Independent real-world application of a clinical-grade automated prostate cancer detection system.

Artificial intelligence (AI)-based systems applied to histopathology whole-slide images have the potential to improve patient care through mitigation of challenges posed by diagnostic variability, histopathology caseload, and shortage of pathologists. We sought to define the performance of an AI-based automated prostate cancer detection system, Paige Prostate, when applied to independent real-world data. The algorithm was employed to classify slides into two categories: benign (no further review needed) or suspicious (additional histologic and/or immunohistochemical analysis required). We assessed the sensitivity, specificity, positive predictive values (PPVs), and negative predictive values (NPVs) of a local pathologist, two central pathologists, and Paige Prostate in the diagnosis of 600 transrectal ultrasound-guided prostate needle core biopsy regions ('part-specimens') from 100 consecutive patients, and to ascertain the impact of Paige Prostate on diagnostic accuracy and efficiency. Paige Prostate displayed high sensitivity (0.99; CI 0.96-1.0), NPV (1.0; CI 0.98-1.0), and specificity (0.93; CI 0.90-0.96) at the part-specimen level. At the patient level, Paige Prostate displayed optimal sensitivity (1.0; CI 0.93-1.0) and NPV (1.0; CI 0.91-1.0) at a specificity of 0.78 (CI 0.64-0.89). The 27 part-specimens considered by Paige Prostate as suspicious, whose final diagnosis was benign, were found to comprise atrophy (n = 14), atrophy and apical prostate tissue (n = 1), apical/benign prostate tissue (n = 9), adenosis (n = 2), and post-atrophic hyperplasia (n = 1). Paige Prostate resulted in the identification of four additional patients whose diagnoses were upgraded from benign/suspicious to malignant. Additionally, this AI-based test provided an estimated 65.5% reduction of the diagnostic time for the material analyzed. Given its optimal sensitivity and NPV, Paige Prostate has the potential to be employed for the automated identification of patients whose histologic slides could forgo full histopathologic review. In addition to providing incremental improvements in diagnostic accuracy and efficiency, this AI-based system identified patients whose prostate cancers were not initially diagnosed by three experienced histopathologists. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Point Mutations in Exon 1B of APC Reveal Gastric Adenocarcinoma and Proximal Polyposis of the Stomach as a Familial Adenomatous Polyposis Variant.

Gastric adenocarcinoma and proximal polyposis of the stomach (GAPPS) is an autosomal-dominant cancer-predisposition syndrome with a significant risk of gastric, but not colorectal, adenocarcinoma. We mapped the gene to 5q22 and found loss of the wild-type allele on 5q in fundic gland polyps from affected individuals. Whole-exome and -genome sequencing failed to find causal mutations but, through Sanger sequencing, we identified point mutations in APC promoter 1B that co-segregated with disease in all six families. The mutations reduced binding of the YY1 transcription factor and impaired activity of the APC promoter 1B in luciferase assays. Analysis of blood and saliva from carriers showed allelic imbalance of APC, suggesting that these mutations lead to decreased allele-specific expression in vivo. Similar mutations in APC promoter 1B occur in rare families with familial adenomatous polyposis (FAP). Promoter 1A is methylated in GAPPS and sporadic FGPs and in normal stomach, which suggests that 1B transcripts are more important than 1A in gastric mucosa. This might explain why all known GAPPS-affected families carry promoter 1B point mutations but only rare FAP-affected families carry similar mutations, the colonic cells usually being protected by the expression of the 1A isoform. Gastric polyposis and cancer have been previously described in some FAP-affected individuals with large deletions around promoter 1B. Our finding that GAPPS is caused by point mutations in the same promoter suggests that families with mutations affecting the promoter 1B are at risk of gastric adenocarcinoma, regardless of whether or not colorectal polyps are present.

Mixed ductal-lobular carcinomas: evidence for progression from ductal to lobular morphology.

Mixed ductal-lobular carcinomas (MDLs) show both ductal and lobular morphology, and constitute an archetypal example of intratumoural morphological heterogeneity. The mechanisms underlying the coexistence of these different morphological entities are poorly understood, although theories include that these components either represent 'collision' of independent tumours or evolve from a common ancestor. We performed comprehensive clinicopathological analysis of a cohort of 82 MDLs, and found that: (1) MDLs more frequently coexist with ductal carcinoma in situ (DCIS) than with lobular carcinoma in situ (LCIS); (2) the E-cadherin-catenin complex was normal in the ductal component in 77.6% of tumours; and (3) in the lobular component, E-cadherin was almost always aberrantly located in the cytoplasm, in contrast to invasive lobular carcinoma (ILC), where E-cadherin is typically absent. Comparative genomic hybridization and multiregion whole exome sequencing of four representative cases revealed that all morphologically distinct components within an individual case were clonally related. The mutations identified varied between cases; those associated with a common clonal ancestry included BRCA2, TBX3, and TP53, whereas those associated with clonal divergence included CDH1 and ESR1. Together, these data support a model in which separate morphological components of MDLs arise from a common ancestor, and lobular morphology can arise via a ductal pathway of tumour progression. In MDLs that present with LCIS and DCIS, the clonal divergence probably occurs early, and is frequently associated with complete loss of E-cadherin expression, as in ILC, whereas, in the majority of MDLs, which present with DCIS but not LCIS, direct clonal divergence from the ductal to the lobular phenotype occurs late in tumour evolution, and is associated with aberrant expression of E-cadherin. The mechanisms driving the phenotypic change may involve E-cadherin-catenin complex deregulation, but are yet to be fully elucidated, as there is significant intertumoural heterogeneity, and each case may have a unique molecular mechanism. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

An epithelial to mesenchymal transition programme does not usually drive the phenotype of invasive lobular carcinomas.

Epithelial to mesenchymal transition (EMT) is a cellular phenotype switching phenomenon which occurs during normal development and is proposed to promote tumour cell invasive capabilities during tumour progression. Invasive lobular carcinoma (ILC) is a histological special type of breast cancer with a peculiar aetiology - the tumour cells display an invasive growth pattern, with detached, single cells or single files of cells, and a canonical feature is the loss of E-cadherin expression. These characteristics are indicative of an EMT or at the very least that they represent some plasticity between phenotypes. While some gene expression profiling data support this view, the tumour cells remain epithelial and limited immunohistochemistry data suggest that EMT markers may not feature prominently in ILC. We assessed the expression of a panel of EMT markers (fibronectin, vimentin, N-cadherin, smooth muscle actin, osteonectin, Snail, Twist) in 148 ILCs and performed a meta-analysis of publically available molecular data from 154 ILCs. Three out of 148 (2%) ILCs demonstrated an early and coordinated alteration of multiple EMT markers (down-regulation of E-cadherin, nuclear TWIST, and up-regulation of vimentin, osteonectin, and smooth muscle actin). However, the data overall do not support a role for EMT in defining the phenotypic peculiarities of the majority of ILCs. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Integrated genomic and transcriptomic analysis of human brain metastases identifies alterations of potential clinical significance.

Treatment options for patients with brain metastases (BMs) have limited efficacy and the mortality rate is virtually 100%. Targeted therapy is critically under-utilized, and our understanding of mechanisms underpinning metastatic outgrowth in the brain is limited. To address these deficiencies, we investigated the genomic and transcriptomic landscapes of 36 BMs from breast, lung, melanoma and oesophageal cancers, using DNA copy-number analysis and exome- and RNA-sequencing. The key findings were as follows. (a) Identification of novel candidates with possible roles in BM development, including the significantly mutated genes DSC2, ST7, PIK3R1 and SMC5, and the DNA repair, ERBB-HER signalling, axon guidance and protein kinase-A signalling pathways. (b) Mutational signature analysis was applied to successfully identify the primary cancer type for two BMs with unknown origins. (c) Actionable genomic alterations were identified in 31/36 BMs (86%); in one case we retrospectively identified ERBB2 amplification representing apparent HER2 status conversion, then confirmed progressive enrichment for HER2-positivity across four consecutive metastatic deposits by IHC and SISH, resulting in the deployment of HER2-targeted therapy for the patient. (d) In the ERBB/HER pathway, ERBB2 expression correlated with ERBB3 (r(2) = 0.496; p < 0.0001) and HER3 and HER4 were frequently activated in an independent cohort of 167 archival BM from seven primary cancer types: 57.6% and 52.6% of cases were phospho-HER3(Y1222) or phospho-HER4(Y1162) membrane-positive, respectively. The HER3 ligands NRG1/2 were barely detectable by RNAseq, with NRG1 (8p12) genomic loss in 63.6% breast cancer-BMs, suggesting a microenvironmental source of ligand. In summary, this is the first study to characterize the genomic landscapes of BM. The data revealed novel candidates, potential clinical applications for genomic profiling of resectable BMs, and highlighted the possibility of therapeutically targeting HER3, which is broadly over-expressed and activated in BMs, independent of primary site and systemic therapy.

In situ carcinoma - can we predict which patient will come back with a recurrence?

The frequency of in situ carcinomas has been rising since the introduction of mammographic screening. The management of patients with preinvasive disease remains difficult due to our lack of ability to accurately predict which patients will recur and progress to invasive carcinoma. Although some factors, such as lesion size and extent of margin clearance, are strong predictors of recurrence, many patients are still under- or overtreated. In this issue of Cancer Cell, Gauthier and colleagues suggest that abrogated response to cell stress measured by analysis of p16 and the proliferation marker Ki67 accurately predicts recurrence in ductal carcinoma in situ.

Crossing the Andes: Challenges and opportunities for digital pathology in Latin America.

The most widely accepted and used type of digital pathology (DP) is whole-slide imaging (WSI). The USFDA granted two WSI system approvals for primary diagnosis, the first in 2017. In Latin America, DP has the potential to reshape healthcare by enhancing diagnostic capabilities through artificial intelligence (AI) and standardizing pathology reports. Yet, we must tackle regulatory hurdles, training, resource availability, and unique challenges to the region. Collectively addressing these hurdles can enable the region to harness DP's advantages-enhancing disease diagnosis, medical research, and healthcare accessibility for its population. Americas Health Foundation assembled a panel of Latin American pathologists who are experts in DP to assess the hurdles to implementing it into pathologists' workflows in the region and provide recommendations for overcoming them. Some key steps recommended include creating a Latin American Society of Digital Pathology to provide continuing education, developing AI models trained on the Latin American population, establishing national regulatory frameworks for protecting the data, and standardizing formats for DP images to ensure that pathologists can collaborate and validate specimens across the various DP platforms.

Claudin 18.2 as a New Biomarker in Gastric Cancer-What Should We Know?

Gastric cancer (GC) remains a formidable global health challenge, ranking among the top-five causes of cancer-related deaths worldwide. The majority of patients face advanced stages at diagnosis, with a mere 6% five-year survival rate. First-line treatment for metastatic GC typically involves a fluoropyrimidine and platinum agent combination; yet, predictive molecular markers have proven elusive. This review navigates the evolving landscape of GC biomarkers, with a specific focus on Claudin 18.2 (CLDN18.2) as an emerging and promising target. Recent phase III trials have unveiled the efficacy of Zolbetuximab, a CLDN18.2-targeting antibody, in combination with oxaliplatin-based chemotherapy for CLDN18.2-positive metastatic GC. As this novel therapeutic avenue unfolds, understanding the nuanced decision making regarding the selection of anti-CLDN18.2 therapies over other targeted agents in metastatic GC becomes crucial. This manuscript reviews the evolving role of CLDN18.2 as a biomarker in GC and explores the current status of CLDN18.2-targeting agents in clinical development. The aim is to provide concise insights into the potential of CLDN18.2 as a therapeutic target and guide future clinical decisions in the management of metastatic GC.

DNA methylome of familial breast cancer identifies distinct profiles defined by mutation status.

It is now understood that epigenetic alterations occur frequently in sporadic breast carcinogenesis, but little is known about the epigenetic alterations associated with familial breast tumors. We performed genome-wide DNA-methylation profiling on familial breast cancers (n = 33) to identify patterns of methylation specific to the different mutation groups (BRCA1, BRCA2, and BRCAx) or intrinsic subtypes of breast cancer (basal, luminal A, luminal B, HER2-amplified, and normal-like). We used methylated DNA immunoprecipitation (MeDIP) on Affymetrix promoter chips to interrogate methylation profiles across 25,500 distinct transcripts. Using a support vector machine classification algorithm, we demonstrated that genome-wide methylation profiles predicted tumor mutation status with estimated error rates of 19% (BRCA1), 31% (BRCA2), and 36% (BRCAx) but did not accurately predict the intrinsic subtypes defined by gene expression. Furthermore, using unsupervised hierarchical clustering, we identified a distinct subgroup of BRCAx tumors defined by methylation profiles. We validated these findings in the 33 tumors in the test set, as well as in an independent validation set of 47 formalin-fixed, paraffin-embedded familial breast tumors, by pyrosequencing and Epityper. Finally, gene-expression profiling and SNP CGH array previously performed on the same samples allowed full integration of methylation, gene-expression, and copy-number data sets, revealing frequent hypermethylation of genes that also displayed loss of heterozygosity, as well as of genes that show copy-number gains, providing a potential mechanism for expression dosage compensation. Together, these data show that methylation profiles for familial breast cancers are defined by the mutation status and are distinct from the intrinsic subtypes.

Complete Response to Immunotherapy in a Patient with MUTYH-Associated Polyposis and Gastric Cancer: A Case Report.

MUTYH-associated polyposis syndrome is an uncommon, autosomal recessive colorectal polyposis syndrome caused by biallelic inactivation of MUTYH. Most patients present with multiple colorectal polyps. However, other primary tumor sites have been described as less frequent. In this report, we describe the case of a young patient with a germline biallelic pathogenic MUTYH mutation with three different primary tumors. We focused on a metastatic gastric adenocarcinoma that presented with complete bowel obstruction secondary to extensive peritoneal carcinomatosis and achieved complete response upon treatment with immunotherapy. The patient's tumor presented with a high tumor mutational burden and a 100% combined positive score, which certainly contributed to the complete response to immunotherapy. To date, no studies have described the association of MUTYH-related tumors with high PD-L1 expression, but we hypothesized that it may be linked to the increased antigenicity of these cancers.

Gene expression profiling of tumour epithelial and stromal compartments during breast cancer progression.

The progression of ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) marks a critical step in the evolution of breast cancer. There is some evidence to suggest that dynamic interactions between the neoplastic cells and the tumour microenvironment play an important role. Using the whole-genome cDNA-mediated annealing, selection, extension and ligation assay (WG-DASL, Illumina), we performed gene expression profiling on 87 formalin-fixed paraffin-embedded (FFPE) samples from 17 patients consisting of matched IDC, DCIS and three types of stroma: IDC-S (<3 mm from IDC), DCIS-S (<3 mm from DCIS) and breast cancer associated-normal stroma (BC-NS; >10 mm from IDC or DCIS). Differential gene expression analysis was validated by quantitative real time-PCR, immunohistochemistry and immunofluorescence. The expression of several genes was down-regulated in stroma from cancer patients relative to normal stroma from reduction mammoplasties. In contrast, neoplastic epithelium underwent more gene expression changes during progression, including down regulation of SFRP1. In particular, we observed that molecules related to extracellular matrix (ECM) remodelling (e.g. COL11A1, COL5A2 and MMP13) were differentially expressed between DCIS and IDC. COL11A1 was overexpressed in IDC relative to DCIS and was expressed by both the epithelial and stromal compartments but was enriched in invading neoplastic epithelial cells. The contributions of both the epithelial and stromal compartments to the clinically important scenario of progression from DCIS to IDC. Gene expression profiles, we identified differential expression of genes related to ECM remodelling, and specifically the elevated expression of genes such as COL11A1, COL5A2 and MMP13 in epithelial cells of IDC. We propose that these expression changes could be involved in facilitating the transition from in situ disease to invasive cancer and may thus mark a critical point in disease development.

CT-X antigen expression in human breast cancer.

Cancer/testis (CT) genes are predominantly expressed in human germ line cells, but not somatic tissues, and frequently become activated in different cancer types. Several CT antigens have already proved to be useful biomarkers and are promising targets for therapeutic cancer vaccines. The aim of the present study was to investigate the expression of CT antigens in breast cancer. Using previously generated massively parallel signature sequencing (MPSS) data, together with 9 publicly available gene expression datasets, the expression pattern of CT antigens located on the X chromosome (CT-X) was interrogated. Whereas a minority of unselected breast cancers was found to contain CT-X transcripts, a significantly higher expression frequency was detected in estrogen and progesterone receptor (ER) negative breast cancer cell lines and primary breast carcinomas. A coordinated pattern of CT-X antigen expression was observed, with MAGEA and NY-ESO-1/CTAG1B being the most prevalent antigens. Immunohistochemical staining confirmed the correlation of CT-X antigen expression and ER negativity in breast tumors and demonstrated a trend for their coexpression with basal cell markers. Because of the limited therapeutic options for ER-negative breast cancers, vaccines based on CT-X antigens might prove to be useful.

Splicing and multifactorial analysis of intronic BRCA1 and BRCA2 sequence variants identifies clinically significant splicing aberrations up to 12 nucleotides from the intron/exon boundary.

Clinical management of breast cancer families is complicated by identification of BRCA1 and BRCA2 sequence alterations of unknown significance. Molecular assays evaluating the effect of intronic variants on native splicing can help determine their clinical relevance. Twenty-six intronic BRCA1/2 variants ranging from the consensus dinucleotides in the splice acceptor or donor to 53 nucleotides into the intron were identified in multiple-case families. The effect of the variants on splicing was assessed using HSF matrices, MaxEntScan and NNsplice, followed by analysis of mRNA from lymphoblastoid cell lines. A total of 12 variants were associated with splicing aberrations predicted to result in production of truncated proteins, including a variant located 12 nucleotides into the intron. The posterior probability of pathogenicity was estimated using a multifactorial likelihood approach, and provided a pathogenic or likely pathogenic classification for seven of the 12 spliceogenic variants. The apparent disparity between experimental evidence and the multifactorial predictions is likely due to several factors, including a paucity of likelihood information and a nonspecific prior probability applied for intronic variants outside the consensus dinucleotides. Development of prior probabilities of pathogenicity incorporating bioinformatic prediction of splicing aberrations should improve identification of functionally relevant variants and enhance multifactorial likelihood analysis of intronic variants.

Rare variants in the ATM gene and risk of breast cancer.

INTRODUCTION: The ataxia-telangiectasia mutated (ATM) gene (MIM ID 208900) encodes a protein kinase that plays a significant role in the activation of cellular responses to DNA double-strand breaks through subsequent phosphorylation of central players in the DNA damage-response pathway. Recent studies have confirmed that some specific variants in the ATM gene are associated with increased breast cancer (BC) risk. However, the magnitude of risk and the subset of variants that are pathogenic for breast cancer remain unresolved. METHODS: To investigate the role of ATM in BC susceptibility, we studied 76 rare sequence variants in the ATM gene in a case-control family study of 2,570 cases of breast cancer and 1,448 controls. The variants were grouped into three categories based on their likely pathogenicity, as determined by in silico analysis and analyzed by conditional logistic regression. Likely pathogenic sequence variants were genotyped in 129 family members of 27 carrier probands (15 of which carried c.7271T > G), and modified segregation analysis was used to estimate the BC penetrance associated with these rare ATM variants. RESULTS: In the case-control analysis, we observed an odds ratio of 2.55 and 95% confidence interval (CI, 0.54 to 12.0) for the most likely deleterious variants. In the family-based analyses, the maximum-likelihood estimate of the increased risk associated with these variants was hazard ratio (HR) = 6.88 (95% CI, 2.33 to 20.3; P = 0.00008), corresponding to a 60% cumulative risk of BC by age 80 years. Analysis of loss of heterozygosity (LOH) in 18 breast tumors from women carrying likely pathogenic rare sequence variants revealed no consistent pattern of loss of the ATM variant. CONCLUSIONS: The risk estimates from this study suggest that women carrying the pathogenic variant, ATM c.7271T > G, or truncating mutations demonstrate a significantly increased risk of breast cancer with a penetrance that appears similar to that conferred by germline mutations in BRCA2.

Gene expression profiling of formalin-fixed, paraffin-embedded familial breast tumours using the whole genome-DASL assay.

Tissue sample acquisition is a limiting step in many studies. There are many thousands of formalin-fixed, paraffin-embedded archival blocks collected around the world, but in contrast relatively few fresh frozen samples in tumour banks. Once samples are fixed in formalin, the RNA is degraded and traditional methods for gene expression profiling are not suitable. In this study, we have evaluated the ability of the whole genome DASL (cDNA-mediated Annealing, Selection, extension, and Ligation) assay from Illumina to perform transcriptomic analysis of archived breast tumour tissue in formalin-fixed, paraffin-embedded (FFPE) blocks. We profiled 76 familial breast tumours from cases carrying a BRCA1, BRCA2 or ATM mutation, or from non-BRCA1/2 families. We found that replicate samples correlated well with each other (r(2) = 0.9-0.98). In 12/15 cases, the matched formalin-fixed and frozen samples predicted the same tumour molecular subtypes with confidence. These results demonstrate that the whole genome DASL assay is a valuable tool to profile degraded RNA from archival FFPE material. This assay will enable transcriptomic analysis of a large number of archival samples that are stored in pathology archives around the globe and consequently will have the potential to improve our understanding and characterization of many diseases.

Intra-operative four-stranded hamstring tendon graft diameter evaluation.

PURPOSE: To evaluate whether clinical factors enable prediction of the diameter of hamstring tendons harvested for anterior cruciate ligament (ACL) reconstruction. METHODS: Eighty patients were submitted for reconstruction of the ACL with hamstring tendons in a quadruple manner. During surgery the diameter of the graft was measured. The variables analyzed were: age, gender, weight, height, operated side, dominant side, leg length, thigh length, thigh diameter, body mass index (BMI), and sports activity. The data was collected pre-operatively and correlated with the diameter of the graft. RESULTS: The diameter of the graft was strongly related to gender, height, leg length, thigh length, weight, and thigh diameter. Women presented significantly smaller graft diameter than men; as well as weight, height, leg length, and thigh length. Men with height equal to or greater than 1.80 m showed average graft diameter greater than the total group, and greater percentage of 9 mm grafts. CONCLUSION: The diameter of the hamstring graft is significantly associated to weight, height, leg length, thigh length, thigh diameter, and gender. The variable that had most influence was height, followed by gender and leg length. The variables BMI, age, sports activity, and dominant side did not present correlation. Tendon diameter was larger in men than in women. Men with a height equal to or greater than 1.80 m had a higher prevalence of 9 mm grafts and had a larger average tendon diameter. LEVEL OF EVIDENCE: Prospective cross sectional collection of data, Level IV.

Bayes analysis provides evidence of pathogenicity for the BRCA1 c.135-1G>T (IVS3-1) and BRCA2 c.7977-1G>C (IVS17-1) variants displaying in vitro splicing results of equivocal clinical significance.

Although in vitro splicing assays can provide useful information about the clinical interpretation of sequence variants in high-risk cancer genes such as BRCA1 and BRCA2, results can sometimes be difficult to interpret. The BRCA1 c.135-1G>T (IVS3-1G>T) variant has been shown to give rise to an in-frame deletion of exon 5 (BRCA1 c.135_212del) that is predicted to encode 26 amino acids. BRCA2 c.7977-1G>C (IVS17-1G>C) was shown to increase the expression of two naturally occurring transcripts that contain frameshifts (BRCA2, c.7977_8311del (exon 18 deletion); BRCA2, c.7806_8331del (exon 17&18 deletion)). In this study we conducted multifactorial likelihood analysis to evaluate the clinical significance of these two variants, including assessing variant segregation in families by Bayes analysis, and breast tumor pathology features suggestive of positive mutation status. Multifactorial analysis provided strong evidence for causality for both of these variants. The Bayes scores from a single family with BRCA1 c.135-1G>T was 9528:1, and incorporation of pathology features gave an overall likelihood of causality of 28108:1. The Bayes scores from five informative families with BRCA2 c.7977-1G>C was 47401:1, and the combined Bayes-pathology odds of causality was 29389:1. Multifactorial likelihood analysis indicates that the BRCA1 c.135-1G>T and BRCA2 c.7977-1G>C variants are disease-associated mutations which should be managed clinically in the same fashion as classical truncating mutations.

Detection of splicing aberrations caused by BRCA1 and BRCA2 sequence variants encoding missense substitutions: implications for prediction of pathogenicity.

Missense substitutions in high-risk cancer susceptibility genes create clinical uncertainty in the genetic counseling process. Multifactorial likelihood classification approaches and in vitro assays are useful for the classification of exonic sequence variants in BRCA1 and BRCA2, but these currently rely on the assumption that changes in protein function are the major biological mechanism of pathogenicity. This study investigates the potentially pathogenic role of aberrant splicing for exonic variants predicted to encode missense substitutions using patient-derived RNA. No splicing aberrations were identified for BRCA1c.5054C>T and BRCA2c.7336A>G, c.8839G>A, and c.9154C>T. However, RT-PCR analysis identified a major splicing aberration for BRCA1c.4868C>G(p.Ala1623Gly), a variant encoding a missense substitution considered likely to be neutral. Splicing aberrations were also observed for BRCA2c.7988A>T(p.Glu2663Val) and c.8168A>G(p.Asp2723Gly), but both variant and wildtype alleles were shown to be present in full-length mRNA transcripts, suggesting that variant protein may be translated. BRCA2 protein function assays indicated that BRCA2p.Glu2663Val, p.Asp2723Gly and p.Arg3052Trp missense proteins have abrogated function consistent with pathogenicity. Multifactorial likelihood analysis provided evidence for pathogenicity for BRCA1 c.5054C>T(p.Thr1685Ile) and BRCA2c.7988A>T(p.Glu2663Val), c.8168A>G(p.Asp2723Gly) and c.9154C>T(p.Arg3052Trp), supporting experimentally derived evidence. These findings highlight the need for improved bioinformatic prediction of splicing aberrations and to refine multifactorial likelihood models used to assess clinical significance.

Dissecting the transcriptional networks underlying breast cancer: NR4A1 reduces the migration of normal and breast cancer cell lines.

INTRODUCTION: Breast cancer currently accounts for more than one-quarter of all female cancers and, despite the great progress in treatment observed in the past few years, the need for identification of new gene targets that can be used for diagnosis, prognosis and therapy is evident. A previous study identified the transcription factor NR4A1 as a gene upregulated in primary breast cancer compared with normal tissue by microarray analysis and sequencing technologies. The purpose of the study was to identify the role of NR4A1 in normal mammary epithelial and breast cancer cell biology. METHODS: NR4A1 expression in breast tumours was assessed by semiquantitative and real-time PCR using RNA from normal and tumour samples or breast cancer cell lines. Immunohistochemistry on tissue microarrays was performed to check NR4A1 protein expression in breast tumours. MCF-10A and 226L normal mammary epithelial cells as well as the tumour lines PMC42, ZR-75-1 and MDA-MB-231 were transduced with full-length NR4A1, and the ability of NR4A1-overexpressing cells to migrate was tested using scratch wound or transwell migration assays. Proliferation was measured using the MTT and BrdU assays, while apoptosis was determined by the Annexin V assay. The ability of the cells to adhere to extracellular matrix was tested by adhesion assays and integrin cell surface expression was measured by flow cytometry. Activation of the FAK as well as ERK1/2 and PI3K pathways was checked by western blotting. RESULTS: Breast tissue microarray analysis showed NR4A1 expression in primary tumours, which was reduced in higher grade and metastatic tumours. Ectopic expression of NR4A1 in MCF-10A, 226L, PMC42 and ZR-75-1 cells led to reduced ability of the cells to migrate, while no differences were observed in their proliferation and apoptotic index. NR4A1 expression altered the ability of the MCF-10A cells to adhere to the extracellular matrix and affected cell surface expression of integrins. CONCLUSIONS: NR4A1 acts as an antimigratory factor in two normal mammary epithelial and two breast cancer cell lines tested. It is therefore possible that NR4A1 acts as an antimigratory factor in breast tumours, and further studies should be conducted to understand the mechanisms involved.

HER3 and downstream pathways are involved in colonization of brain metastases from breast cancer.

INTRODUCTION: Metastases to the brain from breast cancer have a high mortality, and basal-like breast cancers have a propensity for brain metastases. However, the mechanisms that allow cells to colonize the brain are unclear. METHODS: We used morphology, immunohistochemistry, gene expression and somatic mutation profiling to analyze 39 matched pairs of primary breast cancers and brain metastases, 22 unmatched brain metastases of breast cancer, 11 non-breast brain metastases and 6 autopsy cases of patients with breast cancer metastases to multiple sites, including the brain. RESULTS: Most brain metastases were triple negative and basal-like. The brain metastases over-expressed one or more members of the HER family and in particular HER3 was significantly over-expressed relative to matched primary tumors. Brain metastases from breast and other primary sites, and metastases to multiple organs in the autopsied cases, also contained somatic mutations in EGFR, HRAS, KRAS, NRAS or PIK3CA. This paralleled the frequent activation of AKT and MAPK pathways. In particular, activation of the MAPK pathway was increased in the brain metastases compared to the primary tumors. CONCLUSIONS: Deregulated HER family receptors, particularly HER3, and their downstream pathways are implicated in colonization of brain metastasis. The need for HER family receptors to dimerize for activation suggests that tumors may be susceptible to combinations of anti-HER family inhibitors, and may even be effective in the absence of HER2 amplification (that is, in triple negative/basal cancers). However, the presence of activating mutations in PIK3CA, HRAS, KRAS and NRAS suggests the necessity for also specifically targeting downstream molecules.