Differences in somatic TP53 mutation type in breast tumors by race and receptor status.

PURPOSE: Somatic driver mutations in TP53 are associated with triple-negative breast cancer (TNBC) and poorer outcomes. Breast cancers in women of African ancestry (AA) are more likely to be TNBC and have somatic TP53 mutations than cancers in non-Hispanic White (NHW) women. Missense driver mutations in TP53 have varied functional impact including loss-of-function (LOF) or gain-of-function (GOF) activity, and dominant negative (DNE) effects. We aimed to determine if there were differences in somatic TP53 mutation types by patient ancestry or TNBC status. METHODS: We identified breast cancer datasets with somatic TP53 mutation data, ancestry, age, and hormone receptor status. Mutations were classified for functional impact using published data and type of mutation. We assessed differences using Fisher's exact test. RESULTS: From 96 breast cancer studies, we identified 2964 women with somatic TP53 mutations: 715 (24.1%) Asian, 258 (8.7%) AA, 1931 (65.2%) NHW, and 60 (2%) Latina. The distribution of TP53 mutation type was similar by ancestry. However, 35.8% of tumors from NHW individuals had GOF mutations compared to 29% from AA individuals (p = 0.04). Mutations with DNE activity were positively associated with TNBC (OR 1.37, p = 0.03) and estrogen receptor (ER) negative status (OR 1.38; p = 0.005). CONCLUSIONS: Somatic TP53 mutation types did not differ by ancestry overall, but GOF mutations were more common in NHW women than AA women. ER-negative and TNBC tumors are less likely to have DNE+ TP53 mutations which could reflect biological processes. Larger cohorts and functional studies are needed to further elucidate these findings.

Reproductive characteristics are associated with gene-specific promoter methylation status in breast cancer.

BACKGROUND: Reproductive characteristics are well-established risk factors for breast cancer, but the underlying mechanisms are not fully resolved. We hypothesized that altered DNA methylation, measured in tumor tissue, could act in concert with reproductive factors to impact breast carcinogenesis. METHODS: Among a population-based sample of women newly diagnosed with first primary breast cancer, reproductive history was assessed using a life-course calendar approach in an interviewer-administered questionnaire. Methylation-specific polymerase chain reaction and Methyl Light assays were used to assess gene promotor methylation status (methylated vs. unmethylated) for 13 breast cancer-related genes in archived breast tumor tissue. We used case-case unconditional logistic regression to estimate adjusted odds ratios (ORs) and 95% confidence intervals (CIs) for associations with age at menarche and parity (among 855 women), and age at first birth and lactation (among a subset of 736 parous women) in association with methylation status. RESULTS: Age at first birth > 27 years, compared with < 23 years, was associated with lower odds of methylation of CDH1 (OR = 0.44, 95% CI = 0.20-0.99) and TWIST1 (OR = 0.48, 95% CI = 0.28-0.82), and higher odds of methylation of BRCA1 (OR = 1.63, 95% CI = 1.14-2.35). Any vs. no lactation was associated with higher odds of methylation of the PGR gene promoter (OR = 1.59, 95% CI = 1.01-2.49). No associations were noted for parity and methylation in any of the genes assayed. CONCLUSIONS: Our findings indicate that age at first birth, lactation and, perhaps age at menarche, are associated with gene promoter methylation in breast cancer, and should be confirmed in larger studies with robust gene coverage.

Utility of TERT Promoter Mutations for Cutaneous Primary Melanoma Diagnosis.

Telomerase reverse transcriptase (TERT) promoter mutations are commonly found in malignant melanomas but rare in melanocytic nevi. To assess its potential diagnostic utility for the distinction of melanoma from nevus, we determined the TERT promoter mutation status of 86 primary melanomas, 72 melanocytic nevi, and 40 diagnostically problematic melanocytic proliferations. Of the 86 melanomas, 67 (77.9%) were TERT-positive, defined as harboring a hotspot TERT promoter mutation at positions -124C>T, -124_125CC>TT, -138_139CC>TT, or -146C>T. Of the 72 nevi, only 1 (1.4%) was TERT-positive. Of the 40 diagnostically uncertain melanocytic proliferations, 2 (5.0%) were TERT-positive. TERT positivity as a test for melanoma versus nevus had an accuracy of 87.3% [95% confidence interval (CI), 81.1-92.1], a sensitivity of 77.9% (95% CI, 68.9-85.4), a specificity of 98.6% (95% CI, 95.8-100), a positive predictive value of 98.5% (95% CI, 95.6-100), and a negative predictive value of 78.9% (95% CI, 72.6-85.4). Our results indicate that hotspot TERT promoter mutation status may be a useful ancillary parameter for the diagnosis of melanoma. In particular, the high specificity of these mutations for melanoma indicates the presence of a TERT promoter mutation in a melanocytic neoplasm associated with diagnostic controversy, or uncertainty should increase concern for a melanoma.

Breast tumor DNA methylation patterns associated with smoking in the Carolina Breast Cancer Study.

PURPOSE: Tobacco smoking is a risk factor in several cancers, yet its roles as a putative etiologic exposure or poor prognostic factor in breast cancer are less clear. Altered DNA methylation contributes to breast cancer development and may provide a mechanistic link between smoking and gene expression changes leading to cancer development or progression. METHODS: Using a cancer-focused array, we examined methylation at 933 CpGs in 517 invasive breast tumors in the Carolina Breast Cancer Study to determine whether methylation patterns differ by exposure to tobacco smoke. Multivariable generalized linear regression models were used to compare tumor methylation profiles between smokers and never smokers, overall, or stratified on hormone receptor (HR) status. RESULTS: Modest differences in CpG methylation were detected at p < 0.05 in breast tumors from current or ever smokers compared with never smokers. In stratified analyses, HR- tumors from smokers exhibited primarily hypomethylation compared with tumors from never smokers; hypomethylation was similarly detected within the more homogeneous basal-like subtype. Most current smoking-associated CpG loci exhibited methylation levels in former smokers that were intermediate between those in current and never smokers and exhibited progressive changes in methylation with increasing duration of smoking. Among former smokers, restoration of methylation toward baseline (never smoking) levels was observed with increasing time since quitting. Moreover, smoking-related hypermethylation was stronger in HR+ breast tumors from blacks than in whites. CONCLUSIONS: Our results suggest that breast tumor methylation patterns differ with tobacco smoke exposure; however, additional studies are needed to confirm these findings.

Active smoking and survival following breast cancer among African American and non-African American women in the Carolina Breast Cancer Study.

PURPOSE: To examine racial differences in smoking rates at the time of breast cancer diagnosis and subsequent survival among African American and non-African American women in the Carolina Breast Cancer Study (Phases I/II), a large population-based North Carolina study. METHODS: We interviewed 788 African American and 1,020 Caucasian/non-African American women diagnosed with invasive breast cancer from 1993 to 2000, to assess smoking history. After a median follow-up of 13.56 years, we identified 717 deaths using the National Death Index; 427 were breast cancer-related. We used Cox regression to examine associations between self-reported measures of smoking and breast cancer-specific survival within 5 years and up to 18 years after diagnosis conditional on 5-year survival. We examined race and estrogen receptor status as potential modifiers. RESULTS: Current (vs never) smoking was not associated with 5-year survival; however, risk of 13 year conditional breast cancer-specific mortality was elevated among women who were current smokers at diagnosis (HR 1.54, 95% CI 1.06-2.25), compared to never smokers. Although smoking rates were similar among African American (22.0%) and non-African American (22.1%) women, risk of breast cancer-specific mortality was elevated among African American (HR 1.69, 95% CI 1.00-2.85), but only weakly elevated among non-African American (HR 1.22, 95% CI 0.70-2.14) current (vs. never) smokers (P Interaction = 0.30). Risk of breast cancer-specific mortality was also elevated among current (vs never) smokers diagnosed with ER- (HR 2.58, 95% CI 1.35-4.93), but not ER+ (HR 1.11, 95% CI 0.69-1.78) tumors (P Interaction = 0.17). CONCLUSIONS: Smoking may negatively impact long-term survival following breast cancer. Racial differences in long-term survival, as related to smoking, may be driven by ER status, rather than by differences in smoking patterns.

Polycyclic aromatic hydrocarbons and postmenopausal breast cancer: An evaluation of effect measure modification by body mass index and weight change.

BACKGROUND: Polycyclic aromatic hydrocarbons (PAHs) have been linked to breast cancer in many, but not all, previous studies. PAHs are lipophilic and stored in fat tissue, which we hypothesized may result in constant low-dose exposure to these carcinogens. No previous studies have evaluated whether obesity modifies associations between multiple measures of PAHs and breast cancer incidence. METHODS: This population-based study included 1,006 postmenopausal women with first primary in situ or invasive breast cancer and 990 age-frequency matched controls. To evaluate effect modification by obesity (adult body mass index (BMI, kg/m2) and weight change) on multiple PAH measures (the biomarker PAH-DNA adducts, and long-term sources active cigarette smoking, living with a smoking spouse, grilled/smoked meat intake, residential synthetic log burning, and vehicular traffic), interaction contrast ratios (ICRs) for the additive scale, and ratio of odds ratios (RORs) with log-likelihood ratio tests (LRT) for the multiplicative scale, were determined using unconditional logistic regression. RESULTS: BMI modified the PAH-DNA adduct and postmenopausal breast cancer association on the additive (ICR: 0.49; 95% CI: 0.01, 0.96) and multiplicative (ROR: 1.56; 95% CI: 0.91, 2.68) scales. The odds ratio for detectable vs. non-detectable adducts was increased among women with BMI ≥25 (OR=1.34; 95% CI: 0.94, 1.92), but not in those with BMI <25 (OR=0.86; 95% CI: 0.57, 1.28) (LRT p=0.1). For most other PAH measures, the pattern of modification by BMI/weight gain was similar, but estimates were imprecise. CONCLUSIONS: The association between PAH-DNA adducts and breast cancer incidence may be elevated among overweight/obese women.

Active smoking and risk of Luminal and Basal-like breast cancer subtypes in the Carolina Breast Cancer Study.

PURPOSE: Growing evidence suggests an association between active cigarette smoking and increased breast cancer risk. However, the weak magnitude of association and conflicting results have yielded uncertainty and it is unknown whether associations differ by breast cancer subtype. METHODS: Using population-based case-control data from phases I and II of the Carolina Breast Cancer Study, we examined associations between self-reported measures of smoking and risk of Luminal and Basal-like breast cancers. We used logistic regression models to estimate case-control odds ratios (OR) and 95 % confidence intervals (CI). RESULTS: Ever smoking (current and former) was associated with a weakly increased risk of Luminal breast cancer (OR 1.12, 95 % CI 0.92-1.36) and was not associated with risk of Basal-like breast cancer (OR 0.96, 95 % CI 0.69-1.32). Similarly, smoking duration of more than 20 years was associated with increased risk of Luminal (OR 1.51, 95 % CI 1.19-1.93), but not Basal-like breast cancer (OR 0.90, 95 % CI 0.57-1.43). When stratified by race, elevated odds ratios between smoking and Luminal breast cancer risk were found among black women across multiple exposure measures (ever smoking, duration, and dose); conversely, among white women odds ratios were attenuated or null. CONCLUSIONS: Results from our study demonstrate a positive association between smoking and Luminal breast cancer risk, particularly among black women and women with long smoking histories. Addressing breast cancer heterogeneity in studies of smoking and breast cancer risk may elucidate associations masked in prior studies.

Sources of polycyclic aromatic hydrocarbons are associated with gene-specific promoter methylation in women with breast cancer.

BACKGROUND: Tobacco smoke, diet and indoor/outdoor air pollution, all major sources of polycyclic aromatic hydrocarbons (PAHs), have been associated with breast cancer. Aberrant methylation may be an early event in carcinogenesis, but whether PAHs influence the epigenome is unclear, particularly in breast tissue where methylation may be most relevant. We aimed to evaluate the role of methylation in the association between PAHs and breast cancer. METHODS: In a population-based case-control study, we measured promoter methylation of 13 breast cancer-related genes in breast tumor tissue (n=765-851 cases) and global methylation in peripheral blood (1055 cases/1101 controls). PAH sources (current active smoking, residential environmental tobacco smoke (ETS), vehicular traffic, synthetic log burning, and grilled/smoked meat intake) were evaluated separately. Logistic regression was used to estimate adjusted odds ratios (ORs) and 95% confidence intervals (CIs). RESULTS: When comparing methylated versus unmethylated genes, synthetic log use was associated with increased ORs for CDH1 (OR=2.26, 95%CI=1.06-4.79), HIN1 (OR=2.14, 95%CI=1.34-3.42) and RARß (OR=1.80, 95%CI=1.16-2.78) and decreased ORs for BRCA1 (OR=0.44, 95%CI=0.30-0.66). Residential ETS was associated with decreased ORs for ESR1 (OR=0.74, 95%CI=0.56-0.99) and CCND2 methylation (OR=0.65, 95%CI=0.44-0.96). Current smoking and vehicular traffic were associated with decreased ORs for DAPK (OR=0.53, 95%CI=0.28-0.99) and increased ORs for TWIST1 methylation (OR=2.79, 95%CI=1.24-6.30), respectively. In controls, synthetic log use was inversely associated with LINE-1 (OR=0.59, 95%CI=0.41-0.86). DISCUSSION: PAH sources were associated with hypo- and hypermethylation at multiple promoter regions in breast tumors and LINE-1 hypomethylation in blood of controls. Methylation may be a potential biologic mechanism for the associations between PAHs and breast cancer incidence.

Body mass index associated with genome-wide methylation in breast tissue.

Gene expression studies indicate that body mass index (BMI) is associated with molecular pathways involved in inflammation, insulin-like growth factor activation, and other carcinogenic processes in breast tissue. The goal of this study was to determine whether BMI is associated with gene methylation in breast tissue and to identify pathways that are commonly methylated in association with high BMI. Epigenome-wide methylation profiles were determined using the Illumina HumanMethylation450 BeadChip array in the non-diseased breast tissue of 81 women undergoing breast surgery between 2009 and 2013 at the University of North Carolina Hospitals. Multivariable, robust linear regression was performed to identify methylation sites associated with BMI at a false discovery rate q value <0.05. Gene expression microarray data was used to identify which of the BMI-associated methylation sites also showed correlation with gene expression. Gene set enrichment analysis was conducted to assess which pathways were enriched among the BMI-associated methylation sites. Of the 431,568 methylation sites analyzed, 2573 were associated with BMI (q value <0.05), 57 % of which showed an inverse correlation with BMI. Pathways enriched among the 2573 probe sites included those involved in inflammation, insulin receptor signaling, and leptin signaling. We were able to map 1251 of the BMI-associated methylation sites to gene expression data, and, of these, 226 (18 %) showed substantial correlations with gene expression. Our results suggest that BMI is associated with genome-wide methylation in non-diseased breast tissue and may influence epigenetic pathways involved in inflammatory and other carcinogenic processes.

DNA methylation profiling in the Carolina Breast Cancer Study defines cancer subclasses differing in clinicopathologic characteristics and survival.

INTRODUCTION: Breast cancer is a heterogeneous disease, with several intrinsic subtypes differing by hormone receptor (HR) status, molecular profiles, and prognosis. However, the role of DNA methylation in breast cancer development and progression and its relationship with the intrinsic tumor subtypes are not fully understood. METHODS: A microarray targeting promoters of cancer-related genes was used to evaluate DNA methylation at 935 CpG sites in 517 breast tumors from the Carolina Breast Cancer Study, a population-based study of invasive breast cancer. RESULTS: Consensus clustering using methylation (ß) values for the 167 most variant CpG loci defined four clusters differing most distinctly in HR status, intrinsic subtype (luminal versus basal-like), and p53 mutation status. Supervised analyses for HR status, subtype, and p53 status identified 266 differentially methylated CpG loci with considerable overlap. Genes relatively hypermethylated in HR+, luminal A, or p53 wild-type breast cancers included FABP3, FGF2, FZD9, GAS7, HDAC9, HOXA11, MME, PAX6, POMC, PTGS2, RASSF1, RBP1, and SCGB3A1, whereas those more highly methylated in HR-, basal-like, or p53 mutant tumors included BCR, C4B, DAB2IP, MEST, RARA, SEPT5, TFF1, THY1, and SERPINA5. Clustering also defined a hypermethylated luminal-enriched tumor cluster 3 that gene ontology analysis revealed to be enriched for homeobox and other developmental genes (ASCL2, DLK1, EYA4, GAS7, HOXA5, HOXA9, HOXB13, IHH, IPF1, ISL1, PAX6, TBX1, SOX1, and SOX17). Although basal-enriched cluster 2 showed worse short-term survival, the luminal-enriched cluster 3 showed worse long-term survival but was not independently prognostic in multivariate Cox proportional hazard analysis, likely due to the mostly early stage cases in this dataset. CONCLUSIONS: This study demonstrates that epigenetic patterns are strongly associated with HR status, subtype, and p53 mutation status and may show heterogeneity within tumor subclass. Among HR+ breast tumors, a subset exhibiting a gene signature characterized by hypermethylation of developmental genes and poorer clinicopathologic features may have prognostic value and requires further study. Genes differentially methylated between clinically important tumor subsets have roles in differentiation, development, and tumor growth and may be critical to establishing and maintaining tumor phenotypes and clinical outcomes.

Pathway Alterations in Stage II/III Primary Melanoma.

PURPOSE: Genomic classification of melanoma has thus far focused on the mutational status of BRAF, NRAS, and NF1. The clinical utility of this classification remains limited, and the landscape of alterations in other oncogenic signaling pathways is underexplored. METHODS: Using primary samples from the InterMEL study, a retrospective cohort of cases with specimens collected from an international consortium with participating institutions throughout the United States and Australia, with oversampling of cases who ultimately died of melanoma, we examined mutual exclusivity and co-occurrence of genomic alterations in 495 stage II/III primary melanomas across 11 cancer pathways. Somatic mutation and copy number alterations were analyzed from next-generation sequencing using a clinical sequencing panel. RESULTS: Mutations in the RTK-RAS pathway were observed in 81% of cases. Other frequently occurring pathways were TP53 (31%), Cell Cycle (30%), and PI3K (18%). These frequencies are generally lower than was observed in The Cancer Genome Atlas, where the specimens analyzed were predominantly obtained from metastases. Overall, 81% of the cases had at least one targetable mutation. The RTK-RAS pathway was the only pathway that demonstrated strong and statistically significant mutual exclusivity. However, this strong mutual exclusivity signal was evident only for the three common genes in the pathway (BRAF, NRAS, and NF1). Analysis of co-occurrence of different pathways exhibited no positive significant trends. However, interestingly, a high frequency of cases with none of these pathways represented was observed, 8.4% of cases versus 4.0% expected (P < .001). A higher frequency of RTK-RAS singletons (with no other pathway alteration) was observed compared with The Cancer Genome Atlas. Clonality analyses suggest strongly that both the cell cycle and RTK-RAS pathways represent early events in melanogenesis. CONCLUSION: Our results confirm the dominance of mutations in the RTK-RAS pathway. The presence of many mutations in several well-known, actionable pathways suggests potential avenues for targeted therapy in these early-stage cases.

Association of functional, inherited vitamin D-binding protein variants with melanoma-specific death.

BACKGROUND: It is unclear whether genetic variants affecting vitamin D metabolism are associated with melanoma prognosis. Two functional missense variants in the vitamin D-binding protein gene (GC), rs7041 and rs4588, determine 3 common haplotypes, Gc1s, Gc1f, and Gc2, of which Gc1f may be associated with decreased all-cause death among melanoma patients based on results of a prior study, but the association of Gc1f with melanoma-specific death is unclear. METHODS: We investigated the association of the Gc1s, Gc1f, and Gc2 haplotypes with melanoma-specific and all-cause death among 4490 individuals with incident, invasive primary melanoma in 2 population-based studies using multivariable Cox-proportional hazards regression. RESULTS: In the pooled analysis of both datasets, the patients with the Gc1f haplotype had a 37% lower risk of melanoma-specific death than the patients without Gc1f (hazard ratio [HR] = 0.63, 95% confidence interval [CI] = 0.47 to 0.83, P = .001), with adjustments for age, sex, study center, first- or higher-order primary melanoma, tumor site, pigmentary phenotypes, and Breslow thickness. Associations were similar in both studies. In pooled analyses stratified by Breslow thickness, the corresponding melanoma-specific death HRs for those patients with the Gc1f haplotype compared with those without Gc1f were 0.89 (95% CI = 0.63 to 1.27) among participants with tumor Breslow thickness equal to or less than 2.0 mm and 0.40 (95% CI = 0.25 to 0.63) among participants with tumor Breslow thickness greater than 2.0 mm (Pinteraction = .003). CONCLUSIONS: Our findings suggest that individuals with the GC haplotype Gc1f may have a lower risk of dying from melanoma-specifically from thicker, higher-risk melanoma-than individuals without this Gc1f haplotype.

Methylation of nonessential genes in cutaneous melanoma - Rule Out hypothesis.

Differential methylation plays an important role in melanoma development and is associated with survival, progression and response to treatment. However, the mechanisms by which methylation promotes melanoma development are poorly understood. The traditional explanation of selective advantage provided by differential methylation postulates that hypermethylation of regulatory 5'-cytosine-phosphate-guanine-3' dinucleotides (CpGs) downregulates the expression of tumor suppressor genes and therefore promotes tumorigenesis. We believe that other (not necessarily alternative) explanations of the selective advantages of methylation are also possible. Here, we hypothesize that melanoma cells use methylation to shut down transcription of nonessential genes - those not required for cell survival and proliferation. Suppression of nonessential genes allows tumor cells to be more efficient in terms of energy and resource usage, providing them with a selective advantage over the tumor cells that transcribe and subsequently translate genes they do not need. We named the hypothesis the Rule Out (RO) hypothesis. The RO hypothesis predicts higher methylation of CpGs located in regulatory regions (CpG islands) of nonessential genes. It also predicts the higher methylation of regulatory CpGs linked to nonessential genes in melanomas compared to nevi and lower expression of nonessential genes in malignant (derived from melanoma) versus normal (derived from nonaffected skin) melanocytes. The analyses conducted using in-house and publicly available data found that all predictions derived from the RO hypothesis hold, providing observational support for the hypothesis.

InterMEL: An international biorepository and clinical database to uncover predictors of survival in early-stage melanoma.

INTRODUCTION: We are conducting a multicenter study to identify classifiers predictive of disease-specific survival in patients with primary melanomas. Here we delineate the unique aspects, challenges, and best practices for optimizing a study of generally small-sized pigmented tumor samples including primary melanomas of at least 1.05mm from AJTCC TNM stage IIA-IIID patients. We also evaluated tissue-derived predictors of extracted nucleic acids' quality and success in downstream testing. This ongoing study will target 1,000 melanomas within the international InterMEL consortium. METHODS: Following a pre-established protocol, participating centers ship formalin-fixed paraffin embedded (FFPE) tissue sections to Memorial Sloan Kettering Cancer Center for the centralized handling, dermatopathology review and histology-guided coextraction of RNA and DNA. Samples are distributed for evaluation of somatic mutations using next gen sequencing (NGS) with the MSK-IMPACTTM assay, methylation-profiling (Infinium MethylationEPIC arrays), and miRNA expression (Nanostring nCounter Human v3 miRNA Expression Assay). RESULTS: Sufficient material was obtained for screening of miRNA expression in 683/685 (99%) eligible melanomas, methylation in 467 (68%), and somatic mutations in 560 (82%). In 446/685 (65%) cases, aliquots of RNA/DNA were sufficient for testing with all three platforms. Among samples evaluated by the time of this analysis, the mean NGS coverage was 249x, 59 (18.6%) samples had coverage below 100x, and 41/414 (10%) failed methylation QC due to low intensity probes or insufficient Meta-Mixed Interquartile (BMIQ)- and single sample (ss)- Noob normalizations. Six of 683 RNAs (1%) failed Nanostring QC due to the low proportion of probes above the minimum threshold. Age of the FFPE tissue blocks (p<0.001) and time elapsed from sectioning to co-extraction (p = 0.002) were associated with methylation screening failures. Melanin reduced the ability to amplify fragments of 200bp or greater (absent/lightly pigmented vs heavily pigmented, p<0.003). Conversely, heavily pigmented tumors rendered greater amounts of RNA (p<0.001), and of RNA above 200 nucleotides (p<0.001). CONCLUSION: Our experience with many archival tissues demonstrates that with careful management of tissue processing and quality control it is possible to conduct multi-omic studies in a complex multi-institutional setting for investigations involving minute quantities of FFPE tumors, as in studies of early-stage melanoma. The study describes, for the first time, the optimal strategy for obtaining archival and limited tumor tissue, the characteristics of the nucleic acids co-extracted from a unique cell lysate, and success rate in downstream applications. In addition, our findings provide an estimate of the anticipated attrition that will guide other large multicenter research and consortia.

Characterization of the CpG Island Hypermethylated Phenotype Subclass in Primary Melanomas.

Cutaneous melanoma can be lethal even if detected at an early stage. Epigenetic profiling may facilitate the identification of aggressive primary melanomas with unfavorable outcomes. We performed clustering of whole-genome methylation data to identify subclasses that were then assessed for survival, clinical features, methylation patterns, and biological pathways. Among 89 cutaneous primary invasive melanomas, we identified three methylation subclasses exhibiting low methylation, intermediate methylation, or hypermethylation of CpG islands, known as the CpG island methylator phenotype (CIMP). CIMP melanomas occurred as early as tumor stage 1b and, compared with low-methylation melanomas, were associated with age at diagnosis ≥65 years, lentigo maligna melanoma histologic subtype, presence of ulceration, higher American Joint Committee on Cancer stage and tumor stage, and lower tumor-infiltrating lymphocyte grade (all P < 0.05). Patients with CIMP melanomas had worse melanoma-specific survival (hazard ratio = 11.84; confidence interval = 4.65‒30.20) than those with low-methylation melanomas, adjusted for age, sex, American Joint Committee on Cancer stage, and tumor-infiltrating lymphocyte grade. Genes hypermethylated in CIMP compared with those in low-methylation melanomas included PTEN, VDR, PD-L1, TET2, and gene sets related to development/differentiation, the extracellular matrix, and immunity. CIMP melanomas exhibited hypermethylation of genes important in melanoma progression and tumor immunity, and although present in some early melanomas, CIMP was associated with worse survival independent of known prognostic factors.

Prognostic significance of RNA-based TP53 pathway function among estrogen receptor positive and negative breast cancer cases.

TP53 and estrogen receptor (ER) are essential in breast cancer development and progression, but TP53 status (by DNA sequencing or protein expression) has been inconsistently associated with survival. We evaluated whether RNA-based TP53 classifiers are related to survival. Participants included 3213 women in the Carolina Breast Cancer Study (CBCS) with invasive breast cancer (stages I-III). Tumors were classified for TP53 status (mutant-like/wildtype-like) using an RNA signature. We used Cox proportional hazards models to estimate covariate-adjusted hazard ratios (HRs) and 95% confidence intervals (CIs) for breast cancer-specific survival (BCSS) among ER- and TP53-defined subtypes. RNA-based results were compared to DNA- and IHC-based TP53 classification, as well as Basal-like versus non-Basal-like subtype. Findings from the diverse (50% Black), population-based CBCS were compared to those from the largely white METABRIC study. RNA-based TP53 mutant-like was associated with BCSS among both ER-negatives and ER-positives (HR (95% CI) = 5.38 (1.84-15.78) and 4.66 (1.79-12.15), respectively). Associations were attenuated when using DNA- or IHC-based TP53 classification. In METABRIC, few ER-negative tumors were TP53-wildtype-like, but TP53 status was a strong predictor of BCSS among ER-positives. In both populations, the effect of TP53 mutant-like status was similar to that for Basal-like subtype. RNA-based measures of TP53 status are strongly associated with BCSS and may have value among ER-negative cancers where few prognostic markers have been robustly validated. Given the role of TP53 in chemotherapeutic response, RNA-based TP53 as a prognostic biomarker could address an unmet need in breast cancer.

TP53 Pathway Function, Estrogen Receptor Status, and Breast Cancer Risk Factors in the Carolina Breast Cancer Study.

BACKGROUND: TP53 and estrogen receptor (ER) both play essential roles in breast cancer development and progression, with recent research revealing cross-talk between TP53 and ER signaling pathways. Although many studies have demonstrated heterogeneity of risk factor associations across ER subtypes, associations by TP53 status have been inconsistent. METHODS: This case-case analysis included incident breast cancer cases (47% Black) from the Carolina Breast Cancer Study (1993-2013). Formalin-fixed paraffin-embedded tumor samples were classified for TP53 functional status (mutant-like/wild-type-like) using a validated RNA signature. For IHC-based TP53 status, mutant-like was classified as at least 10% positivity. We used two-stage polytomous logistic regression to evaluate risk factor heterogeneity due to RNA-based TP53 and/or ER, adjusting for each other and for PR, HER2, and grade. We then compared this with the results when using IHC-based TP53 classification. RESULTS: The RNA-based classifier identified 55% of tumors as TP53 wild-type-like and 45% as mutant-like. Several hormone-related factors (oral contraceptive use, menopausal status, age at menopause, and pre- and postmenopausal body mass index) were associated with TP53 mutant-like status, whereas reproductive factors (age at first birth and parity) and smoking were associated with ER status. Multiparity was associated with both TP53 and ER. When classifying TP53 status using IHC methods, no associations were observed with TP53. Associations observed with RNA-based TP53 remained after accounting for basal-like subtype. CONCLUSIONS: This case-case study found breast cancer risk factors associated with RNA-based TP53 and ER. IMPACT: RNA-based TP53 and ER represent an emerging etiologic schema of interest in breast cancer prevention research.

Landscape of mutations in early stage primary cutaneous melanoma: An InterMEL study.

It is unclear why some melanomas aggressively metastasize while others remain indolent. Available studies employing multi-omic profiling of melanomas are based on large primary or metastatic tumors. We examine the genomic landscape of early-stage melanomas diagnosed prior to the modern era of immunological treatments. Untreated cases with Stage II/III cutaneous melanoma were identified from institutions throughout the United States, Australia and Spain. FFPE tumor sections were profiled for mutation, methylation and microRNAs. Preliminary results from mutation profiling and clinical pathologic correlates show the distribution of four driver mutation sub-types: 31% BRAF; 18% NRAS; 21% NF1; 26% Triple Wild Type. BRAF mutant tumors had younger age at diagnosis, more associated nevi, more tumor infiltrating lymphocytes, and fewer thick tumors although at generally more advanced stage. NF1 mutant tumors were frequent on the head/neck in older patients with severe solar elastosis, thicker tumors but in earlier stages. Triple Wild Type tumors were predominantly male, frequently on the leg, with more perineural invasion. Mutations in TERT, TP53, CDKN2A and ARID2 were observed often, with TP53 mutations occurring particularly frequently in the NF1 sub-type. The InterMEL study will provide the most extensive multi-omic profiling of early-stage melanoma to date. Initial results demonstrate a nuanced understanding of the mutational and clinicopathological landscape of these early-stage tumors.

Disease-Associated Risk Variants in ANRIL Are Associated with Tumor-Infiltrating Lymphocyte Presence in Primary Melanomas in the Population-Based GEM Study.

BACKGROUND: Genome-wide association studies have reported that genetic variation at ANRIL (CDKN2B-AS1) is associated with risk of several chronic diseases including coronary artery disease, coronary artery calcification, myocardial infarction, and type 2 diabetes mellitus. ANRIL is located at the CDKN2A/B locus, which encodes multiple melanoma tumor suppressors. We investigated the association of these variants with melanoma prognostic characteristics. METHODS: The Genes, Environment, and Melanoma Study enrolled 3,285 European origin participants with incident invasive primary melanoma. For each of ten disease-associated SNPs at or near ANRIL, we used linear and logistic regression modeling to estimate, respectively, the per allele mean changes in log of Breslow thickness and ORs for presence of ulceration and tumor-infiltrating lymphocytes (TIL). We also assessed effect modification by tumor NRAS/BRAF mutational status. RESULTS: Rs518394, rs10965215, and rs564398 passed false discovery and were each associated (P ≤ 0.005) with TILs, although only rs564398 was independently associated (P = 0.0005) with TILs. Stratified by NRAS/BRAF mutational status, rs564398*A was significantly positively associated with TILs among NRAS/BRAF mutant, but not wild-type, cases. We did not find SNP associations with Breslow thickness or ulceration. CONCLUSIONS: ANRIL rs564398 was associated with TIL presence in primary melanomas, and this association may be limited to NRAS/BRAF-mutant cases. IMPACT: Pathways related to ANRIL variants warrant exploration in relationship to TILs in melanoma, especially given the impact of TILs on immunotherapy and survival.

Targeting the IL-2 inducible kinase in melanoma; a phase 2 study of ibrutinib in systemic treatment-refractory distant metastatic cutaneous melanoma: preclinical rationale, biology, and clinical activity (NCI9922).

BACKGROUND: IL-2 inducible kinase (ITK) is highly expressed in metastatic melanomas and its inhibition suppresses melanoma cell proliferation. We hypothesize that ibrutinib has a direct antitumor effect in melanoma cell lines and that treatment of metastatic melanomas with ibrutinib induces antitumor responses. METHODS: We assessed the ibrutinib effect on melanoma cell proliferation, apoptosis, and motility. Patients with metastatic melanoma refractory to PD-1 and MAPK inhibitors (if BRAFV600-mutant) were treated with ibrutinib, 840 mg PO QD, as part of a phase II clinical trial (clinicaltrials.gov NCT02581930). RESULTS: Melanoma cell lines frequently express ITK, YES1, and EGFR. Ibrutinib suppressed cell motility and proliferation in most cell lines. Eighteen patients (13 male; median age 63.5 years, range 37-82; 12 with ipilimumab resistance) were enrolled. The most frequent side effects were fatigue (61%), anorexia (50%), hyponatremia (28%), nausea, and vomiting (22% each). No antitumor responses were seen. At a median follow-up of 6 months (0.3-35.8 months), the median progression-free survival was 1.3 months (range 0.2-5.5 months). Fifteen patients were discontinued from the study due to progression, and 14 patients had died from metastatic melanoma. All archived tumors expressed ITK, 41% had no expression of p16 and PTEN, and 61% had absent tumor-infiltrating lymphocytes (TILs). Ibrutinib significantly suppressed proliferating (Ki67+) CD19+ peripheral blood mononuclear cells and had no significant effect on other lymphocyte subsets. CONCLUSION: Ibrutinib did not induce any meaningful clinical benefit. ITK expression may not be clinically relevant. Treatment-refractory metastatic melanomas have other fundamental defects (i.e. absent PTEN and p16 expression, absent TILs) that may contribute to an adverse prognosis.