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Abstract Background Up to 70% of suspected Lynch syndrome patients harboring MMR deficient tumors lack identifiable germline pathogenic variants in MMR genes, being referred to as Lynch‐like syndrome (LLS). Previous studies have reported biallelic somatic MMR inactivation in a variable range of LLS‐associated tumors. Moreover, translating tumor testing results into patient management remains controversial. Our aim is to assess the challenges associated with the implementation of tumoral MMR gene testing in routine workflows. Methods Here, we present the clinical characterization of 229 LLS patients. MMR gene testing was performed in 39 available tumors, and results were analyzed using two variant allele frequency (VAF) thresholds (≥5% and ≥10%). Results and Discussion More biallelic somatic events were identified at VAF ≥ 5% than ≥10% (35.9% vs. 25.6%), although the rate of nonconcordant results regarding immunohistochemical pattern increased (30.8% vs. 20.5%). Interpretation difficulties question the current utility of the identification of MMR somatic hits in the diagnostic algorithm of suspected LS cases.

Abstract Background Germline variants affecting the proofreading activity of polymerases epsilon and delta cause a hereditary cancer and adenomatous polyposis syndrome characterized by tumors with a high mutational burden and a specific mutational spectrum. In addition to the implementation of multiple pieces of evidence for the classification of gene variants, POLE and POLD1 variant classification is particularly challenging given that non-disruptive variants affecting the proofreading activity of the corresponding polymerase are the ones associated with cancer. In response to an evident need in the field, we have developed gene-specific variant classification recommendations, based on the ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology) criteria, for the assessment of non-disruptive variants located in the sequence coding for the exonuclease domain of the polymerases. Methods A training set of 23 variants considered pathogenic or benign was used to define the usability and strength of the ACMG/AMP criteria. Population frequencies, computational predictions, co-segregation data, phenotypic and tumor data, and functional results, among other features, were considered. Results Gene-specific variant classification recommendations for non-disruptive variants located in the exonuclease domain of POLE and POLD1 were defined. The resulting recommendations were applied to 128 exonuclease domain variants reported in the literature and/or public databases. A total of 17 variants were classified as pathogenic or likely pathogenic, and 17 as benign or likely benign. Conclusions Our recommendations, with room for improvement in the coming years as more information become available on carrier families, tumor molecular characteristics and functional assays, are intended to serve the clinical and scientific communities and help improve diagnostic performance, avoiding variant misclassifications.

Abstract Case–control studies have shown an association of BARD1 with hereditary breast and/or ovarian cancer (HBOC) predisposition. BARD1 alternatively spliced isoforms are abundant and some are highly expressed in different cancer types. In addition, a number of BARD1 germline pathogenic variants have been reported among HBOC patients. In previous reports, BARD1 c.1977A>G variant has been classified as pathogenic since it produces a frameshift transcript lacking exons 2 to 9. In the present study, we sought to validate the mRNA splicing results previously published and to contribute with new evidence to refine the classification of this substitution according to ACMG/AMP guidelines. The presence of the variant was screened in patients and controls. RT-PCR was performed in order to compare the transcriptional profiles of two variant carriers and ten non-carrier controls. In addition, allele-specific expression was assessed. No differences in variant frequency were detected between patients and controls. The RNA assay confirmed the presence of the shorter transcript lacking exons 2–9, but it was detected both in carriers and non-carriers. Furthermore, allelic imbalance was discarded and no significant differences in the proportion of full-length and shorter transcript were detected between carriers and controls. The shorter transcript detected corresponds to BARD1 isoform η, constituted by exons 1, 10 and 11. Our results support that this transcript is a constitutive splicing product rather than an aberrant transcript caused by BARD1 c.1977A>G variant, and for this reason this variant should be considered as likely benign following ACMG/AMP guidelines.

Abstract Individuals with Lynch syndrome (LS), have an increased risk of developing cancer. Common genetic variants of telomerase reverse transcriptase (TERT) have been associated with a wide range of cancers, including colorectal cancer (CRC) in LS. We combined genotype data from 1881 LS patients, carrying pathogenic variants in MLH1, MSH2 or MSH6, for rs2075786 (G>A, intronic variant), 1207 LS patients for rs2736108 (C>T, upstream variant) and 1201 LS patients for rs7705526 (C>A, intronic variant). The risk of cancer was estimated by heterozygous/homozygous odds ratio (OR) with mixed-effects logistic regression to adjust for gene/gender/country of sample origin considering family identity. The AA genotype of SNP rs2075786 is associated with 85% higher odds at developing cancer compared to GG genotype in MSH2 pathogenic variant carriers (p = 0.0160). Kaplan–Meier analysis also shows an association for rs2075786; the AA allele for MSH2 variant carriers confers risk for earlier diagnosis of LS cancer (log-rank p = 0.0011). We report a polymorphism in TERT to be a possible modifier of disease risk in MSH2 pathogenic variant carriers. The rs2075786 SNP in TERT is associated with a differential risk of developing cancer for MSH2 pathogenic variant carriers. Use of this information has the potential to personalise screening protocols for LS patients.

Background VCN-01 is an oncolytic adenovirus (Ad5 based) designed to replicate in cancer cells with dysfunctional RB1 pathway, express hyaluronidase to enhance virus intratumoral spread and facilitate chemotherapy and immune cells extravasation into the tumor. This phase I clinical trial was aimed to find the maximum tolerated dose/recommended phase II dose (RP2D) and dose-limiting toxicity (DLT) of the intravenous delivery of the replication-competent VCN-01 adenovirus in patients with advanced cancer.Methods Part I: patients with advanced refractory solid tumors received one single dose of VCN-01. Parts II and III: patients with pancreatic adenocarcinoma received VCN-01 (only in cycle 1) and nab-paclitaxel plus gemcitabine (VCN-concurrent on day 1 in Part II, and 7 days before chemotherapy in Part III). Patients were required to have anti-Ad5 neutralizing antibody (NAbs) titers lower than 1/350 dilution. Pharmacokinetic and pharmacodynamic analyses were performed.Results 26% of the patients initially screened were excluded based on high NAbs levels. Sixteen and 12 patients were enrolled in Part I and II, respectively: RP2D were 1×1013 viral particles (vp)/patient (Part I), and 3.3×1012 vp/patient (Part II). Fourteen patients were included in Part III: there were no DLTs and the RP2D was 1×1013 vp/patient. Observed DLTs were grade 4 aspartate aminotransferase increase in one patient (Part I, 1×1013 vp), grade 4 febrile neutropenia in one patient and grade 5 thrombocytopenia plus enterocolitis in another patient (Part II, 1×1013 vp). In patients with pancreatic adenocarcinoma overall response rate were 50% (Part II) and 50% (Part III). VCN-01 viral genomes were detected in tumor tissue in five out of six biopsies (day 8). A second viral plasmatic peak and increased hyaluronidase serum levels suggested replication after intravenous injection in all patients. Increased levels of immune biomarkers (interferon-γ, soluble lymphocyte activation gene-3, interleukin (IL)-6, IL-10) were found after VCN-01 administration.Conclusions Treatment with VCN-01 is feasible and has an acceptable safety. Encouraging biological and clinical activity was observed when administered in combination with nab-paclitaxel plus gemcitabine to patients with pancreatic adenocarcinoma.Trial registration number NCT02045602.

IntroductionLoss-of-function TLR7 variants have been recently reported in a small number of males to underlie strong predisposition to severe COVID-19. We aimed to determine the presence of these rare variants in young men with severe COVID-19.MethodsWe prospectively studied males between 18 and 50 years-old without predisposing comorbidities that required at least high-flow nasal oxygen to treat COVID-19. The coding region of TLR7 was sequenced to assess the presence of potentially deleterious variants.ResultsTLR7 missense variants were identified in two out of 14 patients (14.3%). Overall, the median age was 38 (IQR 30-45) years. Both variants were not previously reported in population control databases and were predicted to be damaging by in silico predictors. In a 30-year-old patient a maternally inherited variant [c.644A>G; p.(Asn215Ser)] was identified, co-segregating in his 27-year-old brother who also contracted severe COVID-19. A second variant [c.2797T>C; p.(Trp933Arg)] was found in a 28-year-old patient, co-segregating in his 24-year-old brother who developed mild COVID-19. Functional testing of this variant revealed decreased type I and II interferon responses in peripheral mononuclear blood cells upon stimulation with the TLR7 agonist imiquimod, confirming a loss-of-function effect.ConclusionsThis study supports a rationale for the genetic screening for TLR7 variants in young men with severe COVID-19 in the absence of other relevant risk factors. A diagnosis of TLR7 deficiency could not only inform on treatment options for the patient, but also enables pre-symptomatic testing of at-risk male relatives with the possibility of instituting early preventive and therapeutic interventions.

MicroRNAs from the miR-200 family are commonly associated with the inhibition of the metastatic potential of cancer cells, following inhibition of ZEB transcription factors expression and epithelial-to-mesenchymal transition. However, previous studies performed in pancreatic adenocarcinoma revealed a more complex picture challenging this canonical model. To gain better insights into the role of miR-200 family members in this disease, we analyzed the expression of miR-200a, miR-200b, miR-200c, miR-141, miR-429, and miR-205, and ZEB1, ZEB2, and CDH1 in pancreatic tumors and matching normal adjacent parenchyma and patient-derived xenografts. We found that miR-200a, miR-429, and miR-205 are frequently overexpressed in pancreatic tumors, whereas CDH1 is downregulated, and ZEB1 and ZEB2 levels remain unchanged. Furthermore, we measured a positive correlation between miR-200 family members and CDH1 expression, and a negative correlation between ZEB1 and miR-200c, miR-141, and miR-205 expression, respectively. Interestingly, we identified significant changes in expression of epithelial-to-mesenchymal transition regulators and miR-200 members in patient-derived xenografts. Lastly, functional studies revealed that miR-141 and miR-429 inhibit the tumorigenic potential of pancreatic cancer cells. Taken together, this comprehensive analysis strongly suggests that miRNAs from the miR-200 family, and in particular miR-429, may act as a tumor suppressor gene in pancreatic cancer. Keywords: pancreatic cancer, patient-derived xenograft, miR-200 family, miR-429

The ALFRED (Allelic Loss Featuring Rare Damaging) in silico method was developed to identify cancer predisposition genes through the identification of somatic second hits. By applying ALFRED to ~10,000 tumor exomes, 49 candidate genes were identified. We aimed to assess the causal association of the identified genes with colorectal cancer (CRC) predisposition. Of the 49 genes, NSD1, HDAC10, KRT24, ACACA and TP63 were selected based on specific criteria relevant for hereditary CRC genes. Gene sequencing was performed in 736 patients with familial/early onset CRC or polyposis without germline pathogenic variants in known genes. Twelve (predicted) damaging variants in 18 patients were identified. A gene-based burden test in 1596 familial/early-onset CRC patients, 271 polyposis patients, 543 TCGA CRC patients and >134,000 controls (gnomAD, non-cancer), revealed no clear association with CRC for any of the studied genes. Nevertheless, (non-significant) over-representation of disruptive variants in NSD1, KRT24 and ACACA in CRC patients compared to controls was observed. A somatic second hit was identified in one of 20 tumors tested, corresponding to an NSD1 carrier. In conclusion, most genes identified through the ALFRED in silico method were not relevant for CRC predisposition, although a possible association was detected for NSD1, KRT24 and ACACA.

Abstract Germline mutations in BUB1 and BUB3 have been reported to increase the risk of developing colorectal cancer (CRC) at young age, in presence of variegated aneuploidy and reminiscent dysmorphic traits of mosaic variegated aneuploidy syndrome. We performed a mutational analysis of BUB1 and BUB3 in 456 uncharacterized mismatch repair-proficient hereditary non-polyposis CRC families and 88 polyposis cases. Four novel or rare germline variants, one splice-site and three missense, were identified in four families. Neither variegated aneuploidy nor dysmorphic traits were observed in carriers. Evident functional effects in the heterozygous form were observed for c.1965-1G>A, but not for c.2296G>A (p.E766K), in spite of the positive co-segregation in the family. BUB1 c.2473C>T (p.P825S) and BUB3 c.77C>T (p.T26I) remained as variants of uncertain significance. As of today, the rarity of functionally relevant mutations identified in familial and/or early onset series does not support the inclusion of BUB1 and BUB3 testing in routine genetic diagnostics of familial CRC.

A large proportion of familial and/or early-onset cancer patients do not carry pathogenic variants in known cancer predisposing genes. We aimed to assess the contribution of previously validated low-risk colorectal cancer (CRC) alleles to familial/early-onset CRC (fCRC) and to serrated polyposis. We estimated the association of CRC with a 92-variant-based weighted polygenic risk score (wPRS) using 417 fCRC patients, 80 serrated polyposis patients, 1077 hospital-based incident CRC patients, and 1642 controls. The mean wPRS was significantly higher in fCRC than in controls or sporadic CRC patients. fCRC patients in the highest (20th) wPRS quantile were at four-fold greater CRC risk than those in the middle quantile (10th). Compared to low-wPRS fCRC, a higher number of high-wPRS fCRC patients had developed multiple primary CRCs, had CRC family history, and were diagnosed at age ≥50. No association with wPRS was observed for serrated polyposis. In conclusion, a relevant proportion of mismatch repair (MMR)-proficient fCRC cases might be explained by the accumulation of low-risk CRC alleles. Validation in independent cohorts and development of predictive models that include polygenic risk score (PRS) data and other CRC predisposing factors will determine the implementation of PRS into genetic testing and counselling in familial and early-onset CRC.

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Only a small fraction of hereditary breast and/or ovarian cancer (HBOC) cases are caused by germline variants in the high-penetrance breast cancer 1 and 2 genes (BRCA1 and BRCA2). BRCA1-associated ring domain 1 (BARD1), nuclear partner of BRCA1, has been suggested as a potential HBOC risk gene, although its prevalence and penetrance are variable according to populations and type of tumor. We aimed to investigate the prevalence of BARD1 truncating variants in a cohort of patients with clinical suspicion of HBOC. A comprehensive BARD1 screening by multigene panel analysis was performed in 4015 unrelated patients according to our regional guidelines for genetic testing in hereditary cancer. In addition, 51,202 Genome Aggregation Database (gnomAD) non-Finnish, non-cancer European individuals were used as a control population. In our patient cohort, we identified 19 patients with heterozygous BARD1 truncating variants (0.47%), whereas the frequency observed in the gnomAD controls was 0.12%. We found a statistically significant association of truncating BARD1 variants with overall risk (odds ratio (OR) = 3.78; CI = 2.10–6.48; p = 1.16 × 10−5). This association remained significant in the hereditary breast cancer (HBC) group (OR = 4.18; CI = 2.10–7.70; p = 5.45 × 10−5). Furthermore, deleterious BARD1 variants were enriched among triple-negative BC patients (OR = 5.40; CI = 1.77–18.15; p = 0.001) compared to other BC subtypes. Our results support the role of BARD1 as a moderate penetrance BC predisposing gene and highlight a stronger association with triple-negative tumors.

Background: Colorectal (CRC) and endometrial cancer (EC) are the most common types of cancer in Lynch syndrome (LS). Risk reducing surgeries (RRS) might impact cancer incidence and mortality. Our objectives were to evaluate cumulative incidences of CRC, gynecological cancer and all-cause mortality after RRS in LS individuals. Methods: Retrospective analysis of 976 LS carriers from a single-institution registry. Primary endpoints were cumulative incidence at 75 years of cancer (metachronous CRC in 425 individuals; EC and ovarian cancer (OC) in 531 individuals) and all-cause mortality cumulative incidence, comparing extended (ES) vs. segmental surgery (SS) in the CRC cohort and risk reducing gynecological surgery (RRGS) vs. surveillance in the gynecological cohort. Results: Cumulative incidence at 75 years of metachronous CRC was 12.5% vs. 44.7% (p = 0.04) and all-cause mortality cumulative incidence was 38.6% vs. 55.3% (p = 0.31), for ES and SS, respectively. Cumulative, incidence at 75 years was 11.2% vs. 46.3% for EC (p = 0.001) and 0% vs. 12.7% for OC (p N/A) and all-cause mortality cumulative incidence was 0% vs. 52.7% (p N/A), for RRGS vs. surveillance, respectively. Conclusions: RRS in LS reduces the incidence of metachronous CRC and gynecological neoplasms, also indicating a reduction in all-cause mortality cumulative incidence in females undergoing RRGS.

The causal mechanism for cancer predisposition in Lynch-like syndrome (LLS) remains unknown. Our aim was to elucidate the constitutional basis of mismatch repair (MMR) deficiency in LLS patients throughout a comprehensive (epi)genetic analysis. One hundred and fifteen LLS patients harboring MMR-deficient tumors and no germline MMR mutations were included. Mutational analysis of 26 colorectal cancer (CRC)-associated genes was performed. Pathogenicity of MMR variants was assessed by splicing and multifactorial likelihood analyses. Genome-wide methylome analysis was performed by the Infinium Human Methylation 450K Bead Chip. The multigene panel analysis revealed the presence of two MMR gene truncating mutations not previously found. Of a total of 15 additional MMR variants identified, five -present in 6 unrelated individuals- were reclassified as pathogenic. In addition, 13 predicted deleterious variants in other CRC-predisposing genes were found in 12 probands. Methylome analysis detected one constitutional MLH1 epimutation, but no additional differentially methylated regions were identified in LLS compared to LS patients or cancer-free individuals. In conclusion, the use of an ad-hoc designed gene panel combined with pathogenicity assessment of variants allowed the identification of deleterious MMR mutations as well as new LLS candidate causal genes. Constitutional epimutations in non-LS-associated genes are not responsible for LLS.

In the past two decades, multiple studies have been undertaken to elucidate the genetic cause of the predisposition to mismatch repair (MMR)-proficient nonpolyposis colorectal cancer (CRC). Here, we present the proposed candidate genes according to their involvement in specific pathways considered relevant in hereditary CRC and/or colorectal carcinogenesis. To date, only pathogenic variants in RPS20 may be convincedly linked to hereditary CRC. Nevertheless, accumulated evidence supports the involvement in the CRC predisposition of other genes, including MRE11, BARD1, POT1, BUB1B, POLE2, BRF1, IL12RB1, PTPN12, or the epigenetic alteration of PTPRJ. The contribution of the identified candidate genes to familial/early onset MMR-proficient nonpolyposis CRC, if any, is extremely small, suggesting that other factors, such as the accumulation of low risk CRC alleles, shared environmental exposures, and/or gene–environmental interactions, may explain the missing heritability in CRC.

Fanconi anemia (FA) is caused by biallelic mutations in FA genes. Monoallelic mutations in five of these genes (BRCA1, BRCA2, PALB2, BRIP1 and RAD51C) increase the susceptibility to breast/ovarian cancer and are used in clinical diagnostics as bona-fide hereditary cancer genes. Increasing evidence suggests that monoallelic mutations in other FA genes could predispose to tumor development, especially breast cancer. The objective of this study is to assess the mutational spectrum of 14 additional FA genes (FANCA, FANCB, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCL, FANCM, FANCP, FANCQ, FANCR and FANCU) in a cohort of hereditary cancer patients, to compare with local cancer-free controls as well as GnomAD. A total of 1021 hereditary cancer patients and 194 controls were analyzed using our next generation custom sequencing panel. We identified 35 pathogenic variants in eight genes. A significant association with the risk of breast cancer/breast and ovarian cancer was found for carriers of FANCA mutations (odds ratio (OR) = 3.14 95% confidence interval (CI) 1.4–6.17, p = 0.003). Two patients with early-onset cancer showed a pathogenic FA variant in addition to another germline mutation, suggesting a modifier role for FA variants. Our results encourage a comprehensive analysis of FA genes in larger studies to better assess their role in cancer risk.

In silico tools for splicing defect prediction have a key role to assess the impact of variants of uncertain significance. Our aim was to evaluate the performance of a set of commonly used splicing in silico tools comparing the predictions against RNA in vitro results. This was done for natural splice sites of clinically relevant genes in hereditary breast/ovarian cancer (HBOC) and Lynch syndrome. A study divided into two stages was used to evaluate SSF-like, MaxEntScan, NNSplice, HSF, SPANR, and dbscSNV tools. A discovery dataset of 99 variants with unequivocal results of RNA in vitro studies, located in the 10 exonic and 20 intronic nucleotides adjacent to exon–intron boundaries of BRCA1, BRCA2, MLH1, MSH2, MSH6, PMS2, ATM, BRIP1, CDH1, PALB2, PTEN, RAD51D, STK11, and TP53, was collected from four Spanish cancer genetic laboratories. The best stand-alone predictors or combinations were validated with a set of 346 variants in the same genes with clear splicing outcomes reported in the literature. Sensitivity, specificity, accuracy, negative predictive value (NPV) and Mathews Coefficient Correlation (MCC) scores were used to measure the performance. The discovery stage showed that HSF and SSF-like were the most accurate for variants at the donor and acceptor region, respectively. The further combination analysis revealed that HSF, HSF+SSF-like or HSF+SSF-like+MES achieved a high performance for predicting the disruption of donor sites, and SSF-like or a sequential combination of MES and SSF-like for predicting disruption of acceptor sites. The performance confirmation of these last results with the validation dataset, indicated that the highest sensitivity, accuracy, and NPV (99.44%, 99.44%, and 96.88, respectively) were attained with HSF+SSF-like or HSF+SSF-like+MES for donor sites and SSF-like (92.63%, 92.65%, and 84.44, respectively) for acceptor sites.We provide recommendations for combining algorithms to conduct in silico splicing analysis that achieved a high performance. The high NPV obtained allows to select the variants in which the study by in vitro RNA analysis is mandatory against those with a negligible probability of being spliceogenic. Our study also shows that the performance of each specific predictor varies depending on whether the natural splicing sites are donors or acceptors.

Abstract Malignant peripheral nerve sheath tumors (MPNSTs) are soft‐tissue sarcomas that can arise either sporadically or in association with neurofibromatosis type 1 (NF1). These aggressive malignancies confer poor survival, with no effective therapy available. We present the generation and characterization of five distinct MPNST orthoxenograft models for preclinical testing and personalized medicine. Four of the models are patient‐derived tumor xenografts (PDTX), two independent MPNSTs from the same NF1 patient and two from different sporadic patients. The fifth model is an orthoxenograft derived from an NF1‐related MPNST cell line. All MPNST orthoxenografts were generated by tumor implantation, or cell line injection, next to the sciatic nerve of nude mice, and were perpetuated by 7–10 mouse‐to‐mouse passages. The models reliably recapitulate the histopathological properties of their parental primary tumors. They also mimic distal dissemination properties in mice. Human stroma was rapidly lost after MPNST engraftment and replaced by murine stroma, which facilitated genomic tumor characterization. Compatible with an origin in a catastrophic event and subsequent genome stabilization, MPNST contained highly altered genomes that remained remarkably stable in orthoxenograft establishment and along passages. Mutational frequency and type of somatic point mutations were highly variable among the different MPNSTs modeled, but very consistent when comparing primary tumors with matched orthoxenografts generated. Unsupervised cluster analysis and principal component analysis (PCA) using an MPNST expression signature of ~1,000 genes grouped together all primary tumor–orthoxenograft pairs. Our work points to differences in the engraftment process of primary tumors compared with the engraftment of established cell lines. Following standardization and extensive characterization and validation, the orthoxenograft models were used for initial preclinical drug testing. Sorafenib (a BRAF inhibitor), in combination with doxorubicin or rapamycin, was found to be the most effective treatment for reducing MPNST growth. The development of genomically well‐characterized preclinical models for MPNST allowed the evaluation of novel therapeutic strategies for personalized medicine.

Aberrant telomere length measured in blood has been associated with increased risk of several cancer types. In the field of hereditary non-polyposis colorectal cancer (CRC), and more particularly in Lynch syndrome, caused by germline mutations in the mismatch repair (MMR) genes, we recently found that cancer-affected MMR gene mutation carriers had shorter telomeres and more pronounced shortening of telomere length with age than controls and unaffected MMR gene mutation carriers. Here we evaluate blood telomere length in MMR-proficient hereditary non-polyposis CRC, i.e. familial CRC type X (fCRC-X). A total of 57 cancer-affected and 57 cancer-free individuals from 34 Amsterdam-positive fCRC-X families were analyzed and compared to the data previously published on 144 cancer-affected and 100 cancer-free MMR gene mutation carriers, and 234 controls. Relative telomere length was measured using a monochrome multiplex quantitative PCR method, following strict measures to avoid sources of bias and adjusting by age. Despite the retrospective nature of our study, the results show that longer telomeres associate with cancer risk in fCRC-X, thus identifying different patterns of telomere length according to the status of the MMR system.

Telomere length variation has been associated with increased risk of several types of tumors, and telomere shortening, with genetic anticipation in a number of genetic diseases including hereditary cancer syndromes. No conclusive studies have been performed for Lynch syndrome, a hereditary colorectal cancer syndrome caused by germline mutations in the DNA mismatch repair genes. Here we evaluate telomere length in Lynch syndrome, both as a cancer risk factor and as a mechanism associated with anticipation in the age of cancer onset observed in successive generations of Lynch syndrome families. Leukocyte telomere length was measured in 244 mismatch repair gene mutation carriers from 96 Lynch syndrome families and in 234 controls using a monochrome multiplex quantitative PCR method. Cancer-affected mutation carriers showed significantly shorter telomeres than cancer-free mutation carriers. In addition, cancer-affected carriers showed the most pronounced shortening of telomere length with age, compared with unaffected carriers. The anticipation in the age of cancer onset observed in successive generations was not associated with telomere shortening, although, interestingly, all mother-son pairs showed telomere shortening. In conclusion, cancer-affected mismatch repair gene mutation carriers have distinct telomere-length pattern and dynamics. However, anticipation in the age of onset is not explained by telomere shortening. Pending further study, our findings suggest that telomere attrition might explain the previously reported dependence of cancer risk on the parent-of-origin of mismatch repair gene mutations.

Germline inactivating mutations in BRCA1 and BRCA2 genes are responsible for Hereditary Breast and Ovarian Cancer Syndrome (HBOCS). Genetic testing of these genes is available, although approximately 15% of tests identify variants of uncertain significance (VUS). Classification of these variants into pathogenic or non-pathogenic type is an important challenge in genetic diagnosis and counseling. The aim of the present study is to functionally assess a set of 7 missense VUS (Q1409L, S1473P, E1586G, R1589H, Y1703S, W1718L and G1770V) located in the C-terminal region of BRCA1 by combining in silico prediction tools and structural analysis with a transcription activation (TA) assay. The in silico prediction programs gave discrepant results making its interpretation difficult. Structural analysis of the three variants located in the BRCT domains (Y1703S, W1718L and G1770V) reveals significant alterations of BRCT structure. The TA assay shows that variants Y1703S, W1718L and G1770V dramatically compromise the transcriptional activity of BRCA1, while variants Q1409L, S1473P, E1586G and R1589H behave like wild-type BRCA1. In conclusion, our results suggest that variants Y1703S, W1718L and G1770V can be classified as likely pathogenic BRCA1 mutations.

Differentiated mammary epithelium shows apicobasal polarity, and loss of tissue organization is an early hallmark of breast carcinogenesis. In BRCA1 mutation carriers, accumulation of stem and progenitor cells in normal breast tissue and increased risk of developing tumors of basal-like type suggest that BRCA1 regulates stem/progenitor cell proliferation and differentiation. However, the function of BRCA1 in this process and its link to carcinogenesis remain unknown. Here we depict a molecular mechanism involving BRCA1 and RHAMM that regulates apicobasal polarity and, when perturbed, may increase risk of breast cancer. Starting from complementary genetic analyses across families and populations, we identified common genetic variation at the low-penetrance susceptibility HMMR locus (encoding for RHAMM) that modifies breast cancer risk among BRCA1, but probably not BRCA2, mutation carriers: n = 7,584, weighted hazard ratio ((w)HR) = 1.09 (95% CI 1.02-1.16), p(trend) = 0.017; and n = 3,965, (w)HR = 1.04 (95% CI 0.94-1.16), p(trend) = 0.43; respectively. Subsequently, studies of MCF10A apicobasal polarization revealed a central role for BRCA1 and RHAMM, together with AURKA and TPX2, in essential reorganization of microtubules. Mechanistically, reorganization is facilitated by BRCA1 and impaired by AURKA, which is regulated by negative feedback involving RHAMM and TPX2. Taken together, our data provide fundamental insight into apicobasal polarization through BRCA1 function, which may explain the expanded cell subsets and characteristic tumor type accompanying BRCA1 mutation, while also linking this process to sporadic breast cancer through perturbation of HMMR/RHAMM.

Gene expression profiling has identified cancer prognostic and predictive signatures with superior performance to conventional histopathological or clinical parameters. Consequently, signatures are being incorporated into clinical practice and will soon influence everyday decisions in oncology. However, the slight overlap in the gene identity between signatures for the same cancer type or condition raises questions about their biological and clinical implications. To clarify these issues, better understanding of the molecular properties and possible interactions underlying apparently dissimilar signatures is needed. Here, we evaluated whether the signatures of 24 independent studies are related at the genome, transcriptome or proteome levels. Significant associations were consistently observed across these molecular layers, which suggest the existence of a common cancer cell phenotype. Convergence on cell proliferation and death supports the pivotal involvement of these processes in prognosis, metastasis and treatment response. In addition, functional and molecular associations were identified with the immune response in different cancer types and conditions that complement the contribution of cell proliferation and death. Examination of additional, independent, cancer datasets corroborated our observations. This study proposes a comprehensive strategy for interpreting cancer signatures that reveals common design principles and systems-level properties.