Single-cell mutation analysis of clonal evolution in myeloid malignancies.

Myeloid malignancies, including acute myeloid leukaemia (AML), arise from the expansion of haematopoietic stem and progenitor cells that acquire somatic mutations. Bulk molecular profiling has suggested that mutations are acquired in a stepwise fashion: mutant genes with high variant allele frequencies appear early in leukaemogenesis, and mutations with lower variant allele frequencies are thought to be acquired later1-3. Although bulk sequencing can provide information about leukaemia biology and prognosis, it cannot distinguish which mutations occur in the same clone(s), accurately measure clonal complexity, or definitively elucidate the order of mutations. To delineate the clonal framework of myeloid malignancies, we performed single-cell mutational profiling on 146 samples from 123 patients. Here we show that AML is dominated by a small number of clones, which frequently harbour co-occurring mutations in epigenetic regulators. Conversely, mutations in signalling genes often occur more than once in distinct subclones, consistent with increasing clonal diversity. We mapped clonal trajectories for each sample and uncovered combinations of mutations that synergized to promote clonal expansion and dominance. Finally, we combined protein expression with mutational analysis to map somatic genotype and clonal architecture with immunophenotype. Our findings provide insights into the pathogenesis of myeloid transformation and how clonal complexity evolves with disease progression.

Author Correction: Tumour lineage shapes BRCA-mediated phenotypes.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Tumour lineage shapes BRCA-mediated phenotypes.

Mutations in BRCA1 and BRCA2 predispose individuals to certain cancers1-3, and disease-specific screening and preventative strategies have reduced cancer mortality in affected patients4,5. These classical tumour-suppressor genes have tumorigenic effects associated with somatic biallelic inactivation, although haploinsufficiency may also promote the formation and progression of tumours6,7. Moreover, BRCA1/2-mutant tumours are often deficient in the repair of double-stranded DNA breaks by homologous recombination8-13, and consequently exhibit increased therapeutic sensitivity to platinum-containing therapy and inhibitors of poly-(ADP-ribose)-polymerase (PARP)14,15. However, the phenotypic and therapeutic relevance of mutations in BRCA1 or BRCA2 remains poorly defined in most cancer types. Here we show that in the 2.7% and 1.8% of patients with advanced-stage cancer and germline pathogenic or somatic loss-of-function alterations in BRCA1/2, respectively, selective pressure for biallelic inactivation, zygosity-dependent phenotype penetrance, and sensitivity to PARP inhibition were observed only in tumour types associated with increased heritable cancer risk in BRCA1/2 carriers (BRCA-associated cancer types). Conversely, among patients with non-BRCA-associated cancer types, most carriers of these BRCA1/2 mutation types had evidence for tumour pathogenesis that was independent of mutant BRCA1/2. Overall, mutant BRCA is an indispensable founding event for some tumours, but in a considerable proportion of other cancers, it appears to be biologically neutral-a difference predominantly conditioned by tumour lineage-with implications for disease pathogenesis, screening, design of clinical trials and therapeutic decision-making.

Universal germline genetic testing in patients with hematologic malignancies using DNA isolated from nail clippings.

Not available.

Cell-free DNA from nail clippings as source of normal control for genomic studies in hematologic malignancies.

Comprehensive genomic sequencing is becoming a critical component in the assessment of hematologic malignancies, with broad implications for patient management. In this context, unequivocally discriminating somatic from germline events is challenging but greatly facilitated by matched analysis of tumor:normal pairs. In contrast to solid tumors, conventional sources of normal control (peripheral blood, buccal swabs, saliva) could be highly involved by the neoplastic process, rendering them unsuitable. In this work we describe our real-world experience using cell free DNA (cfDNA) isolated from nail clippings as an alternate source of normal control, through the dedicated review of 2,610 tumor:nail pairs comprehensively sequenced by MSK-IMPACT-heme. Overall, we find nail cfDNA is a robust source of germline control for paired genomic studies. In a subset of patients, nail DNA may have tumor DNA contamination, reflecting unique attributes of the hematologic disease and transplant history. Contamination is generally low level, but significantly more common among patients with myeloid neoplasms (20.5%; 304/1482) compared to lymphoid diseases (5.4%; 61/1128) and particularly enriched in myeloproliferative neoplasms with marked myelofibrosis. When identified in patients with lymphoid and plasma-cell neoplasms, mutations commonly reflected a myeloid profile and correlated with a concurrent/evolving clonal myeloid neoplasm. For nails collected after allogeneic stem-cell transplantation, donor DNA was identified in 22% (11/50). In this cohort, an association with recent history of graft-vs-host disease was identified. These findings should be considered as a potential limitation for the use of nail as normal control but could also provide important diagnostic information regarding the disease process.

Genomic profiling identifies somatic mutations predicting thromboembolic risk in patients with solid tumors.

Venous thromboembolism (VTE) associated with cancer (CAT) is a well-described complication of cancer and a leading cause of death in patients with cancer. The purpose of this study was to assess potential associations of molecular signatures with CAT, including tumor-specific mutations and the presence of clonal hematopoiesis. We analyzed deep-coverage targeted DNA-sequencing data of >14 000 solid tumor samples using the Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets platform to identify somatic alterations associated with VTE. End point was defined as the first instance of cancer-associated pulmonary embolism and/or proximal/distal lower extremity deep vein thrombosis. Cause-specific Cox proportional hazards regression was used, adjusting for pertinent clinical covariates. Of 11 695 evaluable individuals, 72% had metastatic disease at time of analysis. Tumor-specific mutations in KRAS (hazard ratio [HR], 1.34; 95% confidence interval (CI), 1.09-1.64; adjusted P = .08), STK11 (HR, 2.12; 95% CI, 1.55-2.89; adjusted P < .001), KEAP1 (HR, 1.84; 95% CI, 1.21-2.79; adjusted P = .07), CTNNB1 (HR, 1.73; 95% CI, 1.15-2.60; adjusted P = .09), CDKN2B (HR, 1.45; 95% CI, 1.13-1.85; adjusted P = .07), and MET (HR, 1.83; 95% CI, 1.15-2.92; adjusted P = .09) were associated with a significantly increased risk of CAT independent of tumor type. Mutations in SETD2 were associated with a decreased risk of CAT (HR, 0.35; 95% CI, 0.16-0.79; adjusted P = .09). The presence of clonal hematopoiesis was not associated with an increased VTE rate. This is the first large-scale analysis to elucidate tumor-specific genomic events associated with CAT. Somatic tumor mutations of STK11, KRAS, CTNNB1, KEAP1, CDKN2B, and MET were associated with an increased risk of VTE in patients with solid tumors. Further analysis is needed to validate these findings and identify additional molecular signatures unique to individual tumor types.

Disparities in cancer genetics care by race/ethnicity among pan-cancer patients with pathogenic germline variants.

BACKGROUND: Germline risk assessment is increasing as part of cancer care; however, disparities in subsequent genetic counseling are unknown. METHODS: Pan-cancer patients were prospectively consented to tumor-normal sequencing via custom next generation sequencing panel (Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets) inclusive of germline analysis of ≥76 genes from January 2015 through December 2019 (97.5% research nonbillable) with protocol for genetics referral. Rates of pathogenic/likely pathogenic germline variants (PVs) and downstream counseling were compared across ancestry groups (mutually exclusive groups based on self-reported race/ethnicity and Ashkenazi Jewish [AJ] heritage) using nonparametric tests and multivariable logistic regression models. RESULTS: Among 15,775 patients (59.6%, non-Hispanic [NH]-White; 15.7%, AJ; 20.5%, non-White [6.9%, Asian; 6.8%, Black/African American (AA); 6.7%, Hispanic; 0.1%, Other], and 4.2%, unknown), 2663 (17%) had a PV. Non-White patients had a lower PV rate (n = 433, 13.4%) compared to NH-Whites (n = 1451, 15.4%) and AJ patients (n = 683, 27.6%), p < .01, with differences in mostly moderate and low/recessive/uncertain penetrance variants. Among 2239 patients with new PV, 1652 (73.8%) completed recommended genetic counseling. Non-White patients had lower rates of genetic counseling (67.7%) than NH-White (73.7%) and AJ patients (78.8%), p < .01, with lower rates occurring in Black/AA (63%) compared to NH-White patients, even after adjustment for confounders (odds ratio, 0.60; 95% confidence interval, 0.37-0.97; p = .036). Non-White, particularly Black/AA and Asian, probands had a trend toward lower rates and numbers of at-risk family members being seen for counseling/genetic testing. CONCLUSIONS: Despite minimizing barriers to genetic testing, non-White patients were less likely to receive recommended cancer genetics follow-up, with potential implications for oncologic care, cancer risk reduction, and at-risk family members. LAY SUMMARY: Genetic testing is becoming an important part of cancer care, and we wanted to see if genetics care was different between individuals of different backgrounds. We studied 15,775 diverse patients with cancer who had genetic testing using a test called MSK-IMPACT that was covered by research funding. Clinically important genetic findings were high in all groups. However, Black patients were less likely to get recommended counseling compared to White patients. Even after removing many roadblocks, non-White and especially Black patients were less likely to get recommended genetics care, which may affect their cancer treatments and families.

Evaluation of a Combined Multilocus Sequence Typing and Whole-Genome Sequencing Two-Step Algorithm for Routine Typing of Clostridioides difficile.

Multilocus sequence typing (MLST) is a low-resolution but rapid genotyping method for Clostridioides difficile Whole-genome sequencing (WGS) has emerged as the new gold standard for C. difficile typing, but cost and lack of standardization still limit broad utilization. In this study, we evaluated the potential to combine the portability of MLST with the increased resolution of WGS for a cost-saving approach to routine C. difficile typing. C. difficile strains from two New York City hospitals (hospital A and hospital B) were selected. WGS single-nucleotide polymorphism (wgSNP) was performed using established methods. Sequence types (ST) were determined using PubMLST, while wgSNP analysis was performed using the Bionumerics software. An additional analysis of a subset of data (hospital A) was made comparing the Bionumerics software to the CosmosID pipeline. Cost and turnaround time to results were compared for the algorithmic approach of MLST followed by wgSNP versus direct wgSNP. Among the 202 C. difficile isolates typed, 91% (n = 185/203) clustered within the representative ST, showing a high agreement between MLST and wgSNP. While clustering was similar between the Bionumerics and CosmosID pipelines, large differences in the overall number of SNPs were noted. A two-step algorithm for routine typing results in significantly lower cost than routine use of WGS. Our results suggest that using MLST as a first step in routine typing of C. difficile followed by WGS for MLST concordant strains is a less technically demanding, cost-saving approach for performing C. difficile typing than WGS alone without loss of discriminatory power.

Mesonephric and mesonephric-like carcinomas of the female genital tract: molecular characterization including cases with mixed histology and matched metastases.

Mesonephric carcinoma of the cervix is a rare tumor derived from Wolffian remnants. Mesonephric-like carcinomas of the ovary and endometrium, while morphologically similar, do not have obvious Wolffian derivation. Here, we sought to characterize the repertoire of genetic alterations in primary mesonephric and mesonephric-like carcinomas, in the distinct histologic components of mixed cases, as well as in matched primary tumors and metastases. DNA from microdissected tumor and normal tissue from mesonephric carcinomas (cervix, n = 8) and mesonephric-like carcinomas (ovarian n = 15, endometrial n = 13) were subjected to sequencing targeting 468 cancer-related genes. The histologically distinct components of four cases with mixed histology and four primary tumors and their matched metastases were microdissected and analyzed separately. Mesonephric-like carcinomas were underpinned by somatic KRAS mutations (25/28, 89%) akin to mesonephric carcinomas (8/8, 100%), but also harbored genetic alterations more frequently reported in Müllerian tumors. Mesonephric-like carcinomas that lacked KRAS mutations harbored NRAS (n = 2, ovary) or BRAF (n = 1, endometrium) hotspot mutations. PIK3CA mutations were identified in both mesonephric-like (8/28, 28%) and mesonephric carcinomas (2/8, 25%). Only mesonephric-like tumors harbored CTNNB1 hotspot (4/28, 14%) and PTEN (3/13, 23%) mutations. Copy number analysis revealed frequent gains of chromosomes 1q and 10 in both mesonephric (87% 1q; 50% chromosome 10) and mesonephric-like tumors (89% 1q; 43% chromosome 10). Chromosome 12 gains were more frequent in ovarian mesonephric-like carcinomas, and losses of chromosome 9 were more frequent in mesonephric than in mesonephric-like carcinomas (both p = 0.01, Fisher's exact test). The histologically distinct components of four mixed cases were molecularly related and shared similar patterns of genetic alterations. The progression from primary to metastatic lesions involved the acquisition of additional mutations, and/or shifts from subclonal to clonal mutations. Our findings suggest that mesonephric-like carcinomas are derived from a Müllerian substrate with differentiation along Wolffian/mesonephric lines.

Next-generation assessment of human epidermal growth factor receptor 2 gene (ERBB2) amplification status in invasive breast carcinoma: a focus on Group 4 by use of the 2018 American Society of Clinical Oncology/College of American Pathologists HER2 testing guideline.

AIMS: The American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) updated the testing guideline in 2018 to address issues arising from uncommon human epidermal growth factor receptor 2 (HER2) fluorescence in-situ hybridisation (FISH) results according to the 2013 guideline. Next-generation sequencing (NGS) may be used to better classify patients. The aim of this study was to assess the ERBB2 amplification status of invasive breast carcinoma with equivocal HER2 immunohistochemistry (IHC) results by using NGS, focusing on Group 4 (HER2/CEP17 ratio of <2.0; average HER2 signals/cell of ≥4.0 and <6.0). METHODS AND RESULTS: We retrospectively reviewed HER2 FISH and NGS data of HER2 IHC-equivocal breast carcinomas at our centre between January 2009 and September 2019, wherein all three assays were performed on the same tissue block, and compared HER2 FISH results, according to the 2018 ASCO/CAP guideline, and the ERBB2 amplification status determined with NGS. A total of 52 HER2 FISH and NGS results from 51 patients with HER2 IHC-equivocal breast carcinomas were reviewed. The cohort included eight cases classified as 2018 ASCO/CAP in-situ hybridisation Group 1, three classified as Group 2, three classified as Group 3, 14 classified as Group 4, and 24 classified as Group 5. Thirteen of 14 (92.9%) Group 4 (HER2-negative) cases were classified as ERBB2-non-amplified by the use of NGS; the discordant case was later classified as Group 1 with alternative sample FISH testing. NGS revealed no significant difference in somatic mutations or copy number alterations between Groups 4 and 5. CONCLUSIONS: Our NGS findings support the reclassification of HER2 FISH-equivocal cases as HER2-negative under the 2018 ASCO/CAP guideline.

Genetic and molecular subtype heterogeneity in newly diagnosed early- and advanced-stage endometrial cancer.

OBJECTIVE: To characterize and compare the molecular subtypes and profiles of prospectively-accrued newly-diagnosed early- and advanced-stage endometrial cancers (ECs). METHODS: EC patients consented to an IRB-approved protocol of massively parallel sequencing of 410-468 cancer-related genes; 175 ECs of 7 histologic types (n = 135 FIGO stages I/II, n = 40 FIGO stages III/IV) were included. Previously reported sequencing data from 99 additional advanced-stage ECs were retrieved for comparisons. RESULTS: Irrespective of histologic type, all 175 ECs could be stratified into the molecular subtypes, with 75 (43%) being of p53 wild-type, 49 (28%) MMR-deficient, 39 (22%) p53 abnormal and 12 (7%) of POLE molecular subtypes. Subtype distribution, mutational and copy number profiles varied according to histologic type. In endometrioid ECs, genetic alterations varied according to histologic grade. Potential therapeutic targets, including high tumor mutational burden, ERBB2 amplification and PIK3CA hotspot mutations, were found across histologic types in 63% (n = 110) of all ECs. Compared to their early-stage counterparts, advanced-stage endometrioid ECs had a significantly higher fraction of genome altered (median 0.1% vs 12%, p < 0.001) and ARID1B mutations (0% vs 11%, p = 0.01), and advanced-stage serous ECs harbored more frequent ERBB2 amplification (18% vs 8%, p > 0.05) and PIK3CA mutations (46% vs 27%, p > 0.05). Whole-genome doubling was found in advanced- but not early-stage carcinosarcomas and clear cell carcinomas. CONCLUSIONS: Our findings demonstrate the molecular heterogeneity within and across histologic types of EC and the increased genomic complexity of advanced-stage ECs. Molecular subtypes are present across EC histologic types and may help stratify EC patients for prognostic and therapeutic purposes.

Fumarate hydratase FH c.1431_1433dupAAA (p.Lys477dup) variant is not associated with cancer including renal cell carcinoma.

Fumarate hydratase (FH) mutations underpin the autosomal recessive syndrome. FH deficiency and the autosomal dominant syndrome hereditary leiomyomatosis and renal cell carcinoma (HLRCC). The FH c.1431_1433dupAAA (p.Lys477dup) genomic alteration has been conclusively shown to contribute to FH deficiency when occurring with another FH germline alteration. However, a sufficiently large dataset has been lacking to conclusively determine its clinical significance to cancer predisposition in the heterozygous state. We reviewed a series of 7,571 patients with cancer who received germline results through MSK-IMPACT testing at the Memorial Sloan Kettering Cancer Center. The FH c.1431_1433dupAAA (p.Lys477dup) variant was detected in 24 individuals, none of whom was affected with renal cancer. Eleven of the 372 patients with renal cancer were identified to carried pathogenic FH variants associated with HLRCC. None of these 372 patients with renal cancer carried the FH c.1431_1433dupAAA variant. Our data indicate the FH c.1431_1433dupAAA is not associated with cancer including renal cell carcinoma.

Molecular profiling and analysis of genetic aberrations aimed at identifying potential therapeutic targets in fibrolamellar carcinoma of the liver.

BACKGROUND: Fibrolamellar carcinoma (FLC) is a rare primary liver cancer of young adults. A functional chimeric transcript resulting from the in-frame fusion of the DNAJ homolog, subfamily B, member 1 (DNAJB1), and the catalytic subunit of protein kinase A (PRKACA) genes on chromosome 19 is believed to be unique in FLC, with a possible role in pathogenesis, yet with no established therapeutic value. The objective of the current study was to understand the molecular landscape of FLC and to identify potential novel therapeutic targets. METHODS: Archival fresh, formalin-fixed, paraffin-embedded samples from patients with FLC who prospectively consented to an institutional review board-approved protocol were analyzed using Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT), a next-generation sequencing assay encompassing up to 468 key cancer genes. Custom targeted RNA-Seq was performed in selected patients. Demographics, treatment, and outcome data were collected prospectively. Survival outcomes were estimated and correlated with mutation and/or copy number alterations. RESULTS: A total of 33 tumor samples from 31 patients with FLC were analyzed. The median age of the patients at the time of diagnosis was 18 years and approximately 53% were women. The DNAJB1-PRKACA fusion transcript was detected in 100% of patients. In 10 of 31 patients in which MSK-IMPACT did not detect the fusion, its presence was confirmed by targeted RNA-Seq. TERT promoter mutation was the second most common, and was detected in 7 patients. The median follow up was 30 months (range, 6-153 months). The 3-year overall survival rate was 84% (95% CI, 61%-93%). CONCLUSIONS: The DNAJB1-PRKACA fusion transcript is nonspecific and nonsensitive to FLC. Its potential therapeutic value currently is under evaluation. Opportunities currently are under development for therapy that may be driven or related to the DNAJB1-PRKACA fusion transcript or any therapeutic target identified from next-generation sequencing in patients with FLC.

Retained mismatch repair protein expression occurs in approximately 6% of microsatellite instability-high cancers and is associated with missense mutations in mismatch repair genes.

Immunohistochemistry for mismatch repair protein expression is widely used as a surrogate for microsatellite instability status-an important signature for immunotherapy and germline testing. There are no systematic analyses examining the sensitivity of immunohistochemistry for microsatellite instability-high status. Mismatch repair immunohistochemistry and microsatellite instability testing were performed routinely as clinically validated assays. We classified germline/somatic mutation types as truncating (nonsense, frameshift, and in/del) versus missense and predicted pathogenicity of the latter. Discordant cases were compared with concordant groups: microsatellite instability-high/mismatch repair-deficient for mutation comparison and microsatellite stable/mismatch repair-proficient for immunohistochemical comparison. 32 of 443 (7%) microsatellite instability-high cases had immunohistochemistry. Four additional microsatellite instability-high research cases had discordant immunohistochemistry. Of 36 microsatellite instability-high cases with discordant immunohistochemistry, 30 were mismatch repair-proficient, while six (five MLH1 and one MSH2) retained expression of the defective mismatch repair protein and lost its partner. In microsatellite instability-high tumors with discordant immunohistochemistry, we observed an enrichment in deleterious missense mutations over truncating mutations, with 69% (25/36) of cases having pathogenic germline or somatic missense mutations, as opposed to only 19% (7/36) in a matched microsatellite instability-high group with concordant immunohistochemistry (p = 0.0007).  In microsatellite instability-high cases with discordant immunohistochemistry and MLH1 or PMS2 abnormalities, less cells showed expression (p = 0.015 and p = 0.00095, respectively) compared with microsatellite stable/mismatch repair-proficient cases. Tumor mutation burden, MSIsensor score, and truncating mismatch repair gene mutations were similar between microsatellite instability-high cases with concordant versus discordant immunohistochemical expression. Approximately 6% of microsatellite instability-high cases have retained mismatch repair protein expression and would be missed by immunohistochemistry-based testing, hindering patient access to immunotherapy. Another 1% of microsatellite instability-high cases show isolated loss of the defective gene's dimerization partner, which may lead to germline testing of the wrong gene. These cases are enriched for pathogenic mismatch repair missense mutations.

NTRK fusion detection across multiple assays and 33,997 cases: diagnostic implications and pitfalls.

With the FDA approval of larotrectinib, NTRK fusion assessment has recently become a standard part of management for patients with locally advanced or metastatic cancers. Unlike somatic mutation assessment, the detection of NTRK fusions is not straightforward, and various assays exist at the DNA, RNA, and protein level. Here, we investigate the performance of immunohistochemistry and DNA-based next-generation sequencing to indirectly or directly detect NTRK fusions relative to an RNA-based next-generation sequencing approach in the largest cohort of NTRK fusion positive solid tumors to date. A retrospective analysis of 38,095 samples from 33,997 patients sequenced by a targeted DNA-based next-generation sequencing panel (MSK-IMPACT), 2189 of which were also examined by an RNA-based sequencing assay (MSK-Fusion), identified 87 patients with oncogenic NTRK1-3 fusions. All available institutional NTRK fusion positive cases were assessed by pan-Trk immunohistochemistry along with a cohort of control cases negative for NTRK fusions by next-generation sequencing. DNA-based sequencing showed an overall sensitivity and specificity of 81.1% and 99.9%, respectively, for the detection of NTRK fusions when compared to RNA-based sequencing. False negatives occurred when fusions involved breakpoints not covered by the assay. Immunohistochemistry showed overall sensitivity of 87.9% and specificity of 81.1%, with high sensitivity for NTRK1 (96%) and NTRK2 (100%) fusions and lower sensitivity for NTRK3 fusions (79%). Specificity was 100% for carcinomas of the colon, lung, thyroid, pancreas, and biliary tract. Decreased specificity was seen in breast and salivary gland carcinomas (82% and 52%, respectively), and positive staining was often seen in tumors with neural differentiation. Both sensitivity and specificity were poor in sarcomas. Selection of the appropriate assay for NTRK fusion detection therefore depends on tumor type and genes involved, as well as consideration of other factors such as available material, accessibility of various clinical assays, and whether comprehensive genomic testing is needed concurrently.

Ampullary cancer: Evaluation of somatic and germline genetic alterations and association with clinical outcomes.

BACKGROUND: Ampullary carcinoma (AC) is a rare gastrointestinal cancer. Pathogenic germline alterations (PGAs) in BRCA2 and potentially targetable somatic alterations (SAs) in ERBB2 and ELF3 have been previously described in AC. Memorial Sloan Kettering Cancer Center has implemented an opt-in strategy for germline testing (GT) and somatic testing (ST) for patients with AC to further evaluate the spectrum of PGAs and SAs. METHODS: Forty-five patients with pathologically confirmed AC prospectively consented with the Memorial Sloan Kettering Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) test (410-468 genes). A subset of the cohort (23 of the 45 patients) also consented to GT with MSK-IMPACT (76-88 genes). Germline data for 21 of the remaining 22 patients who had not consented to GT were obtained in a de-identified fashion without clinical correlation. Clinicopathologic features, treatment histories, and survival data for consenting patients were collected and analyzed. RESULTS: Pancreaticobiliary, intestinal, and mixed features of the 2 types were the primary pathologic subtypes of AC identified in this cohort. No difference in median overall survival was found between pathologic subtypes. Eight of 44 patients (18%) were identified as harboring pathogenic mutations in BRCA2, ATM, RAD50, and MUTYH. In addition, this study found a wide spectrum of SAs in genes such as KRAS, MDM2, ERBB2, ELF3, and PIK3CA. Two patients in the cohort underwent SA-targeted therapy, and 1 had a partial radiographic response. CONCLUSIONS: Mutations in multiple somatic and germline genes were identified in this cohort. Significantly, actionable targets were identified in the tumors, and broader testing for PGAs and SAs should be considered for all patients with AC.

Inherited DNA-Repair Gene Mutations in Men with Metastatic Prostate Cancer.

BACKGROUND: Inherited mutations in DNA-repair genes such as BRCA2 are associated with increased risks of lethal prostate cancer. Although the prevalence of germline mutations in DNA-repair genes among men with localized prostate cancer who are unselected for family predisposition is insufficient to warrant routine testing, the frequency of such mutations in patients with metastatic prostate cancer has not been established. METHODS: We recruited 692 men with documented metastatic prostate cancer who were unselected for family history of cancer or age at diagnosis. We isolated germline DNA and used multiplex sequencing assays to assess mutations in 20 DNA-repair genes associated with autosomal dominant cancer-predisposition syndromes. RESULTS: A total of 84 germline DNA-repair gene mutations that were presumed to be deleterious were identified in 82 men (11.8%); mutations were found in 16 genes, including BRCA2 (37 men [5.3%]), ATM (11 [1.6%]), CHEK2 (10 [1.9% of 534 men with data]), BRCA1 (6 [0.9%]), RAD51D (3 [0.4%]), and PALB2 (3 [0.4%]). Mutation frequencies did not differ according to whether a family history of prostate cancer was present or according to age at diagnosis. Overall, the frequency of germline mutations in DNA-repair genes among men with metastatic prostate cancer significantly exceeded the prevalence of 4.6% among 499 men with localized prostate cancer (P<0.001), including men with high-risk disease, and the prevalence of 2.7% in the Exome Aggregation Consortium, which includes 53,105 persons without a known cancer diagnosis (P<0.001). CONCLUSIONS: In our multicenter study, the incidence of germline mutations in genes mediating DNA-repair processes among men with metastatic prostate cancer was 11.8%, which was significantly higher than the incidence among men with localized prostate cancer. The frequencies of germline mutations in DNA-repair genes among men with metastatic disease did not differ significantly according to age at diagnosis or family history of prostate cancer. (Funded by Stand Up To Cancer and others.).

Immunohistochemical analysis of estrogen receptor in breast cancer with ESR1 mutations detected by hybrid capture-based next-generation sequencing.

Estrogen receptor-α (ER-α), encoded by ESR1, is detected by immunohistochemistry in approximately 70% of invasive breast cancers and serves as a strong predictive biomarker. ESR1-activating mutations in the ligand-binding domain have been reported in up to 35-40% of ER-positive metastatic breast cancers and are associated with endocrine therapy resistance and disease progression. At present, it is unclear whether ESR1 mutations alter the immunohistochemical detection of ER performed in routine clinical practice. In this study, ESR1 mutations in breast cancer were identified utilizing Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT), a Food and Drug Administration-approved hybridization capture-based next-generation sequencing assay. Five hundred and eighty-six breast cancers from patients with locally advanced or metastatic disease were analyzed using MSK-IMPACT in the study period. ESR1 somatic alterations were identified in 67 breast cancer samples from 66 patients. Immunohistochemical analysis of ER, progesterone receptor, and human epidermal growth factor receptor 2 was performed on the primary and treated breast cancers from these patients at the time of diagnosis. Twenty unique ESR1 mutations were identified involving the ligand-binding domain, all in breast cancer samples from patients previously treated with endocrine therapy. The most frequent mutations were D538G (n = 22), Y537S (n = 7), and E380Q (n = 7). All breast cancer samples with an ESR1 mutation were ER-positive by immunohistochemistry. Review of the ER immunohistochemistry in the paired untreated primary tumor and treated tumor from 34 patients showed no detectable change in the ER-positive immunohistochemical status (median percentage of invasive tumor cells with nuclear staining: untreated primary tumor 90%, treated tumor 95%). We conclude that ESR1 mutations do not appreciably diminish ER-positive staining by immunohistochemistry. In addition to standard biomarker testing by immunohistochemistry, the assessment of ESR1 mutations by molecular testing can help guide the clinical management of patients with ER-positive breast cancer in the setting of endocrine resistance and progression of disease.

The value of cell-free DNA for molecular pathology.

Over the past decade, advances in molecular biology and genomics techniques have revolutionized the diagnosis and treatment of cancer. The technological advances in tissue profiling have also been applied to the study of cell-free nucleic acids, an area of increasing interest for molecular pathology. Cell-free nucleic acids are released from tumour cells into the surrounding body fluids and can be assayed non-invasively. The repertoire of genomic alterations in circulating tumour DNA (ctDNA) is reflective of both primary tumours and distant metastatic sites, and ctDNA can be sampled multiple times, thereby overcoming the limitations of the analysis of single biopsies. Furthermore, ctDNA can be sampled regularly to monitor response to treatment, to define the evolution of the tumour genome, and to assess the acquisition of resistance and minimal residual disease. Recently, clinical ctDNA assays have been approved for guidance of therapy, which is an exciting first step in translating cell-free nucleic acid research tests into clinical use for oncology. In this review, we discuss the advantages of cell-free nucleic acids as analytes in different body fluids, including blood plasma, urine, and cerebrospinal fluid, and their clinical applications in solid tumours and haematological malignancies. We will also discuss practical considerations for clinical deployment, such as preanalytical factors and regulatory requirements. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

A FISH assay efficiently screens for BRAF gene rearrangements in pancreatic acinar-type neoplasms.

Approximately 1-2% of pancreatic neoplasms are acinar cell carcinomas. Recently, BRAF gene rearrangements were identified in over 20% of acinar-type neoplasms, which included both pure acinar cell carcinomas and mixed carcinomas with acinar differentiation, using next-generation sequencing-based platforms, providing a potential therapeutic target for patients with these neoplasms. Thus, it is clinically important to develop a rapid, cost- and material-efficient assay to screen for BRAF gene fusions in pancreatic acinar-type neoplasms. We developed a dual color, break-apart FISH assay to detect BRAF gene rearrangements in these neoplasms and evaluated its performance in comparison to next-generation sequencing-based studies. A blinded BRAF rearrangement FISH investigation was performed on 31 acinar-type neoplasms that had been studied previously by next-generation sequencing-based analysis as well as on 18 additional acinar-type neoplasms that were accrued over the past 2 years. In total, BRAF fusions were identified in 12/49 (24%) acinar-type neoplasms by FISH. BRAF fusion partners were uncovered by using targeted next-generation sequencing studies in 11 FISH-positive cases that had sufficient material for next-generation sequencing studies. SND1 was the most frequent fusion partner involved in BRAF-fusion acinar-type neoplasms (50%), followed by HERPUD1 (18%). No BRAF fusions were identified by next-generation sequencing in any of the FISH-negative cases investigated. FISH analysis showed that BRAF rearrangements were diffusely present across tumor-rich areas in BRAF-fusion acinar-type neoplasms, which is consistent with an oncogenic driver alteration pattern. Thus, we demonstrated that, in comparison to targeted next-generation sequencing-based technologies, the FISH assay is highly sensitive and specific as well as time- and cost-efficient in the detection of BRAF fusions in acinar-type neoplasms. The FISH assay can be easily implemented in diagnostic settings to identify acinar-type neoplasms patients potentially suitable for targeted therapy to inhibit MAPK pathway activity.