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PURPOSE To estimate the risk of contralateral breast cancer (CBC) among women with germline pathogenic variants (PVs) in ATM, BRCA1, BRCA2, CHEK2, and PALB2. METHODS The study population included 15,104 prospectively followed women within the CARRIERS study treated with ipsilateral surgery for invasive breast cancer. The risk of CBC was estimated for PV carriers in each gene compared with women without PVs in a multivariate proportional hazard regression analysis accounting for the competing risk of death and adjusting for patient and tumor characteristics. The primary analyses focused on the overall cohort and on women from the general population. Secondary analyses examined associations by race/ethnicity, age at primary breast cancer diagnosis, menopausal status, and tumor estrogen receptor (ER) status. RESULTS Germline BRCA1, BRCA2, and CHEK2 PV carriers with breast cancer were at significantly elevated risk (hazard ratio > 1.9) of CBC, whereas only the PALB2 PV carriers with ER-negative breast cancer had elevated risks (hazard ratio, 2.9). By contrast, ATM PV carriers did not have significantly increased CBC risks. African American PV carriers had similarly elevated risks of CBC as non-Hispanic White PV carriers. Among premenopausal women, the 10-year cumulative incidence of CBC was estimated to be 33% for BRCA1, 27% for BRCA2, and 13% for CHEK2 PV carriers with breast cancer and 35% for PALB2 PV carriers with ER-negative breast cancer. The 10-year cumulative incidence of CBC among postmenopausal PV carriers was 12% for BRCA1, 9% for BRCA2, and 4% for CHEK2. CONCLUSION Women diagnosed with breast cancer and known to carry germline PVs in BRCA1, BRCA2, CHEK2, or PALB2 are at substantially increased risk of CBC and may benefit from enhanced surveillance and risk reduction strategies.

Purpose: To explore the role of NBN as a pan-cancer susceptibility gene. Experimental Design: Matched germline and somatic DNA samples from 34,046 patients were sequenced using Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets and presumed pathogenic germline variants (PGV) identified. Allele-specific and gene-centered analysis of enrichment was conducted and a validation cohort of 26,407 pan-cancer patients was analyzed. Functional studies utilized cellular models with analysis of protein expression, MRN complex formation/localization, and viability assessment following treatment with γ-irradiation. Results: We identified 83 carriers of 32 NBN PGVs (0.25% of the studied series), 40% of which (33/83) carried the Slavic founder p.K219fs. The frequency of PGVs varied across cancer types. Patients harboring NBN PGVs demonstrated increased loss of the wild-type allele in their tumors [OR = 2.7; confidence interval (CI): 1.4–5.5; P = 0.0024; pan-cancer], including lung and pancreatic tumors compared with breast and colorectal cancers. p.K219fs was enriched across all tumor types (OR = 2.22; CI: 1.3–3.6; P = 0.0018). Gene-centered analysis revealed enrichment of PGVs in cases compared with controls in the European population (OR = 1.9; CI: 1.3–2.7; P = 0.0004), a finding confirmed in the replication cohort (OR = 1.8; CI: 1.2–2.6; P = 0.003). Two novel truncating variants, p.L19* and p.N71fs, produced a 45 kDa fragment generated by alternative translation initiation that maintained binding to MRE11. Cells expressing these fragments showed higher sensitivity to γ-irradiation and lower levels of radiation-induced KAP1 phosphorylation. Conclusions: Burden analyses, biallelic inactivation, and functional evidence support the role of NBN as contributing to a broad cancer spectrum. Further studies in large pan-cancer series and the assessment of epistatic and environmental interactions are warranted to further define these associations.

Integrating genomic data into the electronic health record (EHR) is key for optimally delivering genomic medicine.The PennChart Genomics Initiative (PGI) at the University of Pennsylvania is a multidisciplinary collaborative that has successfully linked orders and results from genetic testing laboratories with discrete genetic data in the EHR. We quantified the use of the genomic data within the EHR, performed a time study with genetic counselors, and conducted key informant interviews with PGI members to evaluate the effect of the PGI's efforts on genetics care delivery.The PGI has interfaced with 4 genetic testing laboratories, resulting in the creation of 420 unique computerized genetic testing orders that have been used 4073 times to date. In a time study of 96 genetic testing activities, EHR use was associated with significant reductions in time spent ordering (2 vs 8 minutes, P.001) and managing (1 vs 5 minutes, P.001) genetic results compared with the use of online laboratory-specific portals. In key informant interviews, multidisciplinary collaboration and institutional buy-in were identified as key ingredients for the PGI's success.The PGI's efforts to integrate genomic medicine into the EHR have substantially streamlined the delivery of genomic medicine.

Table S1 has the RNA-Seq sample names and raw counts.

PDF file 311K, Loss of miR-30a-3p promotes UMRC2 tumor growth

Metastatic disease in pheochromocytomas and paragangliomas (PCC/PGL) is not well-understood. The Cancer Genome Atlas discovered recurrent MAML3 fusion genes in a subset of tumors that lacked known germline or somatic driver mutations and were associated with aggressive disease. Here, we aimed to investigate the role of MAML3 in tumorigenesis. Human PCC/PGLs were used for IHC and genetic analysis. Three neuroendocrine tumor cell lines, SK-N-SH, QGP-1, and BON-1, were transiently transfected with MAML3 (FL) or exon 1 deleted MAML3 (dEx1; mimicking the fusion), and biologic effects of overexpression were examined in vitro. We found 7% (4/55) of human PCC/PGL have UBTF∼MAML3 fusions and all were sporadic cases with metastatic disease. Fusion-positive tumors had intense MAML3 nuclear staining and increased β-catenin by IHC and showed increased WNT4 expression. In vitro, overexpression of FL and dEx1 MAML3 increased invasion in SK-N-SH, QGP-1, and BON-1 (all P < 0.05) and increased soft-agar colony formation in QGP-1 and BON-1 (all P < 0.05). Cotransfection with FL or dEx1 MAML3 and β-catenin increased TCF/LEF promoter activation by luciferase activity and coimmunoprecipitation confirmed interaction between MAML3 and β-catenin. These data suggest MAML3 is involved in WNT signaling pathway activation. In summary, UBTF∼MAML3 fusions are present in a subset of PCC/PGL and associated with metastatic disease without other known drivers. MAML3 overexpression led to increased tumorigenicity in neuroendocrine tumor cells and the mechanism of action may involve WNT signaling pathways.Implications:MAML3 increases tumorigenicity and invasion in neuroendocrine tumor cells and may be a prognostic marker for aggressive disease.

MAML3 overexpression has no effect on proliferation or apoptotic signaling. SK-N-SH, QGP-1 and BON-1 transfected with FL or dEx1 MAML3 and showed A) no increase in proliferation by BrdU assay when grown in full serum media for 72 hours and B) no increase in total or cleaved PARP or Caspase 3 when grown in serum free media for 48 or 72 hours. No cleaved caspase 3 was seen at all. The % of cleaved PARP was calculated after normalizing to beta tubulin and no significant differences were found at either time point for any cell line. Experiment performed N=3 with representative blots shown. T-PARP= total PARP; C-PARP=cleaved PARP; T-Caspase 3=total Caspase 3; C-Caspase 3=cleaved Caspase 3

Supplementary Figure 1 PDF file 108K, This document provides the raw data for V1736A genotyping used in LOH experiments

Supplementary Table 1 PDF file 38K, This table summarizes all additional V1736A pedigrees

Supplementary Methods PDF file 61K, This document describes the methods for all supplemental data

Combined BRAF and MEK inhibition is a standard of care in patients with advanced BRAF-mutant melanoma, but acquired resistance remains a challenge that limits response durability. Here, we quantitated in vivo ERK1/2 activity and tumor response associated with resistance to combined BRAF and MEK inhibition in mutant BRAF xenografts. We found that ERK1/2 pathway reactivation preceded the growth of resistant tumors. Moreover, we detected a subset of cells that not only persisted throughout long-term treatment but restored ERK1/2 signaling and grew upon drug removal. Cell lines derived from combination-resistant tumors (CRT) exhibited elevated ERK1/2 phosphorylation, which were sensitive to ERK1/2 inhibition. In some CRTs, we detected a tandem duplication of the BRAF kinase domain. Monitoring ERK1/2 activity in vivo was efficacious in predicting tumor response during intermittent treatment. We observed maintained expression of the mitotic regulator, polo-like kinase 1 (Plk1), in melanoma resistant to BRAF and MEK inhibitors. Plk1 inhibition induced apoptosis in CRTs, leading to slowed growth of BRAF and MEK inhibitor–resistant tumors in vivo. These data demonstrate the utility of in vivo ERK1/2 pathway reporting as a tool to optimize clinical dosing schemes and establish suppression of Plk1 as potential salvage therapy for BRAF inhibitor and MEK inhibitor–resistant melanoma.

Supplementary Figure 1. Electropherogram. Supplementary Figure 2. Ramachandran plot. Supplementary Figure 3. Modeling ERCC3 R109X via CRISPR/Cas9 genome editing. Supplementary Figure 4. Haplotype analyses from the TAGC heterozygote carriers. Supplementary Figure 5. ERCC3 mutations present in the cBioPortal (MSKCC) datasets. Supplementary Figure 6. ERCC3 transcript levels in patient derived whole blood. Supplementary Table 1. Family history and pathology information on R109X carriers. Supplementary Table 2. Identity of mutations in HMLE cell lines generated by CRISPR/Cas9.

Supplementary Figure 2 PDF file 78K, This figure depicts the assay used to visualize repair proteins at DNA double-strand breaks

Supplemental Fig. 1. Combination-resistant and combination tolerant tumors re-activated ERK1/2 before progression. Supplemental Fig. 2. Combination-resistant tumor-derived cell lines progressed through cell cycle during combo treatment. Supplemental Fig 3. CRTs harbored tandem duplication of kinase domain but remained sensitive to ERK1/2 inhibition. Supplemental Fig. 4. Both intermittently and continuously treated tumors reactivated of ERK1/2 in vitro. Supplemental Fig. 5. Pathway analysis identified Plk11 as selective driver of resistance in CRTs.

Supplementary Figure 3 PDF file 2065K, This figure provides functional data for BRCA1 V1736A and an example of tumor for LOH studies

PDF file 162K, Methods and supplementary figure legends

Supplementary Methods and Figure Legends

Supplementary Figure S1. Germline and somatic data from The Cancer Genome Atlas (TCGA) project, representing 10,268 cancer patients with whole exome sequencing data.

Purpose:To explore the role of NBN as a pan-cancer susceptibility gene.Experimental Design:Matched germline and somatic DNA samples from 34,046 patients were sequenced using Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets and presumed pathogenic germline variants (PGV) identified. Allele-specific and gene-centered analysis of enrichment was conducted and a validation cohort of 26,407 pan-cancer patients was analyzed. Functional studies utilized cellular models with analysis of protein expression, MRN complex formation/localization, and viability assessment following treatment with γ-irradiation.Results:We identified 83 carriers of 32 NBN PGVs (0.25% of the studied series), 40% of which (33/83) carried the Slavic founder p.K219fs. The frequency of PGVs varied across cancer types. Patients harboring NBN PGVs demonstrated increased loss of the wild-type allele in their tumors [OR = 2.7; confidence interval (CI): 1.4–5.5; P = 0.0024; pan-cancer], including lung and pancreatic tumors compared with breast and colorectal cancers. p.K219fs was enriched across all tumor types (OR = 2.22; CI: 1.3–3.6; P = 0.0018). Gene-centered analysis revealed enrichment of PGVs in cases compared with controls in the European population (OR = 1.9; CI: 1.3–2.7; P = 0.0004), a finding confirmed in the replication cohort (OR = 1.8; CI: 1.2–2.6; P = 0.003). Two novel truncating variants, p.L19* and p.N71fs, produced a 45 kDa fragment generated by alternative translation initiation that maintained binding to MRE11. Cells expressing these fragments showed higher sensitivity to γ-irradiation and lower levels of radiation-induced KAP1 phosphorylation.Conclusions:Burden analyses, biallelic inactivation, and functional evidence support the role of NBN as contributing to a broad cancer spectrum. Further studies in large pan-cancer series and the assessment of epistatic and environmental interactions are warranted to further define these associations.

Background: Germline genetic testing (GT) for BRCA1/2 is instrumental in identifying patients with breast and ovarian cancers who are eligible for PARP inhibitors (PARPi). Little is known about recent trends and determinants of GT since PARPi were approved for these patients. Patients and Methods: We performed a retrospective cohort study of patients in a nationwide electronic health record (EHR)–derived oncology-specific database with the following GT eligibility criteria: breast cancer diagnosed at age ≤45 years, triple-negative breast cancer diagnosed at age ≤60 years, male breast cancer, or ovarian cancer. GT within 1 year of diagnosis was assessed and stratified by tumor type. Multivariable log-binomial regressions estimated adjusted relative risks (RRs) of GT by patient and tumor characteristics. Results: Among 2,982 eligible patients with breast cancer, 56.4% underwent GT between January 2011 and March 2020, with a significant increase in GT over time (RR, 1.08; 95% CI, 1.05–1.11, for each year), independent of when PARPi were approved for BRCA1/2-mutated metastatic breast cancer in January 2018. In multivariable analyses, older age (RR, 0.93; 95% CI, 0.90–0.96, for every 5 years) and Medicare coverage (RR, 0.69; 95% CI, 0.49–0.96 vs commercial insurance) were associated with less GT. Among 5,563 eligible patients with ovarian cancer, 35.4% underwent GT between January 2011 and March 2020, with a significant increase in GT over time (RR, 1.11; 95% CI, 1.07–1.14, for each year) that accelerated after approval of PARPi for BRCA1/2-mutated, chemotherapy-refractory ovarian cancer in December 2014 (RR, 1.42; 95% CI, 1.19–1.70). Older age (RR, 0.95; 95% CI, 0.93–0.97, for every 5 years) and Black or African American race (RR, 0.80; 95% CI, 0.65–0.98 vs White race) were associated with less GT. Conclusions: GT remains underutilized nationwide among patients with breast and ovarian cancers. Although GT has increased over time, significant disparities by age, race, and insurance status persist. Additional work is needed to design, implement, and evaluate strategies to ensure that all eligible patients receive GT.

Purpose:Breast cancers with BRCA1/2 alterations have a relatively high mutational load, suggesting that immune checkpoint blockade may be a potential treatment option. However, the degree of immune cell infiltration varies widely, and molecular features contributing to this variability remain unknown.Experimental Design:We hypothesized that genomic signatures might predict immunogenicity in BRCA1/2 breast cancers. Using The Cancer Genome Atlas (TCGA) genomic data, we compared breast cancers with (89) and without (770) either germline or somatic BRCA1/2 alterations. We also studied 35 breast cancers with germline BRCA1/2 mutations from Penn using WES and IHC.Results:We found that homologous recombination deficiency (HRD) scores were negatively associated with expression-based immune indices [cytolytic index (P = 0.04), immune ESTIMATE (P = 0.002), type II IFN signaling (P = 0.002)] despite being associated with a higher mutational/neoantigen burden, in BRCA1/2 mutant breast cancers. Further, absence of allele-specific loss of heterozygosity (LOH negative; P = 0.01) or subclonality (P = 0.003) of germline and somatic BRCA1/2 mutations, respectively, predicted for heightened cytolytic activity. Gene set analysis found that multiple innate and adaptive immune pathways that converge on NF-κB may contribute to this heightened immunogenicity. IHC of Penn breast cancers demonstrated increased CD45+ (P = 0.039) and CD8+ infiltrates (P = 0.037) and increased PDL1 expression (P = 0.012) in HRD-low or LOH-negative cancers. Triple-negative cancers with low HRD had far greater CD8+ T cells (P = 0.0011) and Perforin 1 expression (P = 0.014) compared with hormone receptor-positive HRD-high cancers.Conclusions:HRD scores and hormone receptor subtype are predictive of immunogenicity in BRCA1/2 breast cancers and may inform the design of optimal immune therapeutic strategies.

Supplementary Figures 1-4. Supplementary Figure S1: Full Pedigrees from TP53 c.1000G>C;p.G334R clinically ascertained families. Fam ID allows reference to Table 1; Supplementary Figure S2: Constructs encoding p53 wild-type (TP53-WT), p53-R175H (TP53-R175H), and p53-G334R (c.1000G>A and c.1000G>C) were transiently transfected into p53 deficient Saos-2 cells; Supplementary Figure S3: p53 levels after treatment with 10uM cisplatin (CDDP); Supplementary Figure S4: Heatmap showing fold activation of a canonical p53 signature at 24 hours post nutlin with wildtype versus mutant lymphoblastoid cell lines.

Supplementary Figure 1 - PDF file 214K, Copy number in baseline samples from patients with partial response compared to those with stable or progressive disease

Supplemental Table 1: Genes analyzed which are known to be involved in melanoma pathogenesis Supplemental Table 2: Patient demographics and disease characteristics Supplemental Table 3: Clinical outcomes Supplemental Table 4: BRAF gene amplification is associated with BRAF somatic mutations Supplemental Table 5: RAF1 gene amplification is associated with worse PS Supplemental Figure Legends

Supplementary Table 1 - PDF file 54K, Patient description with tumor PTEN status available

Purpose: BRAF inhibitors (BRAFi) extend survival in BRAF-mutant melanoma but can promote the growth of Ras-mutant neoplasms. This study determined if gastrointestinal polyps found in BRAFi-treated patients harbored Ras mutations.Experimental Design: Colonic and gastric polyps were identified and resected from BRAFi-treated melanoma patients. Next-generation sequencing (NGS) was performed on polyps. The ability of BRAFi to promote polyp formation was functionally characterized in Apc Min+/− mice. MAPK and β-catenin pathway activity was assessed by immunohistochemistry in mouse and human polyps.Results: Fourteen patients treated with BRAFi underwent endoscopy to assess for polyps. Seven out of 7 patients >40 years of age and treated for >2 years were found to have colonic tubular adenomas with 4 out of the 7 patients having 5 or more polyps. One patient presented with bleeding from hyperplastic gastric polyps that recurred 6 months after BRAFi rechallenge. NGS performed on polyps found no mutations in MAPK pathway genes, but found APC mutations in all tubular adenomas. A significant increase in the number of polyps was observed in BRAFi-treated compared with control-treated Apc Min+/− mice (20.8 ± 9.2 vs 12.8 ± 0.1; P = 0.016). No polyps were observed in BRAFi-treated wild-type mice.Conclusions: BRAFi may increase the risk of developing hyperplastic gastric polyps and colonic adenomatous polyps. Due to the risk of gastrointestinal bleeding and the possibility of malignant transformation, further studies are needed to determine whether or not endoscopic surveillance should be recommended for patients treated with BRAFi. Clin Cancer Res; 21(23); 5215–21. ©2015 AACR.

PDF file - 121 KB, Seventeen human melanoma cell lines representing major mutational subgroups of cutaneous melanoma were treated with TNF-� at concentrations up to 50ng/ml.

Supplementary Figures S1-S4, Tables S4, S6.

Supplementary Table S1. Associations of Overall Breast Cancer Risk with 74 Breast Cancer Risk Variants in African Americans with Study-specific Results.

Supplementary Table 2. Results of the statistical analyses in BRCA mutation carriers.

Suppementary Table 4 - PDF file 38K, Correlation analysis of selected genes from aCGH data

Supplementary Table 1. The 3,248 SNPs included in the study

Supplemental Figure 3. BRAF and MET gene amplifications are associated with BRAF V600K mutation cohort

Supplementary Figure S1, Supplementary Figure S1. Regional plots of 3p24, 11q13, 12p11, 14q13, 16q12/TOX3, 16q12/FTO, 16q23, and 19p13.

PDF file - 90K

Purpose: Sorafenib is an inhibitor of VEGF receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), and RAF kinases, amongst others. We assessed the association of somatic mutations with clinicopathologic features and clinical outcomes in patients with metastatic melanoma treated on E2603, comparing treatment with carboplatin, paclitaxel ± sorafenib (CP vs. CPS).Experimental Design: Pretreatment tumor samples from 179 unique individuals enrolled on E2603 were analyzed. Genotyping was performed using a custom iPlex panel interrogating 74 mutations in 13 genes. Statistical analysis was performed using Fisher exact test, logistic regression, and Cox proportional hazards models. Progression-free survival (PFS) and overall survival were estimated using Kaplan–Meier methods.Results:BRAF and NRAS mutations were found at frequencies consistent with other metastatic melanoma cohorts. BRAF-mutant melanoma was associated with worse performance status, increased number of disease sites, and younger age at diagnosis. NRAS-mutant melanoma was associated with better performance status, fewer sites of disease, and female gender. BRAF and NRAS mutations were not significantly predictive of response or survival when treated with CPS versus CP. However, patients with NRAS-mutant melanoma trended toward a worse response and PFS on CP than those with BRAF-mutant or WT/WT melanoma, an association that was reversed for this group on the CPS arm.Conclusions: This study of somatic mutations in melanoma is the last prospectively collected phase III clinical trial population before the era of BRAF-targeted therapy. A trend toward improved clinical response in patients with NRAS-mutant melanoma treated with CPS was observed, possibly due to the effect of sorafenib on CRAF. Clin Cancer Res; 20(12); 3328–37. ©2014 AACR.