Background Dihydropyrimidine dehydrogenase (DPD) deficiency is the main known cause of life-threatening fluoropyrimidine (FP)-induced toxicities. We conducted a meta-analysis on individual patient data to assess the contribution of deleterious DPYD variants *2A/D949V/*13/HapB3 (recommended by EMA) and clinical factors, for predicting G4-5 toxicity. Methods Study eligibility criteria included recruitment of Caucasian patients without DPD-based FP-dose adjustment. Main endpoint was 12-week haematological or digestive G4-5 toxicity. The value of DPYD variants *2A/p.D949V/*13 merged, HapB3, and MIR27A rs895819 was evaluated using multivariable logistic models (AUC). Results Among 25 eligible studies, complete clinical variables and primary endpoint were available in 15 studies (8733 patients). Twelve-week G4-5 toxicity prevalence was 7.3% (641 events). The clinical model included age, sex, body mass index, schedule of FP-administration, concomitant anticancer drugs. Adding *2A/p.D949V/*13 variants (at least one allele, prevalence 2.2%, OR 9.5 [95%CI 6.7–13.5]) significantly improved the model ( p < 0.0001). The addition of HapB3 (prevalence 4.0%, 98.6% heterozygous), in spite of significant association with toxicity (OR 1.8 [95%CI 1.2–2.7]), did not improve the model. MIR27A rs895819 was not associated with toxicity, irrespective of DPYD variants. Conclusions FUSAFE meta-analysis highlights the major relevance of DPYD *2A/p.D949V/*13 combined with clinical variables to identify patients at risk of very severe FP-related toxicity.

Background: Recent population-based female breast cancer and prostate cancer polygenic risk scores (PRS) have been developed. We assessed the associations of these PRS with breast and prostate cancer risks for male BRCA1 and BRCA2 pathogenic variant carriers. Methods: 483 BRCA1 and 1318 BRCA2 European ancestry male carriers were available from the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA). A 147-single nucleotide polymorphism (SNP) prostate cancer PRS (PRSPC) and a 313-SNP breast cancer PRS were evaluated. There were 3 versions of the breast cancer PRS, optimized to predict overall (PRSBC), estrogen receptor (ER)-negative (PRSER-), or ER-positive (PRSER+) breast cancer risk. Results: PRSER+ yielded the strongest association with breast cancer risk. The odds ratios (ORs) per PRSER+ standard deviation estimates were 1.40 (95% confidence interval [CI] =1.07 to 1.83) for BRCA1 and 1.33 (95% CI = 1.16 to 1.52) for BRCA2 carriers. PRSPC was associated with prostate cancer risk for BRCA1 (OR = 1.73, 95% CI = 1.28 to 2.33) and BRCA2 (OR = 1.60, 95% CI = 1.34 to 1.91) carriers. The estimated breast cancer odds ratios were larger after adjusting for female relative breast cancer family history. By age 85 years, for BRCA2 carriers, the breast cancer risk varied from 7.7% to 18.4% and prostate cancer risk from 34.1% to 87.6% between the 5th and 95th percentiles of the PRS distributions. Conclusions: Population-based prostate and female breast cancer PRS are associated with a wide range of absolute breast and prostate cancer risks for male BRCA1 and BRCA2 carriers. These findings warrant further investigation aimed at providing personalized cancer risks for male carriers and informing clinical management.

5-Fluorouracil and its oral prodrug capecitabine are two of the most frequently prescribed chemotherapeutic drugs for the adjuvant and palliative treatment of patients with cancers of the gastrointestinal tract, breast and head and neck. In principle, pharmacogenetics-guided dosing will enable the identification of patients at risk of developing severe toxicity prior to the start of a fluoropyrimidine-based therapy and adjustment of the initial fluoropyrimidine dose. Several phenotyping procedures have been developed to screen patients for a dihydropyrimidine dehydrogenase (DPD) deficiency, such as measurement of the uracil/dihydrouracil ratio, assessment of the DPD activity in peripheral blood mononuclear cells, and loading studies with uracil. To date, numerous decreased function variants have been described in dihydropyrimidine dehydrogenase gene (DPYD) together with dose recommendations based on the gene activity score. In patients who are heterozygous for a "loss-of-function" mutation in DPYD, a dose reduction of 50% has been recommended.

Aim BRCA1- associated protein 1 (BAP1) plays a critical role in the tumour suppressor function of BRCA1. Recent studies discussed the role of BAP1 as an oncogene by stimulating metastasis and proliferation in breast carcinogenesis. Given the interaction of BAP1 with BRCA1, we aimed to explore the expression and prognostic potential of BAP1 in BRCA1/2 mutated and Panel-negative tested cases.

In normal physiology, kallikrein-related peptidase 7 (KLK7), together with other members of the kallikrein-related peptidase family, is mainly involved in skin desquamation and keratinization processes. Moreover, expression of KLK7 was shown in various tumor types to be dysregulated and to correlate to patients' survival time. However, there are contradictory reports in breast cancer whether KLK7 represents an unfavorable or favorable prognostic biomarker. In the present study, we examined the prognostic value of KLK7 protein expression in triple-negative breast cancer (TNBC), determined by immunohistochemistry (IHC). A cohort encompassing 133 TNBC specimens, present on tissue microarrays, was analyzed. For quantification of the staining intensity, an automated digital IHC image analysis algorithm was applied. In both Kaplan-Meier and univariate Cox analyses, elevated KLK7 protein levels were significantly linked with prolonged overall survival (OS). In multivariable Cox analysis, addition of KLK7 immunoreactivity scores to the base model (including the clinical parameters age, tumor size, and nodal status) demonstrated that KLK7 protein expression remained as a statistically significant, independent parameter for prolonged OS. These results strongly indicate that KLK7 is a favorable prognostic biomarker in triple-negative breast cancer.

Dihydropyrimidine dehydrogenase (DPD) deficiency is the main cause of severe FP-tox. DPD expression is regulated at post-transcriptional level by miR-27a. MIR27A rs895819A>G has been associated with significant increased grade (G) ≥3 FP-tox only in patients (pts) carrying a deleterious DPYD variant (*2A/D949V/*13/HapB3). French GPCO-UNICANCER and RNPGx groups initiated the FUSAFE IPD-MA to develop predictive models of FP-tox, allowing to test whether MIR27A rs895819 improves the predictive value of DPYD variants. Eligibility criteria included unbiased recruitment of Caucasian pts without FP dose adjustment based on DPD status. Main endpoint was hematological or digestive G4-5 FP-tox at 12 weeks. A clinical logistic regression model including age, sex, body mass index, FP administration (bolus±continuous vs continuous/p.o.) and associated anticancer drug (AAD) was first developed. Combined DPYD variants *2A/D949V/*13 with or without HapB3 (at least one mutated allele) and MIR27A rs895819 (dominant coding) were further included in this model, along with DPYD x MIR27A interaction. Full clinical data, MIR27A, and DPYD variants were assessable in 2724 pts (5 studies): 71% colorectal cancer, 41% metastatic, 95% FP-naïve, 31% 5FU, 55% AAD. MIR27A rs895819G was carried by 53.4% of pts (9.7% homozygotes) and 1.5% of pts (N=41) carried at least one *2A/D949V/*13 DPYD variant. G4-5 FP-tox was 4.0%. Adjusted Odds Ratio were 6.9 [95%CI 3.0-16.0] for DPYD combined variants *2A/D949V/*13, and 1.3 [95%CI 0.9-1.9] for MIR27A rs895819, with no DPYD x MIR27A statistical interaction (p=0.99). Results on MIR27A and its interaction with DPYD variants were similar when considering G3-4-5 tox at cycle 1, or HapB3. This IPD-MA on MIR27A and DPYD variants, including the largest series of pts published so far, do not confirm the previously published relevance of MIR27A rs895819 for toxicity risk stratification in patients carrying DPYD risk variants. The ongoing FUSAFE-2 project including sequencing of DPYD and MIR genes may provide new insights in FP pharmacogenetics.

BRCA1- associated protein 1 (BAP1) interacts with BRCA1 in its tumour suppressor function. Recent studies described the role of BAP1 as an oncogene in breast carcinogenesis. Considering the interaction of BAP1 with BRCA1, we aimed to explore the expression and prognostic potential of BAP1 in BRCA1/2 mutated and BRCA1/2-negative tested cases.

Purpose TP53germline (g) mutations, associated with the Li-Fraumeni syndrome (LFS), have rarely been reported in the context of hereditary breast and ovarian cancer (HBOC). The prevalence and cancer risks in this target group are unknown and counseling remains challenging. Notably an extensive high-risk surveillance program is implemented, which evokes substantial psychological discomfort. Emphasizing the lack of consensus about clinical implications, we aim to further characterize TP53g mutations in HBOC families. Methods Next-generation sequencing was conducted on 1876 breast cancer (BC) patients who fulfilled the inclusion criteria for HBOC. Results (Likely) pathogenic variants in TP53 gene were present in 0.6% of the BC cohort with higher occurrence in early onset BC < 36 years. (1.1%) and bilateral vs. unilateral BC (1.1% vs. 0.3%). Two out of eleven patients with a (likely) pathogenic TP53g variant (c.542G > A; c.375G > A) did not comply with classic LFS/Chompret criteria. Albeit located in the DNA-binding domain of the p53-protein and therefore revealing no difference to LFS-related variants, they only displayed a medium transactivity reduction constituting a retainment of wildtype-like anti-proliferative functionality. Conclusion Among our cohort of HBOC families, we were able to describe a clinical subgroup, which is distinct from the classic LFS-families. Strikingly, two families did not adhere to the LFS criteria, and functional analysis revealed a reduced impact on TP53 activity, which may suit to the attenuated phenotype. This is an approach that could be useful in developing individualized screening efforts for TP53 g mutation carrier in HBOC families. Due to the low incidence, national/international cooperation is necessary to further explore clinical implications. This might allow providing directions for clinical recommendations in the future.

Gestational diabetes mellitus (GDM) is the most common pregnancy-associated metabolic disorder that negatively impacts on the health of both mothers and their offspring in the long-term. The molecular mechanisms involved are not fully understood. As in other states of insulin resistance, a disproportionate immune response in GDM leads to a state of chronic low-grade inflammation. Galectin-2 exerts regulatory effects on different immune cells. This study investigated galectin-2 expression in the placenta of 40 GDM patients and 40 controls, in a sex-specific manner. Immunohistochemistry was used for semi-quantitative analysis of expression strength. The phenotypes of galectin-2 expressing cells were characterized through double immunofluorescence. We found a significant up-regulation of galectin-2 in the fetal syncytiotrophoblast, as well as in the maternal decidua of GDM placentas. Double staining showed a strong galectin-2 expression in extra villous trophoblast cells and fetal endothelial cells in GDM. These findings present the first systematic investigation of galectin-2 in GDM. The findings contribute to the emerging understanding of the role of immunomodulation and inflammation in GDM and of galectin-2 itself. This might also have implications for the long-term cardiovascular health of the offspring.

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.

Background Despite decades of research, performance of DPYD genotyping to predict FP toxicity (tox) is poorly documented. GPCO-UNICANCER and RNPGx groups initiated the FUSAFE MA on individual patient data (IPD) to assess the prognostic value of consensual deleterious DPYD variants on grade (G) 4-5 FP tox. Methods Eligibility criteria included unbiased recruitment of Caucasian patients (pts) without FP dose adjustment based on DPD status. Main endpoint was 12 weeks hematological or digestive G4-5 tox. Age, sex, body mass index (BMI), advanced stage (M- vs M+), FP drug (5FU vs capecitabine), FP administration (bolus±continuous vs continuous alone or p.o.) and associated anticancer drugs (AAD) were collected. Multivariable logistic models were applied. Performance was assessed by AUC and diagnostic indices maximizing Youden index. Results From the 18 identified eligible studies (10230 pts), 14 were included (9030 pts), with complete IPD collected for 6403 pts (84% colorectal, 16% M+, 66% 5FU, 80% AAD). G4-5 tox prevalence was 8% (518 events). DPYD variants *2A, D949V, *13 and HapB3 were carried by 0.9%, 1.2%, 0.2% and 3.9% of pts, respectively. The clinical model (M1) retained age, sex, BMI, FP-administration, AAD. Adding variants *2A/D949V/*13 (at least one mutated allele) significantly (p Table . 569P Model AUC (area under the curve) Sensitivity Specificity Positive predictive value Negative predictive value M1 0.725 69.7% 65.4% 15.1% 96.1% M2 0.750 75.5% 62.8% 15.2% 96.7% M3 0.752 76.4% 61.9% 15.0% 96.8% Conclusions This is the largest MA on DPYD genotyping and toxicity. It shows the relevance of clinical variables and of the 3 consensual DPYD variants. Despite its association with tox, HapB3 does not improve the discriminant ability to identify pts at risk of G4-5 toxicity. Legal entity responsible for the study Centre Antoine Lacassagne, Nice, France (Dr Marie-Christine Etienne-Grimaldi, coordinator). Funding French Cancer Institute (INCa) funding (PHRC-K 14-193 FUSAFE) and French Ligue Nationale Contre le Cancer. Disclosure V. Boige: Honoraria (self), Advisory / Consultancy, Research grant / Funding (self), Travel / Accommodation / Expenses: Merck Serono; Honoraria (self): Amgen; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: Bayer; Honoraria (self), Travel / Accommodation / Expenses: Roche Genentech; Honoraria (self): MSD; Honoraria (self), Advisory / Consultancy: Ipsen; Advisory / Consultancy: Prestizia; Advisory / Consultancy: Eisai; Travel / Accommodation / Expenses: Sanofi. J. Taieb: Advisory / Consultancy: Amgen; Advisory / Consultancy: Lily; Advisory / Consultancy: Sanofi; Advisory / Consultancy: Roche; Advisory / Consultancy: Merck; Advisory / Consultancy: MSD; Advisory / Consultancy: Celgene; Advisory / Consultancy: Servier; Advisory / Consultancy: Sirtex; Advisory / Consultancy: Pierre Fabre. D. Meulendijks: Full / Part-time employment: AstraZeneca UK. C. Palles: Advisory / Consultancy: Oxford Cancer Biomarkers. U. Zanger: Licensing / Royalties: Robert Bosch GmbH. M. Boisdron-Celle: Speaker Bureau / Expert testimony, Leadership role, Shareholder / Stockholder / Stock options, Licensing / Royalties, Officer / Board of Directors: ODPM. A. Marinaki: Full / Part-time employment: Viapath. E. Gross: Licensing / Royalties: Klinikum Rechts der Isar der Technischen Universitat Munchen. All other authors have declared no conflicts of interest.

Laura Kasak | Constantina Bakolitsa | Zhiqiang Hu | Changhua Yu | Jasper Rine | Dago F. Dimster‐Denk | Gaurav Pandey | Greet De Baets | Yana Bromberg | Chen Cao | Emidio Capriotti | Rita Casadio | Joost Van Durme | Manuel Giollo | Rachel Karchin | Panagiotis Katsonis | Emanuela Leonardi | Olivier Lichtarge | Pier Luigi Martelli | David Masica | Sean D. Mooney | Ayodeji Olatubosun | Predrag Radivojac | Frederic Rousseau | Lipika R. Pal | Castrense Savojardo | Joost Schymkowitz | Janita Thusberg | Silvio C.E. Tosatto | Mauno Vihinen | Jouni Väliaho | Susanna Repo | John Moult | Steven E. Brenner | Iddo Friedberg

The tetraspanin and tumor suppressor KAI1 is downregulated or lost in many cancers which correlates with poor prognosis. KAI1 acts via physical/functional crosstalk with other membrane receptors. Also, a splice variant of KAI1 (KAI1-SP) has been identified indicative of poor prognosis. We here characterized differential effects of the two KAI1 variants on tumor biological events involving integrin (αvß3) and/or epidermal growth factor receptor (EGF-R). In MDA-MB-231 and -435 breast cancer cells, differential effects were documented on the expression levels of the tumor biologically relevant integrin αvß3 which colocalized with KAI1-WT but not with KAI1-SP. Cellular motility was assessed by video image processing, including motion detection and vector analysis for the quantification and visualization of cell motion parameters. In MDA-MB-231 cells, KAI1-SP provoked a quicker wound gap closure and higher closure rates than KAI1-WT, also reflected by different velocities and average motion amplitudes of singular cells. KAI1-SP induced highest cell motion adjacent to the wound gap borders, whereas in MDA-MB-435 cells a comparable induction of both KAI1 variants was noticed. Moreover, while KAI1-WT reduced cell growth, KAI1-SP significantly increased it going along with a pronounced EGF-R upregulation. KAI1-SP-induced cell migration and proliferation was accompanied by the activation of the focal adhesion and Src kinase. Our findings suggest that splicing of KAI1 does not only abrogate its tumor suppressive functions, but even more, promotes tumor biological effects in favor of cancer progression and metastasis.

Germline mutations in the BRIP1 gene have been described as conferring a moderate risk for ovarian cancer (OC), while the role of BRIP1 in breast cancer (BC) pathogenesis remains controversial.

Die Hauptursache für Brustkrebs-assoziierte Sterblichkeit ist die Bildung von Fernmetastasen. Während des Prozesses der Metastasierung lösen sich einzelne Zellen, sog. „Circulating Tumor Cells“ (CTCs) vom Primarius ab, verändern sich im Hinblick auf Zelladhäsion und Beweglichkeit, wandern in Blutgefäße ein und verlassen diese an anderen Orten des Körpers wieder, um sich dort anzusiedeln und den Ursprung einer Metastasierung zu bilden. Der hierfür notwendige Prozess der Veränderung der Zelleigenschaften wird als „Epithelial-mesenchymale Transition“ (EMT) bezeichnet. Die EMT geht mit einer Veränderung der Genexpression einher, so dass CTCs mithilfe einer Real-Time PCR EMT-assoziierter Gene nachgewiesen werden können.

The prevalence of germ line mutations in non-BRCA1/2 genes associated with hereditary breast cancer (BC) is low, and the role of some of these genes in BC predisposition and pathogenesis is conflicting. In this study, 5589 consecutive BC index patients negative for pathogenic BRCA1/2 mutations and 2189 female controls were screened for germ line mutations in eight cancer predisposition genes (ATM, CDH1, CHEK2, NBN, PALB2, RAD51C, RAD51D, and TP53). All patients met the inclusion criteria of the German Consortium for Hereditary Breast and Ovarian Cancer for germ line testing. The highest mutation prevalence was observed in the CHEK2 gene (2.5%), followed by ATM (1.5%) and PALB2 (1.2%). The mutation prevalence in each of the remaining genes was 0.3% or lower. Using Exome Aggregation Consortium control data, we confirm significant associations of heterozygous germ line mutations with BC for ATM (OR: 3.63, 95% CI: 2.67-4.94), CDH1 (OR: 17.04, 95% CI: 3.54-82), CHEK2 (OR: 2.93, 95% CI: 2.29-3.75), PALB2 (OR: 9.53, 95% CI: 6.25-14.51), and TP53 (OR: 7.30, 95% CI: 1.22-43.68). NBN germ line mutations were not significantly associated with BC risk (OR: 1.39, 95% CI: 0.73-2.64). Due to their low mutation prevalence, the RAD51C and RAD51D genes require further investigation. Compared with control datasets, predicted damaging rare missense variants were significantly more prevalent in CHEK2 and TP53 in BC index patients. Compared with the overall sample, only TP53 mutation carriers show a significantly younger age at first BC diagnosis. We demonstrate a significant association of deleterious variants in the CHEK2, PALB2, and TP53 genes with bilateral BC. Both, ATM and CHEK2, were negatively associated with triple-negative breast cancer (TNBC) and estrogen receptor (ER)-negative tumor phenotypes. A particularly high CHEK2 mutation prevalence (5.2%) was observed in patients with human epidermal growth factor receptor 2 (HER2)-positive tumors.

Triple-negative breast cancer (TNBC) constitutes a heterogeneous breast cancer subgroup with poor prognosis; survival rates are likely to be lower with TNBC compared to other breast cancer subgroups. For this disease, systemic adjuvant chemotherapy regimens often yield suboptimal clinical results. To improve treatment regimens in TNBC, identification of molecular biomarkers may help to select patients for individualized adjuvant therapy. Evidence has accumulated that determination of the methylation status of the PITX2 gene provides a predictive value in various breast cancer subgroups, either treated with endocrine-based therapy or anthracycline-containing chemotherapy. To further explore the validity of this novel predictive candidate biomarker, in the present exploratory retrospective study, determination of the PITX2 DNA-methylation status was assessed for non-metastatic TNBC patients treated with adjuvant anthracycline-based chemotherapy by molecular analysis of breast cancer tissues. The PITX2 DNA-methylation status was determined in fresh-frozen tumor tissue specimens (n=56) by methylation-specific qRT-PCR (qMSP) and the data related to disease-free and overall survival, applying an optimized DNA-methylation score of 6.35%. For non-metastatic TNBC patients treated with adjuvant systemic anthracycline-based chemotherapy, a low PITX2 DNA-methylation status (<6.35) defines TNBC patients with poor disease-free and overall survival. Univariate and multivariate analyses demonstrate the statistically independent predictive value of PITX2 DNA-methylation. For non-metastatic TNBC patients, selective determination of the PITX2 DNA-methylation status may serve as a cancer biomarker for predicting response to anthracycline-based adjuvant chemotherapy. The assay based on methylation of the PIXT2 gene can be applied to frozen and routinely available formalin-fixed, paraffin-embedded (FFPE) breast cancer tumor tissues that will not only define those TNBC patients who may benefit from anthracycline-based chemotherapy but also those who should be spared the necessity of such potentially toxic treatment. Such patients should be allocated to alternative treatment options.

TP53 germline mutations are associated with the Li-Fraumeni syndrome, an autosomal dominant cancer syndrome, which predisposes to a wide spectrum of malignancies. For the diagnosis of LFS the classic LFS or the Chompret criteria must be met. TP53 mutations have been rarely reported in the context of hereditary breast and ovarian cancer (HBOC). However, the prevalence in this target group is unknown and medical counseling remains challenging, especially concerning the large-scale screening protocol including annual whole body MRI that is implemented for LFS families.