CRC patients. It is of question, why meticulous patient selection, which was undertaken in the current study, did not result in the improvement of outcomes of single-agent cetuximab treatment.

Detection of BRCA1 gross rearrangements by droplet digital PCR.

PURPOSE: Large genomic rearrangements (LGRs) constitute a significant share of pathogenic BRCA1 mutations. Multiplex ligation-dependent probe amplification (MLPA) is a leading method for LGR detection; however, it is entirely based on the use of commercial kits, includes relatively time-consuming hybridization step, and is not convenient for large-scale screening of recurrent LGRs. MATERIALS AND METHODS: We developed and validated the droplet digital PCR (ddPCR) assay, which covers the entire coding region of BRCA1 gene and is capable to precisely quantitate the copy number for each exon. RESULTS: 141 breast cancer (BC) patients, who demonstrated evident clinical features of hereditary BC but turned out to be negative for founder BRCA1/2 mutations, were subjected to the LGR analysis. Four patients with LGR were identified, with three cases of exon 8 deletion and one women carrying the deletion of exons 5-7. Excellent concordance with MLPA test was observed. Exon 8 copy number was tested in additional 720 BC and 184 ovarian cancer (OC) high-risk patients, and another four cases with the deletion were revealed; MLPA re-analysis demonstrated that exon 8 loss was a part of a larger genetic alteration in two cases, while the remaining two patients had isolated defect of exon 8. Long-range PCR and next generation sequencing of DNA samples carrying exon 8 deletion revealed two types of recurrent LGRs. CONCLUSION: Droplet digital PCR is a reliable tool for the detection of large genomic rearrangements.

The role of intradermal proliferation of T-cells in the pathogenesis of psoriasis.

BACKGROUND:: Psoriasis is a common immune-mediated chronic inflammatory disease of the skin and joints, affecting 1-3% of the population. It is generally accepted that the pathogenesis of psoriasis involves accumulation of effector T-cells within lymph nodes and their subsequent migration into the skin through the blood system. Here we provide evidence that psoriatic plaque itself may serve as a source of inflammatory T-cells. OBJECTIVE:: We examined the intradermal proliferation of T-cells and the number of effector/memory (CD45RO+) T-cells in the skin of psoriatic patients at different periods of the disease. METHODS:: Skin samples were obtained from 41 patients with progressive psoriatic lesions; 18 of these patients also donated skin specimens during the remission of the disease. The control group consisted of 16 healthy subjects. Ki-67 immunohistochemical staining was applied to detect proliferating cells, CD3ε served as a T-cell marker, and CD45RA and CD45RO antibodies were utilized to discriminate between naive and effector/memory T-cells, respectively. RESULTS:: Progressive psoriatic lesions demonstrated Ki67 staining both in keratinocytes and in the CD3ε+ cells of dermal infiltrate. Median count of CD45RO+ cells per microscopic field was 15 in healthy controls, 59 in patients in remission and 208 in progressive psoriatic plaques. The observed differences demonstrated high level of statistical significance. STUDY LIMITATIONS:: Limited number of analyzed patients. CONCLUSION:: Progressive phase of psoriasis is characterized by intradermal proliferation of T-cells. Spots of regressed psoriatic lesions contain high number of CD45RO+ cells, which are likely to render an immunological memory.

The role of intradermal proliferation of T-cells in the pathogenesis of psoriasis

Abstract: BACKGROUND: Psoriasis is a common immune-mediated chronic inflammatory disease of the skin and joints, affecting 1-3% of the population. It is generally accepted that the pathogenesis of psoriasis involves accumulation of effector T-cells within lymph nodes and their subsequent migration into the skin through the blood system. Here we provide evidence that psoriatic plaque itself may serve as a source of inflammatory T-cells. OBJECTIVE: We examined the intradermal proliferation of T-cells and the number of effector/memory (CD45RO+) T-cells in the skin of psoriatic patients at different periods of the disease. METHODS: Skin samples were obtained from 41 patients with progressive psoriatic lesions; 18 of these patients also donated skin specimens during the remission of the disease. The control group consisted of 16 healthy subjects. Ki-67 immunohistochemical staining was applied to detect proliferating cells, CD3ε served as a T-cell marker, and CD45RA and CD45RO antibodies were utilized to discriminate between naive and effector/memory T-cells, respectively. RESULTS: Progressive psoriatic lesions demonstrated Ki67 staining both in keratinocytes and in the CD3ε+ cells of dermal infiltrate. Median count of CD45RO+ cells per microscopic field was 15 in healthy controls, 59 in patients in remission and 208 in progressive psoriatic plaques. The observed differences demonstrated high level of statistical significance. STUDY LIMITATIONS: Limited number of analyzed patients. CONCLUSION: Progressive phase of psoriasis is characterized by intradermal proliferation of T-cells. Spots of regressed psoriatic lesions contain high number of CD45RO+ cells, which are likely to render an immunological memory.

Distribution of EGFR Mutations in 10,607 Russian Patients with Lung Cancer.

INTRODUCTION: This study was aimed to evaluate distribution of epidermal growth factor receptor (EGFR) mutations in a large series of Russian lung cancer (LC) patients. METHODS: 10,607 LC samples were considered for EGFR analysis; EGFR status was successfully determined in 10,426 cases (98.3 %), indicating relatively low failure rate. RESULTS: EGFR mutations (ex19del and L858R) were detected in 1759/8716 (20.2 %) adenocarcinomas, 28/669 (4.2 %) squamous cell carcinomas (SCC) and 8/119 (6.7 %) large cell carcinomas. The occurrence of EGFR mutations in adenocarcinomas gradually increased with age, being attributed mainly to the increment of the L858R frequency in non-smokers (patients aged 18-30 years: 1/27 (3.7 %); 31-40 years: 5/98 (5.1 %); 41-50 years: 18/276 (6.5 %); 51-60 years: 102/944 (10.8 %); 61-70 years: 138/1011 (13.7 %); 71-80 years: 85/496 (17.1 %); 81-100 years: 5/27 (18.5 %); p < 0.0001). The EGFR mutation was detected in 804/2107 (38.2 %) non-smoking women versus 125/806 (15.5 %) non-smoking men (p < 0.0001), while the corresponding figures for smokers were 60/273 (22.0 %) versus 147/2214 (6.6 %) (p < 0.0001). The obtained gender-related data differ from the estimates obtained in Asian studies; they indicate that increased prevalence of EGFR mutations in white females may not be entirely attributed to the low prevalence of smoking, but is likely to be related to gender factors per se. CONCLUSION: Biological causes of distinct age- and gender-related distribution of EGFR mutations in LC deserve further investigation.

Identification of novel hereditary cancer genes by whole exome sequencing.

Whole exome sequencing (WES) provides a powerful tool for medical genetic research. Several dozens of WES studies involving patients with hereditary cancer syndromes have already been reported. WES led to breakthrough in understanding of the genetic basis of some exceptionally rare syndromes; for example, identification of germ-line SMARCA4 mutations in patients with ovarian hypercalcemic small cell carcinomas indeed explains a noticeable share of familial aggregation of this disease. However, studies on common cancer types turned out to be more difficult. In particular, there is almost a dozen of reports describing WES analysis of breast cancer patients, but none of them yet succeeded to reveal a gene responsible for the significant share of missing heritability. Virtually all components of WES studies require substantial improvement, e.g. technical performance of WES, interpretation of WES results, mode of patient selection, etc. Most of contemporary investigations focus on genes with autosomal dominant mechanism of inheritance; however, recessive and oligogenic models of transmission of cancer susceptibility also need to be considered. It is expected that the list of medically relevant tumor-predisposing genes will be rapidly expanding in the next few years.

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.

High prevalence of GPRC5A germline mutations in BRCA1-mutant breast cancer patients.

In a search for new breast cancer (BC) predisposing genes, we performed a whole exome sequencing analysis using six patient samples of familial BC and identified a germline inactivating mutation c.183delG [p. Arg61fs] in an orphan G protein-coupled receptor GPRC5A. An extended case-control study revealed a tenfold enrichment for this mutation in BC patients carrying the 5382insC allele of BRCA1, the major founder mutation in the Russian population, compared to wild-type BRCA1 BC cases [6/117 (5.1%) vs. 8/1578 (0.5%), p = 0.0002]. In mammary tumors (n = 60), the mRNA expression of GPRC5A significantly correlated with that of BRCA1 (p = 0.00018). In addition, the amount of GPRC5A transcript was significantly lower in BC obtained from BRCA1 mutation carriers (n = 17) compared to noncarriers (n = 93) (p = 0.026). Accordingly, a siRNA-mediated knockdown of either BRCA1 or GPRC5A in the MDA-MB-231 human BC cell line reduced expression of GPRC5A or BRCA1, respectively. Knockdown of GPRC5A also attenuated radiation-induced BRCA1- and RAD51-containing nuclear DNA repair foci. Taken together, these data suggest that GPRC5A is a modifier of BC risk in BRCA1 mutation carriers and reveals a functional interaction of these genes.

Pattern of clinically relevant mutations in consecutive series of Russian colorectal cancer patients.

One hundred and ninety-five consecutive surgically treated Russian colorectal cancer (CRC) patients were retrospectively analyzed for the presence of mutations in KRAS, NRAS, BRAF and PIK3CA genes as well as for the microsatellite instability status. Comparison between high-resolution melting analysis, co-amplification at lower denaturation temperature PCR, DNA sequencing and allele-specific PCR for the detection of KRAS codon 12/13 mutations revealed that none of these methods alone provided satisfactory results in 100 % of the analyzed cases; this experience supports the use of more than one mutation-detecting technique at least in some circumstances. KRAS codon 12/13 substitutions were detected in 70 (35.9 %) CRC cases. Other mutations in the RAS/RAF genes occurred in 22 (11.3 %) cases and included rare KRAS (n = 6), NRAS (n = 8) and BRAF (n = 8) alterations. 5 BRAF mutations affected codon 600, while the remaining 3 potentially functional substitutions were located in the position 594. Twenty-four (12.3 %) CRC cases carried mutations in the PIK3CA, and 18 of these tumors also contained activating alteration in the RAS/RAF genes (p = 0.007). Only 3 (1.5 %) CRC cases showed high-level microsatellite instability (MSI-H) as determined by a panel of mononucleotide markers. Overall, the distribution of potentially predictive mutations in Russian CRC cases is similar to the one observed in other patient series of European descent. Noticeable occurrence of D594G mutation in BRAF oncogene and low frequency of MSI-H may deserve specific attention.

Evidence for angiogenesis-independent contribution of VEGFR1 (FLT1) in gastric cancer recurrence.

Angiogenesis plays an important role in cancer progression and involves activation of multiple signaling cascades. This study investigated the relationships between microvessel density, expression of VEGF and VEGFR1 (FLT1), and gastric cancer (GC) recurrence. Twenty-nine surgically treated GC cases with similar initial clinical presentation were selected for the study; 11 of these cases recurred within 3 years, while the remaining 18 did not. Microvessel density correlated with VEGF mRNA content, but neither of these parameters was associated with the disease outcome. When tumors were ranked according to the level of expression of angiogenic molecules, 9 out of 10 cases with the highest VEGFR1 expression belonged to the recurrence group, while none of the 10 GC with the lowest content of VEGFR1 mRNA had the disease relapse (p = 0.000). VEGFR1 expression did not show even a trend to correlation with the level of cancer tissue vascularization. Immunofluorescent staining by anti-VEGFR1 antibody revealed VEGFR1 expression in tumor cells but not in other cell types. Our data provide indirect support to the evidence for a non-angiogenic contribution of VEGFR1 in cancer pathogenesis.

Evidence for predictive role of BRCA1 and bTUBIII in gastric cancer.

Sensitivity of gastric cancer (GC) to conventional cytotoxic therapy may be at least in part attributed to molecular features of the tumor cells. We analyzed all patients with metastatic GC treated in the N.N. Petrov Institute of Oncology (St. Petersburg) within years 1999-2010 and identified 65 cases with evaluable treatment response and available biological material. Two of 65 patients (3 %) carried germ-line BRCA1 5382insC mutation and demonstrated particularly pronounced response to the treatment; both of their tumors showed loss of the remaining BRCA1 allele, thus confirming the causative role of BRCA1 heterozygosity in GC predisposition. RNA expression of TS, DPD, BRCA1, ERCC, TOP2A and bTUBIII was analyzed in the remaining 63 tumors. Low BRCA1 expression was associated with increased response rate [6/9 (67 %) vs. 17/54 (32 %), p = 0.04]. Low bTUBIII level correlated with the improved probability of tumor response [21/49 (43 %) vs. 1/13 (8 %), p = 0.02] and prolonged overall survival (10.5 vs. 7.1 months, p = 0.02); this trend was maintained both for taxane-containing and for taxane-free drug combinations. We conclude that GC should be considered as a part of BRCA1-related hereditary cancer syndrome. Tumors with BRCA1 inactivation and low bTUBIII expression demonstrate improved response to cytotoxic therapy.

Value of bilateral breast cancer for identification of rare recessive at-risk alleles: evidence for the role of homozygous GEN1 c.2515_2519delAAGTT mutation.

Virtually all known tumor predisposing genes have been identified via the analysis of familial cancer cases. Here we argue that this approach is likely to miss recessively acting cancer genes and suggest the analysis of family history-negative patients with multiple primary malignancies for identifying homozygous at-risk genotypes. We performed calculations showing that the homozygous carriers of rare recessive cancer predisposing alleles are unlikely to report a family history of the disease. We further revealed that the c.2515_2519delAAGTT homozygous mutation in a Holliday junction resolvase, GEN1, was overrepresented in women with bilateral breast cancer (BC) as compared to healthy controls [11/360 (3.1 %) vs. 18/1305 (1.4 %); odds ratio (OR) = 2.25 (1.02-4.75); p = 0.031], although this trend was not maintained in unilateral BC patients [23/1851 (1.2 %)]. Noticeably, presence of biallelic c.2515_2519delAAGTT mutation was associated with the absence of BC in mother both in bilateral and unilateral BC cases [7/239 (3.0 %) vs. 0/41 (0 %) and 21/1,558 (1.3 %) vs. 0/215 (0 %), respectively; Mantel-Haenszel p = 0.041]. Thus, this study suggests that identification of dominant and recessive cancer predisposing genes may require distinct study groups.

High prevalence and breast cancer predisposing role of the BLM c.1642 C>T (Q548X) mutation in Russia.

The BLM gene belongs to the RecQ helicase family and has been implicated in the maintenance of genomic stability. Its homozygous germline inactivation causes Bloom syndrome, a severe genetic disorder characterized by growth retardation, impaired fertility and highly elevated cancer risk. We hypothesized that BLM is a candidate gene for breast cancer (BC) predisposition. Sequencing of its entire coding region in 95 genetically enriched Russian BC patients identified two heterozygous carriers of the c.1642 C>T (Q548X) mutation. The extended study revealed this allele in 17/1,498 (1.1%) BC cases vs. 2/1,093 (0.2%) healthy women (p = 0.004). There was a suggestion that BLM mutations were more common in patients reporting first-degree family history of BC (6/251 (2.4%) vs. 11/1,247 (0.9%), p = 0.05), early-onset cases (12/762 (1.6%) vs. 5/736 (0.7%), p = 0.14) and women with bilateral appearance of the disease (2/122 (1.6%) vs. 15/1376 (1.1%), p = 0.64). None of the BLM-associated BC exhibited somatic loss of heterozygosity at the BLM gene locus. This study demonstrates that BLM Q548X allele is recurrent in Slavic subjects and may be associated with BC risk.

High level of miR-21, miR-10b, and miR-31 expression in bilateral vs. unilateral breast carcinomas.

We analyzed the expression of several microRNAs (miRs) implicated in breast cancer (BC) pathogenesis (miR-21, miR-10b, miR17-5p, mir-31, miR-155, miR-200c, miR-18a, miR-205, and miR-27a) in 80 breast carcinomas obtained from patients with bilateral BC (biBC) and 40 cases of unilateral BC (uBC). Unexpectedly, three miRs (miR-21, miR-10b and miR-31) demonstrated significantly higher level of expression in biBC vs. uBC (P = 0.0001, 0.00004 and 0.0002, respectively). Increased contents of miR-21, miR-10b and miR-31 were observed in all categories of biBC tumors, i.e., in synchronous biBC as well as in first and second tumors from metachronous biBC cases. Synchronous biBC showed more similarity of miR expression profiles within pairs that the metachronous doublets (P = 0.004). This study suggests that bilateral breast tumors have somewhat distinct pattern of molecular events as compared to the unilateral disease.