Tumor sequencing is useful to refine the analysis of germline variants in unexplained high-risk breast cancer families.

BACKGROUND: Multigene panels are routinely used to assess for predisposing germline mutations in families at high breast cancer risk. The number of variants of unknown significance thereby identified increases with the number of sequenced genes. We aimed to determine whether tumor sequencing can help refine the analysis of germline variants based on second somatic genetic events in the same gene. METHODS: Whole-exome sequencing (WES) was performed on whole blood DNA from 70 unrelated breast cancer patients referred for genetic testing and without a BRCA1, BRCA2, TP53, or CHEK2 mutation. Rare variants were retained in a list of 735 genes. WES was performed on matched tumor DNA to identify somatic second hits (copy number alterations (CNAs) or mutations) in the same genes. Distinct methods (among which immunohistochemistry, mutational signatures, homologous recombination deficiency, and tumor mutation burden analyses) were used to further study the role of the variants in tumor development, as appropriate. RESULTS: Sixty-eight patients (97%) carried at least one germline variant (4.7 ± 2.0 variants per patient). Of the 329 variants, 55 (17%) presented a second hit in paired tumor tissue. Of these, 53 were CNAs, resulting in tumor enrichment (28 variants) or depletion (25 variants) of the germline variant. Eleven patients received variant disclosure, with clinical measures for five of them. Seven variants in breast cancer-predisposing genes were considered not implicated in oncogenesis. One patient presented significant tumor enrichment of a germline variant in the oncogene ERBB2, in vitro expression of which caused downstream signaling pathway activation. CONCLUSION: Tumor sequencing is a powerful approach to refine variant interpretation in cancer-predisposing genes in high-risk breast cancer patients. In this series, the strategy provided clinically relevant information for 11 out of 70 patients (16%), adapted to the considered gene and the familial clinical phenotype.

Identification by array comparative genomic hybridization of a new amplicon on chromosome 17q highly recurrent in BRCA1 mutated triple negative breast cancer.

INTRODUCTION: Triple Negative Breast Cancers (TNBC) represent about 12% to 20% of all breast cancers (BC) and have a worse outcome compared to other BC subtypes. TNBC often show a deficiency in DNA double-strand break repair mechanisms. This is generally related to the inactivation of a repair enzymatic complex involving BRCA1 caused either by genetic mutations, epigenetic modifications or by post-transcriptional regulations. The identification of new molecular biomarkers that would allow the rapid identification of BC presenting a BRCA1 deficiency could be useful to select patients who could benefit from PARP inhibitors, alkylating agents or platinum-based chemotherapy. METHODS: Genomic DNA from 131 formalin-fixed paraffin-embedded (FFPE) tumors (luminal A and B, HER2+ and triple negative BC) with known BRCA1 mutation status or unscreened for BRCA1 mutation were analysed by array Comparative Genomic Hybridization (array CGH). One highly significant and recurrent gain in the 17q25.3 genomic region was analysed by fluorescent in situ hybridization (FISH). Expression of the genes of the 17q25.3 amplicon was studied using customized Taqman low density arrays and single Taqman assays (Applied Biosystems). RESULTS: We identified by array CGH and confirmed by FISH a gain in the 17q25.3 genomic region in 90% of the BRCA1 mutated tumors. This chromosomal gain was present in only 28.6% of the BRCA1 non-mutated TNBC, 26.7% of the unscreened TNBC, 13.6% of the luminal B, 19.0% of the HER2+ and 0% of the luminal A breast cancers. The 17q25.3 gain was also detected in 50% of the TNBC with BRCA1 promoter methylation. Interestingly, BRCA1 promoter methylation was never detected in BRCA1 mutated BC. Gene expression analyses of the 17q25.3 sub-region showed a significant over-expression of 17 genes in BRCA1 mutated TNBC (n = 15) as compared to the BRCA1 non mutated TNBC (n = 13). CONCLUSIONS: In this study, we have identified by array CGH and confirmed by FISH a recurrent gain in 17q25.3 significantly associated to BRCA1 mutated TNBC. Up-regulated genes in the 17q25.3 amplicon might represent potential therapeutic targets and warrant further investigation.

The doublesex homolog Dmrt5 is required for the development of the caudomedial cerebral cortex in mammals.

Regional patterning of the cerebral cortex is initiated by morphogens secreted by patterning centers that establish graded expression of transcription factors within cortical progenitors. Here, we show that Dmrt5 is expressed in cortical progenitors in a high-caudomedial to low-rostrolateral gradient. In its absence, the cortex is strongly reduced and exhibits severe abnormalities, including agenesis of the hippocampus and choroid plexus and defects in commissural and thalamocortical tracts. Loss of Dmrt5 results in decreased Wnt and Bmp in one of the major telencephalic patterning centers, the dorsomedial telencephalon, and in a reduction of Cajal-Retzius cells. Expression of the dorsal midline signaling center-dependent transcription factors is downregulated, including Emx2, which promotes caudomedial fates, while the rostral determinant Pax6, which is inhibited by midline signals, is upregulated. Consistently, Dmrt5(-/-) brains exhibit patterning defects with a dramatic reduction of the caudomedial cortex. Dmrt5 is increased upon the activation of Wnt signaling and downregulated in Gli3(xt/xt) mutants. We conclude that Dmrt5 is a novel Wnt-dependent transcription factor required for early cortical development and that it may regulate initial cortical patterning by promoting dorsal midline signaling center formation and thereby helping to establish the graded expression of the other transcription regulators of cortical identity.

Role of Myeloperoxidase in ROS Generation and Inflammation Response on Prostate Epithelial Cells.

Myeloperoxidase (MPO) has been reported in prostate tissue, and considering its pro-oxidant properties, this location might be linked to prostate pathology. The possibility that the glandular prostatic tissue might be the source of MPO and its potential inflammatory effects must be tested. Human prostate material was obtained from prostate biopsies and radical prostatectomies. Immunohistochemistry was performed using MPO-specific human antibody. In situ hybridization using MPO-specific probes and laser-assisted microdissection for quantitative real-time RT-PCR were performed to observe whether MPO is being produced in prostate tissue. Mass spectrometry on prostate biopsies was used to detect products of MPO activity in nucleic acids (DNA/RNA). MPO contribution to intracellular accumulation of ROS and interleukin-8 in prostatic epithelial cells was monitored in vitro. Immunohistochemistry confirmed cellular localization of MPO in epithelial cells of the prostate. The staining varied from light to high intensity. In situ hybridization did not address the presence of mRNA coding for MPO. No MPO-specific modifications on nucleic acids were detected. Mox-LDL was a major factor inducing ROS and cytokines production in prostatic epithelial cells. We did not demonstrate that MPO was synthetized by prostatic epithelial cells. However, in vitro experiments showed the ability of MPO to potentiate the ROS production and inflammation on prostate epithelial cells. Results do not allow us to demonstrate a role of MPO in prostate to date but further studies are mandatory to focus on the potential impact of MPO in the development of prostatic diseases.

The homeobox leucine zipper gene Homez plays a role in Xenopus laevis neurogenesis.

The Homez gene encodes a protein with three atypical homeodomains and two leucine zipper motifs of unknown function. Here we show that during neurula stages, Xenopus Homez is broadly expressed throughout the neural plate, the strongest expression being detected in the domains where primary neurons arise. At later stages, Homez is maintained throughout the central nervous system in differentiating progenitors. In accordance with this expression, Homez is positively regulated by neural inducers and by Ngnr1 and negatively by Notch signaling. Interference with Homez function in embryos by injection of an antisense morpholino oligonucleotide results in the specific disruption of the expression of late neuronal markers, without affecting the expression of earlier neuronal and early neurectodermal markers. Consistent with this finding, Homez inhibition also interferes with the expression of late neuronal markers in Ngnr1 overexpressing animal cap explants and in Notch inhibited embryos. In gain of function experiments, Homez inhibits the expression of late neuronal markers but has no effect on earlier ones. These data suggest a role for Homez in neuronal development downstream of proneural/neurogenic genes.

TriMix and tumor antigen mRNA electroporated dendritic cell vaccination plus ipilimumab: link between T-cell activation and clinical responses in advanced melanoma.

BACKGROUND: We previously reported that dendritic cell-based mRNA vaccination plus ipilimumab (TriMixDC-MEL IPI) results in an encouraging rate of tumor responses in patients with pretreated advanced melanoma. Here, we report the TriMixDC-MEL IPI-induced T-cell responses detected in the peripheral blood. METHODS: Monocyte-derived dendritic cells electroporated with mRNA encoding CD70, CD40 ligand, and constitutively active TLR4 (TriMix) as well as the tumor-associated antigens tyrosinase, gp100, MAGE-A3, or MAGE-C2 were administered together with IPI for four cycles. For 18/39 patients, an additional vaccine was administered before the first IPI administration. We evaluated tumor-associated antigen specific T-cell responses in previously collected peripheral blood mononuclear cells, available from 15 patients. RESULTS: Vaccine-induced enzyme-linked immunospot assay responses detected after in vitro T-cell stimulation were shown in 12/15 patients. Immune responses detected in patients with a complete or partial response were significantly stronger and broader, and exhibited a higher degree of multifunctionality compared with responses in patients with stable or progressive disease. CD8+ T-cell responses from patients with an ongoing clinical response, either elicited by TriMixDC-MEL IPI or on subsequent pembrolizumab treatment, exhibited the highest degree of multifunctionality. CONCLUSIONS: TriMixDC-MEL IPI treatment results in robust CD8+ T-cell responses in a meaningful portion of stage III or IV melanoma patients, and obviously in patients with a clinical response. The levels of polyfunctional and multiantigen T-cell responses measured in patients with a complete response, particularly in patients evidently cured after 5+ years of follow-up, may provide a benchmark for the level of immune stimulation needed to achieve a durable clinical remission. TRIAL REGISTRATION NUMBER: NCT01302496.

MiR-210 Is Overexpressed in Tumor-infiltrating Plasma Cells in Triple-negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a heterogeneous group of breast cancer and is characterized by aggressiveness and poor prognosis. MicroRNA represents a new class of biomarkers, and accumulating evidence indicates that microRNAs contribute to tumorigenesis and cancer metastasis. It has been described that miR-210 is highly expressed in TNBC, and its overexpression had been linked to poor prognosis. In a previous work, we showed that in TNBC miR-210 is expressed in tumor cells and also in the tumor microenvironment (TME), particularly in inflammatory CD45-LCA positive cells. However, the exact identity of these cells remained unknown. In this study, we performed in situ hybridization and immunohistochemistry using validated antibodies for the different specific immune cell markers on adjacent sections of 23 TNBC infiltrated with immune cells. We found that miR-210 expressing cells in the TME were stained positive with CD79a, a B-cell lineage marker. These tumor-infiltrating cells were negative for CD20 and Ki-67 but positive for MUM1 and CD38 and also expressed immunoglobulins, indicating that they are immunoglobulin-producing plasma cells (PCs). To the best of our knowledge, this is the first study demonstrating miR-210 expression in tumor-infiltrating PCs.

Protocadherin Celsr3 is crucial in axonal tract development.

In the embryonic CNS, the development of axonal tracts is required for the formation of connections and is regulated by multiple genetic and microenvironmental factors. Here we show that mice with inactivation of Celsr3, an ortholog of Drosophila melanogaster flamingo (fmi; also known as starry night, stan) that encodes a seven-pass protocadherin, have marked, selective anomalies of several major axonal fascicles, implicating protocadherins in axonal development in the mammalian CNS for the first time. In flies, fmi controls planar cell polarity (PCP) in a frizzled-dependent but wingless-independent manner. The neural phenotype in Celsr3 mutant mice is similar to that caused by inactivation of Fzd3, a member of the frizzled family. Celsr3 and Fzd3 are expressed together during brain development and may act in synergy. Thus, a genetic pathway analogous to the one that controls PCP is key in the development of the axonal blueprint.

Knockout mice reveal a role for P2Y6 receptor in macrophages, endothelial cells, and vascular smooth muscle cells.

P2Y receptors are G-protein-coupled receptors activated by extracellular nucleotides. The P2Y(6) receptor is selectively activated by UDP, and its transcript has been detected in numerous organs, including the spleen, thymus, intestine, blood leukocytes, and aorta. To investigate the biological functions of this receptor, we generated P2Y(6)-null mice by gene targeting. The P2Y(6) knockout (KO) mice are viable and are not distinguishable from the wild-type (WT) mice in terms of growth or fertility. In thioglycollate-elicited macrophages, the production of inositol phosphate in response to UDP stimulation was lost, indicating that P2Y(6) is the unique UDP-responsive receptor expressed by mouse macrophages. Furthermore, the amount of interleukin-6 and macrophage-inflammatory protein-2, but not tumor necrosis factor-alpha, released in response to lipopolysaccharide stimulation was significantly enhanced in the presence of UDP, and this effect was lost in the P2Y(6) KO macrophages. The endothelium-dependent relaxation of the aorta by UDP was abolished in KO P2Y(6) mice. The contractile effect of UDP on the aorta, observed when endothelial nitric-oxide synthase is blocked, was also abolished in P2Y(6)-null mice. In conclusion, we generated P2Y(6)-deficient mice and have shown that these mice have a defective response to UDP in macrophages, endothelial cells, and vascular smooth muscle cells. These observations might be relevant to several physiopathological conditions such as atherosclerosis or hypertension.

Silencing of casein kinase 1 delta reduces migration and metastasis of triple negative breast cancer cells.

The casein kinase 1 delta (CSNK1D) is a conserved serine/threonine protein kinase that regulates diverse cellular processes including cell cycle progression, circadian rhythm, and neurite outgrowth. Aberrant expression of CSNK1D is described in several cancer types including breast cancer, where it is amplified in about 30% of triple negative breast (TNBC). Here, we have investigated the function of CSNK1D in triple negative cancer cell migration and metastasis. By using immunohistochemistry and in situ hybridization, we found that CNSK1D is highly expressed in primary tumor cells and in tumor cells invading lymphatic nodes compared to non-metastatic tumors. In vitro, knock-down of CSNK1D expression with specific shRNAs in the breast cancer cell line MDA-MB-231 markedly inhibited cancer cell proliferation, invasion and migration and affected the expression of the tight junction proteins claudin 1, occludin and the junction adhesion molecule A. In vivo, the inactivation of CSNK1D reduced lung metastasis in MDA-MB-231 breast cancer xenografts. Altogether, our results indicate that the downregulation of CSNK1D expression inhibits the proliferation and reduces the migration and the metastasis of breast cancer cells. As numerous inhibitors of CSNK1D are currently under development, this might represent an attractive therapeutic target for the treatment of TNBC.

The MicroRNA miR-210 Is Expressed by Cancer Cells but Also by the Tumor Microenvironment in Triple-Negative Breast Cancer.

The triple-negative breast cancer (TNBC) subtype occurs in about 15% of breast cancer and is an aggressive subtype of breast cancer with poor outcome. Furthermore, treatment of patients with TNBC is more challenging due to the heterogeneity of the disease and the absence of well-defined molecular targets. Microribonucleic acid (RNA) represents a new class of biomarkers that are frequently dysregulated in cancer. It has been described that the microRNA miR-210 is highly expressed in TNBC, and its overexpression had been linked to poor prognosis. TNBC are often infiltrated by immune cells that play a key role in cancer progression. The techniques traditionally used to analyze miR-210 expression such as next generation sequencing or quantitative real-time polymerase chain reaction (PCR) do not allow the precise identification of the cellular subtype expressing the microRNA. In this study, we have analyzed miR-210 expression by in situ hybridization in TNBC. The miR-210 signal was detected in tumor cells, but also in the tumor microenvironment, in a region positive for the pan-leucocyte marker CD45-LCA. Taken together, our results demonstrate that miR-210 is expressed in tumor cells but also in the tumor microenvironment. Our results also highlight the utility of using complementary approaches to take into account the cellular context of microRNA expression.

ApolipoproteinL1 is expressed in papillary thyroid carcinomas.

The apolipoprotein L (apoL) family has not yet been ascribed any definite patho-physiological function although the conserved BH3 protein domain suggests a role in programmed cell death. As repression of the regular apoptotic program is considered a hallmark of tumor progression, we investigated apoL expression in cancer. We show that the levels of one member of the family, apolipoprotein L1 (apoL1) is higher in papillary thyroid carcinoma compared to normal tissue. A combination of qRTPCR, immunohistochemistry and in situ hybridization allowed us to ascribe this increase to endogenous overexpression in carcinoma cells. Whether apoL1 plays an instrumental role in refraining cell death is the subject of ongoing molecular biology experiments.

Nuclear localization of glutamate-cysteine ligase is associated with proliferation in head and neck squamous cell carcinoma.

Glutathione (GSH) is the keystone of the cellular response toward oxidative stress. Elevated GSH content correlates with increased resistance to chemotherapy and radiotherapy of head and neck (HN) tumors. The purpose of the present cross-sectional study was to evaluate whether the expression of glutamate-cysteine ligase (GCL) accounts for the increased GSH availability observed in HN squamous cell carcinoma (SCC). For that purpose, the messenger (m)RNA levels of the modifier (M) and catalytic (C) subunits of GCL and its putative regulators (namely, nuclear factor erythroid 2-related factor 2, heme oxygenase-1 and nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha) were monitored in 35 surgical resections of untreated HNSCC. The localization of GCLM was evaluated using in situ hybridization and immunohistochemistry. GCLM expression was significantly increased in tumor samples, compared with normal mucosa, both at the mRNA and protein level (P=0.029), but the pathway of GCLM activation remains to be elucidated. Protein expression of GCLM was detected in the cytoplasm and nucleus. GCLM and the proliferation marker Ki-67 displayed a similar distribution, being both mainly expressed at the periphery of tumor lobules. The present study reported increased expression of GCL and the rate-limiting enzyme of GSH synthesis, within HNSCC. The nuclear localization of GCLM and the concomitant expression of Ki-67 suggested that the localization of GSH synthesis contributes to the protection against oxidative stress within hotspots of cell proliferation.

Early forebrain wiring: genetic dissection using conditional Celsr3 mutant mice.

Development of axonal tracts requires interactions between growth cones and the environment. Tracts such as the anterior commissure and internal capsule are defective in mice with null mutation of Celsr3. We generated a conditional Celsr3 allele, allowing regional inactivation. Inactivation in telencephalon, ventral forebrain, or cortex demonstrated essential roles for Celsr3 in neurons that project axons to the anterior commissure and subcerebral targets, as well as in cells that guide axons through the internal capsule. When Celsr3 was inactivated in cortex, subcerebral projections failed to grow, yet corticothalamic axons developed normally, indicating that besides guidepost cells, additional Celsr3-independent cues can assist their progression. These observations provide in vivo evidence that Celsr3-mediated interactions between axons and guidepost cells govern axonal tract formation in mammals.

SOX9 is up-regulated by the transient expression of SRY specifically in Sertoli cell precursors.

The Y chromosome gene Sry encodes a transcription factor required to initiate testis development. The related autosomal gene Sox9 is up-regulated shortly after the onset of Sry transcription and is thought essential for the differentiation of Sertoli cells. The lineage that gives rise to Sertoli cells has its origins within the coelomic epithelium (CE) of the genital ridge, but from cells also able to give rise to an interstitial cell type. It was not known at what point SRY acts in the derivation of this lineage or how the two genes interact. To investigate the identity of the cells expressing Sry, we designed two transgenes driven by the Sry promoter: one gives expression of a stable reporter, human placental alkaline phosphatase (hPLAP), while the second gives expression of a functional Myc-epitope tagged SRY protein (SRYMYC). Taking advantage of lasting hPLAP activity after transcription of the reporter gene has ceased, we could show that SryhPLAP was expressed exclusively in all cells fated to become Sertoli cells. SRYMYC-single-positive cells were first observed in the gonad and not in the CE. Subsequently, they became SRYMYC/SOX9-double-positive, but only for a few hours before turning into SOX9-single-positive cells. After the coelomic epithelial cells migrate into the gonad, there is first a decision to become interstitial or supporting cells, and then the transient expression of SRY in the latter determines their fate as Sertoli cells by up-regulating Sox9.

The gene encoding disabled-1 (DAB1), the intracellular adaptor of the Reelin pathway, reveals unusual complexity in human and mouse.

The Disabled-1 (Dab1) gene encodes a key regulator of Reelin signaling. Reelin is a large glycoprotein secreted by neurons of the developing brain, particularly Cajal-Retzius cells. The DAB1 protein docks to the intracellular part of the Reelin very low density lipoprotein receptor and apoE receptor type 2 and becomes tyrosine-phosphorylated following binding of Reelin to cortical neurons. In mice, mutations of Dab1 and Reelin generate identical phenotypes. In humans, Reelin mutations are associated with brain malformations and mental retardation; mutations in DAB1 have not been identified. Here, we define the organization of Dab1, which is similar in human and mouse. The Dab1 gene spreads over 1100 kb of genomic DNA and is composed of 14 exons encoding the major protein form, some alternative internal exons, and multiple 5'-exons. Alternative polyadenylation and splicing events generate DAB1 isoforms. Several 5'-untranslated regions (UTRs) correspond to different promoters. Two 5'-UTRs (1A and 1B) are predominantly used in the developing brain. 5'-UTR 1B is composed of 10 small exons spread over 800 kb. With a genomic length of 1.1 Mbp for a coding region of 5.5 kb, Dab1 provides a rare example of genomic complexity, which will impede the identification of human mutations.

The reelin signaling pathway: some recent developments.

The Reelin signaling pathway plays a key role in the architectonic development of the central nervous system. Extracellular Reelin binds to receptors of the lipoprotein receptor family and induces tyrosine phosphorylation of the adaptor Dab1. In this paper, we discuss three recent developments. First, we show that the central part of Reelin is involved in receptor binding and signal activation as reflected in Dab1 phosphorylation. Second, we examine the genomic organization, alternative splicing and promoter use of the Dab1 gene, which hint at a particularly complex regulation. Third, we present preliminary studies by in situ hybridization that demonstrate regulated expression of Reelin receptors and Dab1 by radial precursors in the ventricular zone.