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.

Strong founder effect for BRCA1 c.3629_3630delAG pathogenic variant in Chechen patients with breast or ovarian cancer.

Coding sequences of BRCA1, BRCA2, ATM, TP53, and PALB2 genes were analyzed in 68 consecutive Chechen patients with high-grade serous ovarian cancer (HGSOC). Pathogenic BRCA1/2 variants were identified in 15 (22%) out of 68 HGSOC cases. Nine out of ten patients with BRCA1 pathogenic alleles carried the same deletion (c.3629_3630delAG), and three out of five BRCA2 heterozygotes had Q3299X allele. The analysis of 49 consecutive patients with triple-negative breast cancer (TNBC) revealed 3 (6%) additional BRCA1 heterozygotes. All women with BRCA1 c.3629_3630delAG allele also carried linked c.1067G>A (Q356R) single nucleotide polymorphism, indicating that this is a genuine founder variant but not a mutational hotspot. An ATM truncating allele was detected in one HGSOC patient. There were no women with TP53 or PALB2 germline alterations. Genetic analysis of non-selected HGSOC patients is an efficient tool for the identification of ethnicity-specific BRCA1/2 pathogenic variants.

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.

Molecular predictors of the outcome of paclitaxel plus carboplatin neoadjuvant therapy in high-grade serous ovarian cancer patients.

BACKGROUND: Patients with advanced high-grade serous ovarian cancer (HGSOC) are usually treated with paclitaxel and carboplatin; however, predictive markers for this drug combination are unknown. METHODS: Tumor samples from 71 consecutive HGSOC patients, who received neoadjuvant chemotherapy with paclitaxel and carboplatin, were subjected to molecular analysis. RESULTS: BRCA1/2 germline mutation carriers (n = 22) had longer treatment-free interval (TFI) than non-carriers (n = 49) (9.5 months vs. 3.8 months; P = 0.007). Fifty-one HGSOCs had sufficient quality of tumor DNA for the next-generation sequencing (NGS) analysis by the SeqCap EZ CNV/LOH Backbone Design panel. All 13 tumors obtained from BRCA1/2 germline mutation carriers and 12 sporadic HGSOCs showed a high number of evenly spread chromosomal breaks, which was defined as a BRCAness phenotype; median TFI for this combined group approached 9.5 months. The remaining 26 HGSOCs had similarly high global LOH score (above 20%); however, in contrast to BRCAness tumors, LOH involved large chromosomal segments; these patients had significantly lower TFI (3.7 months; P = 0.006). All patients with CCNE1 amplification (n = 7), TP53 R175H substitution (n = 6), and RB1 mutation (n = 4) had poor response to paclitaxel plus carboplatin. CONCLUSION: This study describes a cost-efficient method of detecting the BRCAness phenotype, which is compatible with the laboratory-scale NGS equipment. Some molecular predictors allow the identification of potential non-responders to paclitaxel plus carboplatin, who may need to be considered for other treatment options.

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.

Neoadjuvant therapy of BRCA1-driven ovarian cancer by combination of cisplatin, mitomycin C and doxorubicin.

BACKGROUND: Cisplatin, mitomycin C and anthracyclines demonstrate high activity in BRCA1-deficient tumors. This study aimed to evaluate the efficacy of the triplet combination of these drugs in BRCA1-driven high-grade serous ovarian carcinomas (HGSOCs). METHODS: Ten HGSOC patients with germ-line BRCA1 mutation received neoadjuvant chemotherapy (NACT) consisting of mitomycin C 10 mg/m2 (day 1), doxorubicin 30 mg/m2 (days 1 and 8) and cisplatin 80 mg/m2 (day 1), given every 4 weeks (MAP regimen). The comparator group included 16 women, who received standard NACT combination of paclitaxel 175 mg/m2 and carboplatin (6 AUC), given every 3 weeks (TCbP scheme). RESULTS: None of the patients treated by the MAP scheme demonstrated complete pathologic response in ovaries, while 4 women showed absence of tumor cells in surgically excised omental specimens. When chemotherapy response scores (CRS) were considered, poor responsiveness (CRS 1) was not observed in the MAP group, but was common for the TCbP regimen (6/16 (38 %) for ovaries and 5/16 (31 %) for omentum; p = 0.05 and 0.12, respectively). Median treatment-free interval (TFI) was not reached in women treated by the MAP, but was 9.5 months for the TCbP scheme (p = 0.1). The rate of the recurrence within 1 year after the completion of the treatment was 4/10 (40 %) for the MAP and 10/13 (77 %) for the TCbP (p = 0.1). CONCLUSIONS: The attempt to intensify NACT by administering combination of 3 drugs did not result in high rate of complete pathologic responses. However, there was a trend towards higher efficacy of the MAP regimen versus conventional TCbP scheme with regard to CRS and clinical outcomes.

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.

Rapid Improvement of the Performance Status and Reduction of the Tumor Size in KRAS-Mutated Colorectal Cancer Patient Receiving Binimetinib, Hydroxychloroquine, and Bevacizumab.

Activating RAS mutations occur in more than a half of colorectal cancers (CRCs). RAS-mutated CRCs are notoriously difficult to treat given that they are characterized by the aggressive disease course and the lack of appropriate targeted therapies. Recent preclinical studies demonstrated that RAS-mutated cells escape from therapeutic MEK inhibition by the development of autophagy, and this escape may be prevented by the administration of an antimalarial drug, hydroxychloroquine. The available clinical data are limited to a single case observation involving a patient with KRAS-mutated pancreatic cancer. Here, we report a woman with KRAS G12D-mutated CRC, whose tumor did not respond to conventional therapy. The combination of binimetinib, hydroxychloroquine, and bevacizumab was administered as a last-hope option. The patient experienced rapid improvement of the performance status. The tumor lumps demonstrated 17% reduction in the size within the first 6 weeks of the therapy. This report calls for evaluation of the efficacy of a combination of MEK inhibitors and hydroxychloroquine, possibly with the addition of bevacizumab, in chemotherapy-resistant patients with RAS-mutated cancers.

Frequency and spectrum of founder and non-founder BRCA1 and BRCA2 mutations in a large series of Russian breast cancer and ovarian cancer patients.

BACKGROUND: The spectrum of BRCA1 and BRCA2 mutations in Slavic countries is characterized by a high prevalence of founder alleles. METHODS: We analyzed a large data set of Russian breast cancer (BC) and ovarian cancer (OC) patients, who were subjected to founder mutation tests or full-length BRCA1 and BRCA2 analysis. RESULTS: The most commonly applied test, which included four founder mutations (BRCA1: 5382insC, 4153delA, 185delAG; BRCA2: 6174delT), identified BRCA1 or BRCA2 heterozygosity in 399/8533 (4.7%) consecutive BC patients, 230/2317 (9.9%) OC patients, and 30/118 (25.4%) women with a combination of BC and OC. The addition of another four recurrent BRCA1 mutations to the test (BRCA1 C61G, 2080delA, 3819del5, 3875del4) resulted in evident increase in the number of identified mutation carriers (BC: 16/993 (1.6%); OC: 34/1289 (2.6%); BC + OC: 2/39 (5.1%)). Full-length sequencing of the entire BRCA1 and BRCA2 coding region was applied to 785 women, very most of whom demonstrated clinical signs of BRCA-driven disease, but turned out negative for all described above founder alleles. This analysis revealed additional BRCA1 or BRCA2 mutation carriers in 54/282 (19.1%) BC, 50/472 (10.6%) OC, and 13/31 (42%) BC + OC patients. The analysis of frequencies of founder and "rare" BRCA1 and BRCA2 pathogenic alleles across various clinical subgroups (BC vs. OC vs. BC + OC; family history positive vs. negative; young vs. late-onset; none vs. single vs. multiple clinical indicators of BRCA1- or BRCA2-associated disease) revealed that comprehensive BRCA1 and BRCA2 analysis increased more than twice the number of identified mutation carriers in all categories of the examined women. CONCLUSION: Full-length BRCA1 and BRCA2 sequencing is strongly advised to Slavic subjects, who have medical indications for BRCA1 and BRCA2 testing but are negative for recurrent BRCA1 and BRCA2 mutations.

The spectrum of Lynch syndrome-associated germ-line mutations in Russia.

Hereditary non-polyposis colorectal cancer (HNPCC), also known as Lynch syndrome (LS), is a common cancer-predisposing syndrome. This study aimed to investigate the spectrum of germ-line mutations in Russian LS patients. LS-related mismatch repair (MMR) genes were analyzed in 16 patients, who were forwarded to genetic testing due to strong clinical features of LS and had high-level microsatellite instability (MSI-H) in the tumor (n = 14) or unknown MSI status (n = 2). In addition, 672 consecutive colorectal cancer (CRC) cases were screened for family history; 15 patients were younger than 50 years and reported 2 or more instances of LS-related cancers in 1st- or 2nd-degree relatives. Seven of these cases demonstrated MSI-H and therefore were subjected to DNA germ-line testing. Overall, 17/23 (74%) subjects carried LS-associated gene variants (MLH1: 10; MSH2: 4; MSH6: 2; PMS2: 1), with 2 alleles (MLH1 c.677G > T and MSH2 с.1906G > C) detected twice. Testing for recurrent mutations of 30 consecutive MSI-H CRCs led to the identification of 2 additional subjects with LS. The analysis of all relevant publications identified 28 unrelated LS patients presented in Russian medical literature and 3 unrelated Russian LS subjects described in international journals. Overall, 15/49 (31%) genetic defects revealed in Russian LS patients were represented by six recurrent alleles (MLH1: c.350C > T, c.677G > T, c.1852_1854del; MSH2: c.942+3A > T, c.1861C > T, с.1906G > C). We conclude that the founder effect for LS in Russia is seemingly less pronounced than the one for hereditary breast-ovarian cancer syndrome, however testing for recurrent LS mutations may be considered feasible in some circumstances.

Exome sequencing study of Russian breast cancer patients suggests a predisposing role for USP39.

PURPOSE: Germline variants in known breast cancer (BC) predisposing genes explain less than half of hereditary BC cases. This study aimed to identify missing genetic determinants of BC. METHODS: Whole exome sequencing (WES) of lymphocyte DNA was performed for 49 Russian patients with clinical signs of genetic BC predisposition, who lacked Slavic founder mutations in BRCA1, BRCA2, CHEK2, and NBS1 genes. RESULTS: Bioinformatic analysis of WES data was allowed to compile a list of 229 candidate mutations. 79 of these mutations were subjected to a three-stage case-control analysis. The initial two stages, which involved up to 797 high-risk BC patients, 1504 consecutive BC cases, and 1081 healthy women, indicated a potentially BC-predisposing role for 6 candidates, i.e., USP39 c.*208G > C, PZP p.Arg680Ter, LEPREL1 p.Pro636Ser, SLIT3 p.Arg154Cys, CREB3 p.Lys157Glu, and ING1 p.Pro319Leu. USP39 c.*208G > C was strongly associated with triple-negative breast tumors (p = 0.0001). In the third replication stage, we genotyped the truncating variant of PZP (rs145240281) and the potential splice variant of USP39 (rs112653307) in three independent cohorts of Russian, Byelorussian, and German ancestry, comprising a total of 3216 cases and 2525 controls. The data obtained for USP39 rs112653307 supported the association identified in the initial stages (the combined OR 1.72, p = 0.035). CONCLUSIONS: This study suggests the role of a rare splicing variant in BC susceptibility. USP39 encodes an ubiquitin-specific peptidase that regulates cancer-relevant tumor suppressors including CHEK2. Further epidemiological and functional studies involving these gene variants are warranted.

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.