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Background: Genetic screening for pathogenic variants (PVs) in cancer predisposition genes can affect treatment strategies, risk prediction and preventive measures for patients and families. For decades, hereditary breast and ovarian cancer (HBOC) has been attributed to PVs in the genes BRCA1 and BRCA2, and more recently other rare alleles have been firmly established as associated with a high or moderate increased risk of developing breast and/or ovarian cancer. Here, we assess the genetic variation and tumor characteristics in a large cohort of women with suspected HBOC in a clinical oncogenetic setting., Methods: Women with suspected HBOC referred from all oncogenetic clinics in Sweden over a six-year inclusion period were screened for PVs in 13 clinically relevant genes. The genetic outcome was compared with tumor characteristics and other clinical data collected from national cancer registries and hospital records., Results: In 4622 women with breast and/or ovarian cancer the overall diagnostic yield (the proportion of women carrying at least one PV) was 16.6%. BRCA1/2 PVs were found in 8.9% of women (BRCA1 5.95% and BRCA2 2.94%) and PVs in the other breast and ovarian cancer predisposition genes in 8.2%: ATM (1.58%), BARD1 (0.45%), BRIP1 (0.43%), CDH1 (0.11%), CHEK2 (3.46%), PALB2 (0.84%), PTEN (0.02%), RAD51C (0.54%), RAD51D (0.15%), STK11 (0) and TP53 (0.56%). Thus, inclusion of the 11 genes in addition to BRCA1/2 increased diagnostic yield by 7.7%. The yield was, as expected, significantly higher in certain subgroups such as younger patients, medullary breast cancer, higher Nottingham Histologic Grade, ER-negative breast cancer, triple-negative breast cancer and high grade serous ovarian cancer. Age and tumor subtype distributions differed substantially depending on genetic finding., Conclusions: This study contributes to understanding the clinical and genetic landscape of breast and ovarian cancer susceptibility. Extending clinical genetic screening from BRCA1 and BRCA2 to 13 established cancer predisposition genes almost doubles the diagnostic yield, which has implications for genetic counseling and clinical guidelines. The very low yield in the syndrome genes CDH1, PTEN and STK11 questions the usefulness of including these genes on routine gene panels., (© 2023. BioMed Central Ltd., part of Springer Nature.)

BOADICEA is a comprehensive risk prediction model for breast and/or ovarian cancer (BC/OC) and for carrying pathogenic variants (PVs) in cancer susceptibility genes. In addition to BRCA1 and BRCA2, BOADICEA version 6 includes PALB2, CHEK2, ATM, BARD1, RAD51C and RAD51D. To validate its predictions for these genes, we conducted a retrospective study including 2033 individuals counselled at clinical genetics departments in Denmark. All counselees underwent comprehensive genetic testing by next generation sequencing on suspicion of hereditary susceptibility to BC/OC. Likelihoods of PVs were predicted from information about diagnosis, family history and tumour pathology. Calibration was examined using the observed-to-expected ratio (O/E) and discrimination using the area under the receiver operating characteristics curve (AUC). The O/E was 1.11 (95% CI 0.97-1.26) for all genes combined. At sub-categories of predicted likelihood, the model performed well with limited misestimation at the extremes of predicted likelihood. Discrimination was acceptable with an AUC of 0.70 (95% CI 0.66-0.74), although discrimination was better for BRCA1 and BRCA2 than for the other genes in the model. This suggests that BOADICEA remains a valid decision-making aid for determining which individuals to offer comprehensive genetic testing for hereditary susceptibility to BC/OC despite suboptimal calibration for individual genes in this population., (© 2023. The Author(s).)

Skipping of BRCA2 exon 3 (∆E3) is a naturally occurring splicing event, complicating clinical classification of variants that may alter ∆E3 expression. This study used multiple evidence types to assess pathogenicity of 85 variants in/near BRCA2 exon 3. Bioinformatically predicted spliceogenic variants underwent mRNA splicing analysis using minigenes and/or patient samples. ∆E3 was measured using quantitative analysis. A mouse embryonic stem cell (mESC) based assay was used to determine the impact of 18 variants on mRNA splicing and protein function. For each variant, population frequency, bioinformatic predictions, clinical data, and existing mRNA splicing and functional results were collated. Variant class was assigned using a gene-specific adaptation of ACMG/AMP guidelines, following a recently proposed points-based system. mRNA and mESC analysis combined identified six variants with transcript and/or functional profiles interpreted as loss of function. Cryptic splice site use for acceptor site variants generated a transcript encoding a shorter protein that retains activity. Overall, 69/85 (81%) variants were classified using the points-based approach. Our analysis shows the value of applying gene-specific ACMG/AMP guidelines using a points-based approach and highlights the consideration of cryptic splice site usage to appropriately assign PVS1 code strength., (© 2022 The Authors. Human Mutation published by Wiley Periodicals LLC.)

Background: We examined whether molecular characterization of high-grade epithelial ovarian cancer can inform the diagnosis and/or identify potential actionable targets., Methods: All of the consecutively sequenced high-grade ovarian tumours with consent between 2014 until 2019 were included. A total of 274 tumours underwent next generation sequencing using a targeted panel., Results: Patients with high-grade ovarian epithelial cancer were consented to prospective molecular characterization. Clinical information was extracted from their medical record. Tumour DNA was subjected to sequencing, and selected patients received PARP inhibitor therapy., Conclusions: Tumours from 274 women were sequenced, including high-grade serous carcinoma ( n = 252), clear cell carcinoma ( n = 4), carcinosarcoma ( n = 9), endometrioid carcinoma ( n = 3), undifferentiated carcinoma ( n = 1), and mixed tumours ( n = 5). Genomic profiling did not influence histologic diagnosis. Mutations were identified in TP53 , BRCA1 , BRCA2 , as well as additional homologous recombination repair pathway genes BARD1 , ATR , CHEK2 , PALB2 , RAD51D , RAD50 , SLX4 , FANCA , RAD51C , and RAD54L . In addition, mutations in PTEN and CDKN2A were identified. Several somatic mutations with implications for germline testing were identified, including RMI1 , STK11 , and CDH1 . Germline testing identified 16 previously unknown BRCA1/2 carriers. Finally, 20 patients were treated with the PARP inhibitor olaparib based on the sequencing results.

Germline protein truncating variants (PTVs) in the FANCM gene have been associated with a 2-4-fold increased breast cancer risk in case-control studies conducted in different European populations. However, the distribution and the frequency of FANCM PTVs in Europe have never been investigated. In the present study, we collected the data of 114 European female breast cancer cases with FANCM PTVs ascertained in 20 centers from 13 European countries. We identified 27 different FANCM PTVs. The p.Gln1701* PTV is the most common PTV in Northern Europe with a maximum frequency in Finland and a lower relative frequency in Southern Europe. On the contrary, p.Arg1931* seems to be the most common PTV in Southern Europe. We also showed that p.Arg658*, the third most common PTV, is more frequent in Central Europe, and p.Gln498Thrfs*7 is probably a founder variant from Lithuania. Of the 23 rare or unique FANCM PTVs, 15 have not been previously reported. We provide here the initial spectrum of FANCM PTVs in European breast cancer cases., Competing Interests: The authors declare no conflict of interest.

The multifactorial likelihood analysis method has demonstrated utility for quantitative assessment of variant pathogenicity for multiple cancer syndrome genes. Independent data types currently incorporated in the model for assessing BRCA1 and BRCA2 variants include clinically calibrated prior probability of pathogenicity based on variant location and bioinformatic prediction of variant effect, co-segregation, family cancer history profile, co-occurrence with a pathogenic variant in the same gene, breast tumor pathology, and case-control information. Research and clinical data for multifactorial likelihood analysis were collated for 1,395 BRCA1/2 predominantly intronic and missense variants, enabling classification based on posterior probability of pathogenicity for 734 variants: 447 variants were classified as (likely) benign, and 94 as (likely) pathogenic; and 248 classifications were new or considerably altered relative to ClinVar submissions. Classifications were compared with information not yet included in the likelihood model, and evidence strengths aligned to those recommended for ACMG/AMP classification codes. Altered mRNA splicing or function relative to known nonpathogenic variant controls were moderately to strongly predictive of variant pathogenicity. Variant absence in population datasets provided supporting evidence for variant pathogenicity. These findings have direct relevance for BRCA1 and BRCA2 variant evaluation, and justify the need for gene-specific calibration of evidence types used for variant classification., (© 2019 Wiley Periodicals, Inc.)

PurposeMonoallelic germ-line mutations in the BRCA1/FANCS, BRCA2/FANCD1 and PALB2/FANCN genes confer high risk of breast cancer. Biallelic mutations in these genes cause Fanconi anemia (FA), characterized by malformations, bone marrow failure, chromosome fragility, and cancer predisposition (BRCA2/FANCD1 and PALB2/FANCN), or an FA-like disease presenting a phenotype similar to FA but without bone marrow failure (BRCA1/FANCS). FANCM monoallelic mutations have been reported as moderate risk factors for breast cancer, but there are no reports of any clinical phenotype observed in carriers of biallelic mutations.MethodsBreast cancer probands were subjected to mutation analysis by sequencing gene panels or testing DNA damage response genes.ResultsFive cases homozygous for FANCM loss-of-function mutations were identified. They show a heterogeneous phenotype including cancer predisposition, toxicity to chemotherapy, early menopause, and possibly chromosome fragility. Phenotype severity might correlate with mutation position in the gene.ConclusionOur data indicate that biallelic FANCM mutations do not cause classical FA, providing proof that FANCM is not a canonical FA gene. Moreover, our observations support previous findings suggesting that FANCM is a breast cancer-predisposing gene. Mutation testing of FANCM might be considered for individuals with the above-described clinical features.

Purpose: To evaluate a simplified method of pre-test information and germline BRCA1/2 mutation testing., Methods: In a prospective, single-arm study, comprehensive BRCA1/2 testing was offered to unselected patients with newly diagnosed breast cancer at three hospitals in south Sweden (BRCAsearch, ClinicalTrials.gov Identifier: NCT02557776). Pre-test information was provided by a standardized invitation letter, but the patients could contact a genetic counselor for telephone genetic counseling if they felt a need for that. Noncarriers were informed about the test result through a letter. Mutation carriers were contacted and offered an appointment for in-person post-test genetic counseling., Results: During the period Feb 2, 2015-Aug 26, 2016, eight hundred and eighteen patients were invited to participate in the study. Through Jan 31, 2017, five hundred and forty-two (66.2%) of them consented to analysis of BRCA1 and BRCA2. Eleven pathogenic mutations were found (BRCA1, n = 2; BRCA2, n = 9), corresponding to a mutation prevalence of 2.0%. Six out of 11 fulfilled the Swedish BRCA testing criteria, and 9 out of 11 fulfilled the NCCN testing criteria. None of the BRCA-associated tumors were of the luminal A-like subtype. Very few patients contacted us for telephone genetic counseling or practical questions, suggesting that a majority felt that the written pre-test information was sufficient for them to make a decision on testing., Conclusions: Streamlining the process of pre-test information, genetic testing, and delivery of test results was feasible and was associated with an uptake of genetic testing in 2/3 of the breast cancer patients.

Purpose: To describe a snapshot of international genetic testing practices, specifically regarding the use of multigene panels, for hereditary breast/ovarian cancers. We conducted a survey through the Evidence-Based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium, covering questions about 16 non- BRCA1 / 2 genes., Methods: Data were collected via in-person and paper/electronic surveys. ENIGMA members from around the world were invited to participate. Additional information was collected via country networks in the United Kingdom and in Italy., Results: Responses from 61 cancer genetics practices across 20 countries showed that 16 genes were tested by > 50% of the centers, but only six ( PALB2 , TP53 , PTEN , CHEK2 , ATM , and BRIP1 ) were tested regularly. US centers tested the genes most often, whereas United Kingdom and Italian centers with no direct ENIGMA affiliation at the time of the survey were the least likely to regularly test them. Most centers tested the 16 genes through multigene panels; some centers tested TP53 , PTEN , and other cancer syndrome-associated genes individually. Most centers reported (likely) pathogenic variants to patients and would test family members for such variants. Gene-specific guidelines for breast and ovarian cancer risk management were limited and differed among countries, especially with regard to starting age and type of imaging and risk-reducing surgery recommendations., Conclusion: Currently, a small number of genes beyond BRCA1 / 2 are routinely analyzed worldwide, and management guidelines are limited and largely based on expert opinion. To attain clinical implementation of multigene panel testing through evidence-based management practices, it is paramount that clinicians (and patients) participate in international initiatives that share panel testing data, interpret sequence variants, and collect prospective data to underpin risk estimates and evaluate the outcome of risk intervention strategies.

Adoptive T-cell therapy (ACT) is a highly intensive immunotherapy regime that has yielded remarkable response rates and many durable responses in clinical trials in melanoma; however, 50-60% of the patients have no clinical benefit. Here, we searched for predictive biomarkers to ACT in melanoma. Whole exome- and transcriptome sequencing and neoantigen prediction were applied to pre-treatment samples from 27 patients recruited to a clinical phase I/II trial of ACT in stage IV melanoma. All patients had previously progressed on other immunotherapies. We report that clinical benefit is associated with significantly higher predicted neoantigen load. High mutation and predicted neoantigen load are significantly associated with improved progression-free and overall survival. Further, clinical benefit is associated with the expression of immune activation signatures including a high MHC-I antigen processing and presentation score. These results improve our understanding of mechanisms behind clinical benefit of ACT in melanoma.

In general, melanoma can be considered as a UV-driven disease with an aggressive metastatic course and high mutational load, with only few tumors (acral, mucosal, and uveal melanomas) not induced by sunlight and possessing a lower mutational load. The most commonly activated pathway in melanoma is the mitogen-activated protein kinase (MAPK) pathway. However, the prognostic significance of mutational stratification is unclear and needs further investigation. Here, in silico we combined mutation data from 162 melanomas subjected to targeted deep sequencing with mutation data from three published studies. Tumors from 870 patients were grouped according to BRAF, RAS, NF1 mutation or triple-wild-type status and correlated with tumor and patient characteristics. We found that the NF1-mutated subtype had a higher mutational burden and strongest UV mutation signature. Searching for co-occurring mutated genes revealed the RASopathy genes PTPN11 and RASA2, as well as another RAS domain-containing gene RASSF2 enriched in the NF1 subtype after adjustment for mutational burden. We found that a larger proportion of the NF1-mutant tumors were from males and with older age at diagnosis. Importantly, we found an increased risk of death from melanoma (disease-specific survival, DSS; HR, 1.9; 95% CI, 1.21-3.10; P = 0.046) and poor overall survival (OS; HR, 2.0; 95% CI, 1.28-2.98; P = 0.01) in the NF1 subtype, which remained significant after adjustment for age, gender, and lesion type (DSS P = 0.03, OS P = 0.06, respectively). Melanoma genomic subtypes display different biological and clinical characteristics. The poor outcome observed in the NF1 subtype highlights the need for improved characterization of this group., (© 2017 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Hereditary syndromes causing colorectal cancer include both polyposis and non-polyposis syndromes. Overlapping phenotypes between the syndromes have been recognized and this make targeted molecular testing for single genes less favorable, instead there is a gaining interest for multi-gene panel-based approaches detecting both SNVs, indels and CNVs in the same assay. We applied a panel including 19 CRC susceptibility genes to 91 individuals of six phenotypic subgroups. Targeted NGS-based sequencing of the whole gene regions including introns of the 19 genes was used. The individuals had a family history of CRC or had a phenotype consistent with a known CRC syndrome. The purpose of the study was to demonstrate the diagnostic difficulties linked to genotype-phenotype diversity and the benefits of using a gene panel. Pathogenicity classification was carried out on 46 detected variants. In total we detected sixteen pathogenic or likely pathogenic variants and 30 variants of unknown clinical significance. Four of the pathogenic or likely pathogenic variants were found in BMPR1A in patients with unexplained familial adenomatous polyposis or atypical adenomatous polyposis, which extends the genotype-phenotype spectrum for this gene. Nine patients had more than one variant remaining after the filtration, including three with truncating mutations in BMPR1A, PMS2 and AXIN2. CNVs were found in three patients, in upstream regions of SMAD4, MSH3 and CTNNB1, and one additional individual harbored a 24.2 kb duplication in CDH1 intron1.

Cancer genome sequencing has shed light on the underlying genetic aberrations that drive tumorigenesis. However, current sequencing-based strategies, which focus on a single tumor biopsy, fail to take into account intratumoral heterogeneity. To address this challenge and elucidate the evolutionary history of melanoma, we performed whole-exome and transcriptome sequencing of 41 multiple melanoma biopsies from eight individual tumors. This approach revealed heterogeneous somatic mutations in the range of 3%-38% in individual tumors. Known mutations in melanoma drivers BRAF and NRAS were always ubiquitous events. Using RNA sequencing, we found that the majority of mutations were not expressed or were expressed at very low levels, and preferential expression of a particular mutated allele did not occur frequently. In addition, we found that the proportion of ultraviolet B (UVB) radiation-induced C>T transitions differed significantly (P < 0.001) between early and late mutation acquisition, suggesting that different mutational processes operate during the evolution of metastatic melanoma. Finally, clinical history reports revealed that patients harboring a high degree of mutational heterogeneity were associated with more aggressive disease progression. In conclusion, our multiregion tumor-sequencing approach highlights the genetic evolution and non-UVB mutational signatures associated with melanoma development and progression, and may provide a more comprehensive perspective of patient outcome. Cancer Res; 76(16); 4765-74. ©2016 AACR., (©2016 American Association for Cancer Research.)

To gain insights into the regulatory mechanisms of hematopoietic stem cells (HSCs), we employed a genome-wide RNAi screen in human cord-blood derived cells and identified candidate genes whose knockdown maintained the HSC phenotype during culture. A striking finding was the identification of members of the cohesin complex (STAG2, RAD21, STAG1, and SMC3) among the top 20 genes from the screen. Upon individual validation of these cohesin genes, we found that their knockdown led to an immediate expansion of cells with an HSC phenotype in vitro. A similar expansion was observed in vivo following transplantation to immunodeficient mice. Transcriptome analysis of cohesin-deficient CD34(+) cells showed an upregulation of HSC-specific genes, demonstrating an immediate shift toward a more stem-cell-like gene expression signature upon cohesin deficiency. Our findings implicate cohesin as a major regulator of HSCs and illustrate the power of global RNAi screens to identify modifiers of cell fate., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Basal-like breast cancer is an aggressive subtype generally characterized as poor prognosis and lacking the expression of the three most important clinical biomarkers, estrogen receptor, progesterone receptor, and HER2. Cell lines serve as useful model systems to study cancer biology in vitro and in vivo. We performed mutational profiling of six basal-like breast cancer cell lines (HCC38, HCC1143, HCC1187, HCC1395, HCC1954, and HCC1937) and their matched normal lymphocyte DNA using targeted capture and next-generation sequencing of 1,237 cancer-associated genes, including all exons, UTRs and upstream flanking regions. In total, 658 somatic variants were identified, of which 378 were non-silent (average 63 per cell line, range 37-146) and 315 were novel (not present in the Catalogue of Somatic Mutations in Cancer database; COSMIC). 125 novel mutations were confirmed by Sanger sequencing (59 exonic, 48 3'UTR and 10 5'UTR, 1 splicing), with a validation rate of 94% of high confidence variants. Of 36 mutations previously reported for these cell lines but not detected in our exome data, 36% could not be detected by Sanger sequencing. The base replacements C/G>A/T, C/G>G/C, C/G>T/A and A/T>G/C were significantly more frequent in the coding regions compared to the non-coding regions (OR 3.2, 95% CI 2.0-5.3, P<0.0001; OR 4.3, 95% CI 2.9-6.6, P<0.0001; OR 2.4, 95% CI 1.8-3.1, P<0.0001; OR 1.8, 95% CI 1.2-2.7, P = 0.024, respectively). The single nucleotide variants within the context of T[C]T/A[G]A and T[C]A/T[G]A were more frequent in the coding than in the non-coding regions (OR 3.7, 95% CI 2.2-6.1, P<0.0001; OR 3.8, 95% CI 2.0-7.2, P = 0.001, respectively). Copy number estimations were derived from the targeted regions and correlated well to Affymetrix SNP array copy number data (Pearson correlation 0.82 to 0.96 for all compared cell lines; P<0.0001). These mutation calls across 1,237 cancer-associated genes and identification of novel variants will aid in the design and interpretation of biological experiments using these six basal-like breast cancer cell lines.

To better understand and characterize chromosomal structural variation during breast cancer progression, we enumerated chromosomal rearrangements for 11 patients by performing low-coverage whole-genome sequencing of 11 primary breast tumors and their 13 matched distant metastases. The tumor genomes harbored a median of 85 (range 18-404) rearrangements per tumor, with a median of 82 (26-310) in primaries compared to 87 (18-404) in distant metastases. Concordance between paired tumors from the same patient was high with a median of 89% of rearrangements shared (range 61-100%), whereas little overlap was found when comparing all possible pairings of tumors from different patients (median 3%). The tumors exhibited diverse genomic patterns of rearrangements: some carried events distributed throughout the genome while others had events mostly within densely clustered chromothripsis-like foci at a few chromosomal locations. Irrespectively, the patterns were highly conserved between the primary tumor and metastases from the same patient. Rearrangements occurred more frequently in genic areas than expected by chance and among the genes affected there was significant enrichment for cancer-associated genes including disruption of TP53, RB1, PTEN, and ESR1, likely contributing to tumor development. Our findings are most consistent with chromosomal rearrangements being early events in breast cancer progression that remain stable during the development from primary tumor to distant metastasis.

Melanoma is currently divided on a genetic level according to mutational status. However, this classification does not optimally predict prognosis. In prior studies, we have defined gene expression phenotypes (high-immune, pigmentation, proliferative and normal-like), which are predictive of survival outcome as well as informative of biology. Herein, we employed a population-based metastatic melanoma cohort and external cohorts to determine the prognostic and predictive significance of the gene expression phenotypes. We performed expression profiling on 214 cutaneous melanoma tumors and found an increased risk of developing distant metastases in the pigmentation (HR, 1.9; 95% CI, 1.05-3.28; P=0.03) and proliferative (HR, 2.8; 95% CI, 1.43-5.57; P=0.003) groups as compared to the high-immune response group. Further genetic characterization of melanomas using targeted deep-sequencing revealed similar mutational patterns across these phenotypes. We also used publicly available expression profiling data from melanoma patients treated with targeted or vaccine therapy in order to determine if our signatures predicted therapeutic response. In patients receiving targeted therapy, melanomas resistant to targeted therapy were enriched in the MITF-low proliferative subtype as compared to pre-treatment biopsies (P=0.02). In summary, the melanoma gene expression phenotypes are highly predictive of survival outcome and can further help to discriminate patients responding to targeted therapy.

Breast cancer type 1 susceptibility protein (BRCA1) has a multitude of functions that contribute to genome integrity and tumor suppression. Its participation in the repair of DNA double-strand breaks (DSBs) during homologous recombination (HR) is well recognized, whereas its involvement in the second major DSB repair pathway, nonhomologous end-joining (NHEJ), remains controversial. Here we have studied the role of BRCA1 in the repair of DSBs in switch (S) regions during immunoglobulin class switch recombination, a physiological, deletion/recombination process that relies on the classical NHEJ machinery. A shift to the use of microhomology-based, alternative end-joining (A-EJ) and increased frequencies of intra-S region deletions as well as insertions of inverted S sequences were observed at the recombination junctions amplified from BRCA1-deficient human B cells. Furthermore, increased use of long microhomologies was found at recombination junctions derived from E3 ubiquitin-protein ligase RNF168-deficient, Fanconi anemia group J protein (FACJ, BRIP1)-deficient, or DNA endonuclease RBBP8 (CtIP)-compromised cells, whereas an increased frequency of S-region inversions was observed in breast cancer type 2 susceptibility protein (BRCA2)-deficient cells. Thus, BRCA1, together with its interaction partners, seems to play an important role in repairing DSBs generated during class switch recombination by promoting the classical NHEJ pathway. This may not only provide a general mechanism underlying BRCA1's function in maintaining genome stability and tumor suppression but may also point to a previously unrecognized role of BRCA1 in B-cell lymphomagenesis.

Diversity between metastatic melanoma tumours in individual patients is known; however, the molecular and genetic differences remain unclear. To examine the molecular and genetic differences between metastatic tumours, we performed gene-expression profiling of 63 melanoma tumours obtained from 28 patients (two or three tumours/patient), followed by analysis of their mutational landscape, using targeted deep sequencing of 1697 cancer genes and DNA copy number analysis. Gene-expression signatures revealed discordant phenotypes between tumour lesions within a patient in 50% of the cases. In 18 of 22 patients (where matched normal tissue was available), we found that the multiple lesions within a patient were genetically divergent, with one or more melanoma tumours harbouring 'private' somatic mutations. In one case, the distant subcutaneous metastasis of one patient occurring 3 months after an earlier regional lymph node metastasis had acquired 37 new coding sequence mutations, including mutations in PTEN and CDH1. However, BRAF and NRAS mutations, when present in the first metastasis, were always preserved in subsequent metastases. The patterns of nucleotide substitutions found in this study indicate an influence of UV radiation but possibly also DNA alkylating agents. Our results clearly demonstrate that metastatic melanoma is a molecularly highly heterogeneous disease that continues to progress throughout its clinical course. The private aberrations observed on a background of shared aberrations within a patient provide evidence of continued evolution of individual tumours following divergence from a common parental clone, and might have implications for personalized medicine strategies in melanoma treatment., (© 2014 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.)

Purpose: For primary melanomas, tumor thickness, mitotic rate, and ulceration are well-laid cornerstones of prognostication. However, a molecular exposition of melanoma aggressiveness is critically missing. We recently uncovered a four-class structure in metastatic melanoma, which predicts outcome and informs biology. This raises the possibility that a molecular structure exists even in the early stages of melanoma and that molecular determinants could underlie histophenotype and eventual patient outcome., Experimental Design: We subjected 223 archival primary melanomas to a horizontally integrated analysis of RNA expression, oncogenic mutations at 238 lesions, histomorphometry, and survival data., Results: Our previously described four-class structure that was elucidated in metastatic lesions was evident within the expression space of primary melanomas. Because these subclasses converged into two larger prognostic and phenotypic groups, we used the metastatic lesions to develop a binary subtype-based signature capable of distinguishing between "high" and "low" grade forms of the disease. The two-grade signature was subsequently applied to the primary melanomas. Compared with low-grade tumors, high-grade primary melanomas were significantly associated with increased tumor thickness, mitotic rate, ulceration (all P < 0.01), and poorer relapse-free (HR = 4.94; 95% CI, 2.84-8.59), and overall (HR = 3.66; 95% CI, 2.40-5.58) survival. High-grade melanomas exhibited elevated levels of proliferation and BRCA1/DNA damage signaling genes, whereas low-grade lesions harbored higher expression of immune genes. Importantly, the molecular-grade signature was validated in two external gene expression data sets., Conclusions: We provide evidence for a molecular organization within melanomas, which is preserved across all stages of disease.

High levels of insulin-like growth factor-1 (IGF-1) have been associated with increased risk of developing several types of cancer including breast cancer. A set of nine haplotype tagging SNPs (htSNPs) in the IGF1 gene were associated with IGF-1 levels and prostate cancer in a Swedish population. We aimed to study the nine htSNPs in three haplotype blocks (block1: rs855211, rs35765, rs2162679; block2: rs1019731, rs7956547, rs5742632; and block3 rs2033178, rs7136446, rs6220) combined into diplotypes, and three additional SNPs (rs5742612, rs35765817, rs35455143) in relation to IGF-1 levels, BRCA status, the IGF1 CA-repeat microsatellite, and breast cancer in a population of 325 Swedish women from breast cancer high-risk families. Questionnaire data and blood samples for IGF-1 and genetic analyses were obtained twice during the menstrual cycle from 269 women aged 40 years or younger. SNP analyses were also performed in 56 BRCA1/2 mutation carriers. Women (n = 14) with any rare variant block1 diplotype had higher odds to be BRCA1 mutation carriers OR 4.1 (95% CI 1.4-12.2), to lack the common IGF1 19 CA-repeat allele OR 33.3 (95% CI 6.6-166.7), and were more likely to develop early-onset breast cancer (Log Rank P < 0.001) than women with common block1 diplotypes. In the subgroup of BRCA1 mutation carriers, block1 rare diplotypes were associated with earlier diagnosis (Log Rank P = 0.031). No association was found between IGF-1 levels and individual SNPs or diplotypes. If confirmed, these rare diplotypes may identify women with particularly high risk for early-onset breast cancer and this group should be included in forthcoming studies.

High testosterone levels have been associated with breast cancer. BRCA1 may function as an androgen receptor (AR) co-regulator. We aimed to examine AR haplotype-tagging single-nucleotide polymorphisms (AR htSNPs) and diplotypes in relation to in vivo androgen levels, combined OC use, CAG and GGC genotypes, and BRCA1/2/X family status in 269 young healthy women from breast cancer high-risk families and 56 additional BRCA1/2 mutation carriers. Testosterone, androstenedione, dehydroepiandrosterone sulfate, and body constitution were measured on cycle days 18-23. Six AR htSNPs and CAG and GGC repeat lengths were genotyped. Most OC users had lower androgen levels than non-users (all Ps<0.0001). Rare variant diplotypes were associated with higher testosterone levels in OC users than in non-users (P(interaction)=0.011). The interaction remained after adjustment for family clustering. Neither individual AR htSNPs nor other diplotypes were significantly associated with androgen levels and did not tag for CAG or GGC genotypes. In the first included woman from each family, the odds of having the most common diplotype was lower in BRCA1 families compared to other families OR 0.41 (95% CI 0.22-0.78). In conclusion, we found few associations between AR htSNPs or diplotypes and androgen levels in women. Diplotypes cannot replace genotyping of microsatellites CAG or GGC. Since testosterone levels are not affected the same way by combined OC use among all women, young women who have higher testosterone levels during combined OC use may belong to the subgroup of women who will not be helped by combined OCs for treatment of androgen-dependent conditions and may be at higher risk for early-onset breast cancer. Whether these women can be identified with AR genotyping needs to be confirmed in an independent cohort., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Given the greatly elevated risks of contralateral breast cancer (CBC) observed in breast cancer patients who carry mutations in BRCA1 and BRCA2, it is critical to determine the effectiveness of standard adjuvant therapies in preventing CBC in mutation carriers. The WECARE study is a matched, case-control study of 708 women with CBC as cases and 1,399 women with unilateral breast cancer (UBC) as controls, including 181 BRCA1/BRCA2 mutation carriers. Interviews and medical record reviews provided detailed information on risk factors and breast cancer therapy. All study participants were screened for BRCA1 and BRCA2 mutations using denaturing high-performance liquid chromatography (DHPLC) to detect genetic variants in the coding and flanking regions of the genes. Conditional logistic regression was used to compare the risk of CBC associated with chemotherapy and tamoxifen in BRCA1/BRCA2 mutation carriers and non-carriers. Chemotherapy was associated with lower CBC risk both in non-carriers (RR = 0.6 [95% CI: 0.5-0.7]) and carriers (RR = 0.5 [95% CI: 0.2-1.0]; P value = 0.04). Tamoxifen was associated with a reduced CBC risk in non-carriers (RR = 0.7 [95% CI: 0.6-1.0]; P value = 0.03). We observed a similar but non-significant reduction associated with tamoxifen in mutation carriers (RR = 0.7 [95% CI: 0.3-1.8]). The tests of heterogeneity comparing carriers to non-carriers did not provide evidence for a difference in the associations with chemotherapy (P value = 0.51) nor with tamoxifen (P value = 0.15). Overall, we did not observe a difference in the relative risk reduction associated with adjuvant treatment between BRCA1/BRCA2 mutation carriers and non-carriers. However, given the higher absolute CBC risk in mutation carriers, the potentially greater impact of adjuvant therapy in reducing CBC risk among mutation carriers should be considered when developing treatment plans for these patients.

Objective: Reproductive factors, such as early age at menarche, late age at menopause, and nulliparity are known risk factors for breast cancer. Previously, we reported these factors to be associated with risk of developing contralateral breast cancer (CBC). In this study, we evaluated the association between these factors and CBC risk among BRCA1 and BRCA2 (BRCA1/2) mutation carriers and non-carriers., Methods: The WECARE Study is a population-based multi-center case-control study of 705 women with CBC (cases) and 1,397 women with unilateral breast cancer (controls). All participants were screened for BRCA1/2 mutations and 181 carriers were identified. Conditional logistic regression models were used to evaluate associations between reproductive factors and CBC for mutation carriers and non-carriers., Results: None of the associations between reproductive factors and CBC risk differed between mutation carriers and non-carriers. The increase in risk with younger age at menarche and decrease in risk in women with more than two full-term pregnancies seen in non-carriers were not significantly different in carriers (adjusted RRs = 1.31, 95% CI 0.65-2.65 and 0.53, 95% CI 0.19-1.51, respectively). No significant associations between the other reproductive factors and CBC risk were observed in mutation carriers or non-carriers., Conclusion: For two reproductive factors previously shown to be associated with CBC risk, we observed similar associations for BRCA1/2 carriers. This suggests that reproductive variables that affect CBC risk may have similar effects in mutation carriers and non-carriers.

BRCA1 and BRCA2 screening in women at high-risk of breast cancer results in the identification of both unambiguously defined deleterious mutations and sequence variants of unknown clinical significance (VUS). We examined a population-based sample of young women with contralateral breast cancer (CBC, n=705) or unilateral breast cancer (UBC, n=1398). We identified 470 unique sequence variants, of which 113 were deleterious mutations. The remaining 357 VUS comprised 185 unique missense changes, 60% were observed only once, while 3% occurred with a frequency of >10%. Deleterious mutations occurred three times more often in women with CBC (15.3%) than in women with UBC (5.2%), whereas combined, VUS were observed in similar frequencies in women with CBC and UBC. A protein alignment algorithm defined 16 rare VUS, occurring at highly conserved residues and/or conferring a considerable biochemical difference, the majority located in the BRCA2 DNA-binding domain. We confirm a multiplicity of BRCA1 and BRCA2 VUS that occur at a wide range of allele frequencies. Although some VUS inflict chemical differences at conserved residues, suggesting a deleterious effect, the majority are not associated with an increased risk of CBC., ((c) 2010 Wiley-Liss, Inc.)

Disease-predisposing germline mutations in cancer susceptibility genes may consist of large genomic rearrangements that are challenging to detect and characterize using standard PCR-based mutation screening methods. Here, we describe a custom-made zoom-in microarray comparative genomic hybridization (CGH) platform of 60mer oligonucleotides. The 4 x 44 K array format provides high-resolution coverage (200-300 bp) of 400-700 kb genomic regions surrounding six cancer susceptibility genes. We evaluate its performance to accurately detect and precisely map earlier described or novel large germline deletions or duplications occurring in BRCA1 (n=11), BRCA2 (n=2), MSH2 (n=7), or MLH1 (n=9). Additionally, we demonstrate its applicability for uncovering complex somatic rearrangements, exemplified by zoom-in analysis of the PTEN and CDKN2A loci in breast cancer cells. The sizes of rearrangements ranged from several 100 kb, including large flanking regions, to <500-bp deletions, including parts of single exons that would be missed by standard multiplex ligation-dependent probe amplification (MLPA) methods. Zoom-in CGH arrays accurately defined the borders of rearrangements, allowing convenient design of primers for sequence determination of the breakpoints. The array platform can be streamlined for a particular application, e.g., focusing on breast cancer susceptibility genes, with increased capacity using multiformat design, and represents a valuable new tool and complement for genetic screening in clinical diagnostics., (Copyright 2008 Wiley-Liss, Inc.)