BACKGROUND: FOXP1, a transcriptional regulator of lymphocyte development, is abnormally expressed in some human tumors. This study investigated FOXP1-mediated regulation of tumor infiltrating lymphocytes (TIL) in untreated primary breast cancer (BC).
METHODS: FOXP1 expression was analyzed in tissues from primary untreated breast tumors, BC cell lines and the METABRIC gene expression BC dataset. Cytokine and chemokine expression and lymphocyte migration in response to primary tumor supernatants (SN) was compared between FOXP1
FINDING: FOXP1 expression was higher in estrogen receptor positive compared to negative BC. FOXP1
INTERPRETATION: These data identify FOXP1 as an important negative regulator of immune responses in BC via its regulation of cytokine and chemokine expression. FUND: Belgian Fund for Scientific Research (FNRS 3.4513.12F) and Opération Télévie (7.4636.13F and 7.4609.15F), Fonds J.C. Heuson and Fonds Lambeau-Marteaux.

A better characterization of T-cell subsets in the microenvironment of classical Hodgkin lymphoma (cHL) would help to develop immunotherapies. Using multicolor flow cytometry, we identified in 6 of 43 cHL tissue samples a previously unrecognized subset of CD8 T cells coexpressing CXCR5 and inducible T-cell costimulator (ICOS) molecules (CD8

Medullary breast carcinoma (MBC) is a rare subtype of triple-negative breast cancer with specific genomic features within the spectrum of basal-like carcinoma (BLC). In this study of 19 MBCs and 36 non-MBC BLCs, we refined the transcriptomic and genomic knowledge about this entity. Unsupervised and supervised analysis of transcriptomic profiles confirmed that MBC clearly differs from non-MBC BLC, with 92 genes overexpressed and 154 genes underexpressed in MBC compared with non-MBC BLC. Immunity-related pathways are the most differentially represented pathways in MBC compared with non-MBC BLC. The proapoptotic gene BCLG (official name BCL2L14) is by far the most intensely overexpressed gene in MBC. A quantitative RT-PCR validation study conducted in 526 breast tumors corresponding to all molecular subtypes documented the specificity of BCLG overexpression in MBC, which was confirmed at the protein level by immunohistochemistry. We also found that most MBCs belong to the immunomodulatory triple-negative breast cancer subtype. Using pan-genomic analysis, it was found that MBC harbors more losses of heterozygosity than non-MBC BLC. These observations corroborate the notion that MBC remains a distinct entity that could benefit from specific treatment strategies (such as deescalation or targeted therapy) adapted to this rare tumor type.

Evidence from mouse chronic viral infection models suggests that CD8

The multifunctional cytokine TGF-β crucially participates in breast cancer (BCa) metastasis and works differently in the disease stages, thus contributing in BCa progression. We address connections between TGF-β and the stem cell-related transcription factor (TF) Oct4 in BCa. In 147 BCa patients with infiltrating duct carcinoma, we identified a significantly higher number of cases with both moderate/high Oct4 expression and high TGF-β in late stages compared to early stages of the disease. In vitro studies showed that TGF-β elevated Oct4 expression, which in turn, regulated Epithelial-to-Mesenchymal transition (EMT)-regulatory gene (Snail and Slug) expression, migratory ability, chemotactic invasiveness and extracellular matrix (ECM) degradation potential of BCa cells. Putative binding sites for Oct4 on the snail, slug and cxcl13 promoters and for Smad3 on the snail and slug promoters were identified. Promoter activities of snail and slug were greater in dual-treated cells than only TGF-β-treated or Oct4-overexpressing cells. CXCL13 mRNA fold changes, however, were low in cells induced with TGF-β, compared to dual-treated or Oct4-overexpressing cells. Our co-IP studies confirmed that Oct4 and Smad3 form heterodimers that recognize specific promoter sequences to promote Snail and Slug expression, but which in turn, indirectly inhibits Smad3-mediated repression of CXCL13 expression, allowing Oct4 to act as a positive TF for CXCL13. Taken together, these data suggest that TGF-β signaling and Oct4 cooperate to induce expression of EMT-related genes Snail, Slug and CXCL13, which accelerates disease progression, particularly in the late stages, and may indicate a poor prognosis for BCa patients.

OBJECTIVE: Tumour-infiltrating lymphocytes (TILs) favour survival in human colorectal cancer (CRC). Chemotactic factors underlying their recruitment remain undefined. We investigated chemokines attracting T cells into human CRCs, their cellular sources and microenvironmental triggers.
DESIGN: Expression of genes encoding immune cell markers, chemokines and bacterial 16S ribosomal RNA (16SrRNA) was assessed by quantitative reverse transcription-PCR in fresh CRC samples and corresponding tumour-free tissues. Chemokine receptor expression on TILs was evaluated by flow cytometry on cell suspensions from digested tissues. Chemokine production by CRC cells was evaluated in vitro and in vivo, on generation of intraperitoneal or intracecal tumour xenografts in immune-deficient mice. T cell trafficking was assessed on adoptive transfer of human TILs into tumour-bearing mice. Gut flora composition was analysed by 16SrRNA sequencing.
RESULTS: CRC infiltration by distinct T cell subsets was associated with defined chemokine gene signatures, including CCL5, CXCL9 and CXCL10 for cytotoxic T lymphocytes and T-helper (Th)1 cells; CCL17, CCL22 and CXCL12 for Th1 and regulatory T cells; CXCL13 for follicular Th cells; and CCL20 and CCL17 for interleukin (IL)-17-producing Th cells. These chemokines were expressed by tumour cells on exposure to gut bacteria in vitro and in vivo. Their expression was significantly higher in intracecal than in intraperitoneal xenografts and was dramatically reduced by antibiotic treatment of tumour-bearing mice. In clinical samples, abundance of defined bacteria correlated with high chemokine expression, enhanced T cell infiltration and improved survival.
CONCLUSIONS: Gut microbiota stimulate chemokine production by CRC cells, thus favouring recruitment of beneficial T cells into tumour tissues.

Tertiary lymphoid structures (TLSs) display phenotypic and functional characteristics of secondary lymphoid organs, and often develop in tissues affected by chronic inflammation, as well as in certain inflammation-associated cancers where they are prognostic of improved patient survival. However, the mechanisms that govern the development of tumour-associated TLSs remain ill-defined. Here, we observed tumour-associated TLSs in a preclinical mouse model (gp130

PURPOSE: The main target of the present research was to examine the antitumor properties of aesculetin in human acute myeloid leukemia cancer cells (THP-1) and peripheral blood mono-nucleated cells (PBMCs) (used as normal cell line model) along with the determination of its effects on induction of apoptosis, inhibition of cancer cell migration and changes in Bcl-2/Bax protein expressions.
METHODS: MTT colorimetric bioassay was performed to study the impact of this natural compound on cytotoxicity of both cell types. Moreover, transmission electron microscopy (TEM), inverted phase contrast and fluorescence microscopic techniques were used to study the effects on cell morphology and cellular ultrastructural details connected with apoptosis. The effects of aesculetin on Bcl-2/Bax protein expressions were assessed by Western blot method.
RESULTS: Selective and dose-dependent antiproliferative activity of aesculetin in human acute myeloid leukemia cancer cells was observed. However, the compound did not induce significant cell growth inhibition of PBMCs, which were used as normal cell controls. Fluorescence and inverted phase contrast microscopic techniques revealed that aesculetin led to morphological changes suggestive of apoptosis (cell shrinkage, chromatin abridgment and membrane blebbing). TEM analysis showed that aesculetin led to fragmented plasma membrane along with appearance of spherical projections (apoptotic bodies). The wound scratch widened after aesculetin treatment, indicating that aesculetin exhibits anticancer effects by suppressing the cancer cell migration. Aesculetin led to significant and dose-dependent reduction in the Bcl-2 expression while the expression of Bax was significantly enhanced resulting in overall reduction of Bcl-2/Bax ratio.
CONCLUSION: The results of the present work revealed that aesculetin exhibits selective anticancer effects in THP-1 human leukemia cells without causing much cytotoxicity in PBMCs. It also led to significant apoptosis induction, inhibition of cancer cell migration and decrease in Blc-2/Bax ratio.

BACKGROUND: Differential co-expression (DCX) signifies change in degree of co-expression of a set of genes among different biological conditions. It has been used to identify differential co-expression networks or interactomes. Many algorithms have been developed for single-factor differential co-expression analysis and applied in a variety of studies. However, in many studies, the samples are characterized by multiple factors such as genetic markers, clinical variables and treatments. No algorithm or methodology is available for multi-factor analysis of differential co-expression.
RESULTS: We developed a novel formulation and a computationally efficient greedy search algorithm called MultiDCoX to perform multi-factor differential co-expression analysis. Simulated data analysis demonstrates that the algorithm can effectively elicit differentially co-expressed (DCX) gene sets and quantify the influence of each factor on co-expression. MultiDCoX analysis of a breast cancer dataset identified interesting biologically meaningful differentially co-expressed (DCX) gene sets along with genetic and clinical factors that influenced the respective differential co-expression.
CONCLUSIONS: MultiDCoX is a space and time efficient procedure to identify differentially co-expressed gene sets and successfully identify influence of individual factors on differential co-expression.

Angioimmunoblastic T-cell lymphoma (AITL) has been classified as a subtype of mature T-cell neoplasms. The recent revision of the WHO classification proposed a new category of nodal T-cell lymphoma with follicular helper T (TFH)-cell phenotype, which was classified into three diseases: AITL, follicular T-cell lymphoma, and nodal peripheral T-cell lymphoma with TFH phenotype. These lymphomas are defined by the expression of TFH-related antigens, CD279/PD-1, CD10, BCL6, CXCL13, ICOS, SAP, and CXCR5. Although recurrent mutations in TET2, IDH2, DNMT3A, RHOA, and CD28, as well as gene fusions, such as ITK-SYK and CTLA4-CD28, were not diagnostic criteria, they may be considered as novel criteria in the near future. Notably, premalignant mutations, tumor-specific mutations, and mutations specific to tumor-infiltrating B cells were identified in AITL. Thus, multi-step and multi-lineage genetic events may lead to the development of AITL.

In solid tumors, the presence of lymph node-like structures called tertiary lymphoid structures (TLS) is associated with improved patient survival. However, little is known about how TLS develop in cancer, how their function affects survival, and whether they are affected by cancer therapy. In this study, we used multispectral microscopy, quantitative pathology, and gene expression profiling to analyze TLS formation in human lung squamous cell carcinoma (LSCC) and in an experimental model of lung TLS induction. We identified a niche of CXCL13

We investigated thoroughly the effect of lncRNA PART1 on prostate cancer cells proliferation and apoptosis, through regulating toll-like receptor (TLR) pathways. LncRNA PART1 expression was also examined by quantitative real-time polymerase chain reactions (qRT-PCR) in human tissues and the cells lines LNCaP and PC3. After transfection with si-PART1 or control constructs, the cell viability was measured by MTS and colony formation assays. In addition, the apoptosis rate of the prostate cancer cells was validated by TUNEL staining. Relationships between lncRNA PART1 expression and TLR pathway genes were demonstrated by qRT-PCR and Western blotting. High levels of lncRNA PART1 expression were correlated with advanced cancer stage and predication of poor survival. LncRNA PART1 levels was increased in PCa cells treated with 5α-dihydrotestosterone (DHT), confirming PART1 was directly induced by androgen. Moreover, down-regulation of lncRNA PART1 inhibited prostate cancer cell proliferation and accelerated cell apoptosis. In addition, lncRNA PART1 induced downstream genes expression in TLR pathways including TLR3, TNFSF10 and CXCL13 to further influence prostate cancer cells, indicating its carcinogenesis on prostate cancer. LncRNA PART1 promoted cell proliferation ability and apoptosis via the inhibition of TLR pathways in prostate cancer. LncRNA PART1 could hence be considered as a new target in the treatment of prostate cancer.

Oral tongue squamous cell carcinoma (OTSCC) has a high incidence and is associated with a high mortality rate. Studies regarding the potential molecular mechanism of OTSCC in the tumor microenvironment (TME) are required. The present study aimed to perform bioinformatic analysis to identify important nodes, clusters and functional pathways during tongue carcinogenesis in the TME. After downloading the gene expression data of GSE42780, differentially expressed genes (DEGs) among carcinoma, dysplastic and normal samples in epithelia and fibroblasts were identified using the affy and limma packages with R version 3.3. Subsequently, the Database for Annotation, Visualization and Integrated Discovery was employed to perform Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Furthermore, a protein‑protein interaction (PPI) network was constructed by using the Search Tool for the Retrieval of Interacting Genes/Proteins and analyzed by Cytoscape software. In total, 85 DEGs were identified for tongue epithelia and 46 DEGs were identified for fibroblasts. Neutrophil chemotaxis and inflammatory response from GO, and cytokine‑cytokine receptor interaction from KEGG were enriched for epithelia and fibroblasts. The PPI network revealed that C‑X‑C motif chemokine ligand (Cxcl)1, Cxcl10, Cxcl13, Cxcl2 and pro‑platelet basic protein were a key cluster for epithelia, and interleukin (Il)1β, Il1 receptor 2, Il1a and Il1 receptor antagonist were a key cluster for fibroblasts. Therefore, the results indicate that fibroblasts and cytokines associated with an inflammatory immune response contributed substantially to tongue carcinogenesis in the TME, which is useful for the development of OTSCC targeted therapy. However, further investigation is required to elucidate the molecular and cellular mechanisms underlying the inflammatory immune network in the TME.

Background: In patients with triple-negative breast cancer (TNBC), the extent of tumor-infiltrating lymphocytes (TILs) in the residual disease after neoadjuvant chemotherapy (NACT) is associated with better prognosis. Our objective was to develop a gene signature from pretreatment samples to predict the extent of TILs after NACT and then to test its prognostic value on survival.
Patients and methods: Using 99 pretreatment samples, we generated a four-gene signature associated with high post-NACT TILs. Prognostic value of the signature on distant relapse-free survival (DRFS) was first assessed on the training set (n = 99) and then on an independent validation set (n = 115).
Results: A four-gene signature combining the expression levels of HLF, CXCL13, SULT1E1, and GBP1 was developed in baseline samples to predict the extent of lymphocytic infiltration after NACT. In a multivariate analysis performed on the training set, this signature was associated with DRFS [hazard ratio (HR): 0.28, for a one-unit increase in the value of the four-gene signature, 95% confidence interval (CI): 0.13-0.63)]. In a multivariate analysis performed on an independent validation set, the four-gene signature was significantly associated with DRFS (HR: 0.17, 95% CI: 0.06-0.43). The four-gene signature added significant prognostic information when compared with the clinicopathologic pretreatment model (likelihood ratio test in the training set P = 0.004 and in the validation set P = 0.002).
Conclusions: A four-gene signature predicts high levels of TILs after anthracycline-containing NACT and outcome in patients with TNBC and adds prognostic information to a clinicopathological model at diagnosis.

BACKGROUND: The role of different subtypes of immune cells is still a matter of debate.
METHODS: We compared the prognostic relevance for metastasis-free survival (MFS) of a B-cell signature (BS), a T-cell signature (TS), and an immune checkpoint signature (CPS) in node-negative breast cancer (BC) using mRNA expression. Microarray-based gene-expression data were analyzed in six previously published cohorts of node-negative breast cancer patients not treated with adjuvant therapy (n = 824). The prognostic relevance of the individual immune markers was assessed using univariate analysis. The amount of independent prognostic information provided by each immune signature was then compared using a likelihood ratio statistic in the whole cohort as well as in different molecular subtypes.
RESULTS: Univariate Cox regression in the whole cohort revealed prognostic significance of CD4 (HR 0.66, CI 0.50-0.87, p = 0.004), CXCL13 (HR 0.86, CI 0.81-0.92, p < 0.001), CD20 (HR 0.76, CI 0.64-0.89, p = 0.001), IgκC (HR 0.81, CI 0.75-0.88, p < 0.001), and CTLA-4 (HR 0.67, CI 0.46-0.97, p = 0.032). Multivariate analyses of the immune signatures showed that both TS (p < 0.001) and BS (p < 0.001) showed a significant prognostic information in the whole cohort. After accounting for clinical-pathological variables, TS (p < 0.001), BS (p < 0.05), and CPS (p < 0.05) had an independent effect for MFS. In subgroup analyses, the prognostic effect of immune cells was most pronounced in HER2+ BC: BS as well as TS showed a strong association with MFS when included first in the model (p < 0.001).
CONCLUSION: Immune signatures provide subtype-specific additional prognostic information over clinical-pathological variables in node-negative breast cancer.

PKCε, an oncogenic member of the PKC family, is aberrantly overexpressed in epithelial cancers. To date, little is known about functional interactions of PKCε with other genetic alterations, as well as the effectors contributing to its tumorigenic and metastatic phenotype. Here, we demonstrate that PKCε cooperates with the loss of the tumor suppressor Pten for the development of prostate cancer in a mouse model. Mechanistic analysis revealed that PKCε overexpression and Pten loss individually and synergistically upregulate the production of the chemokine CXCL13, which involves the transcriptional activation of the CXCL13 gene via the non-canonical nuclear factor κB (NF-κB) pathway. Notably, targeted disruption of CXCL13 or its receptor, CXCR5, in prostate cancer cells impaired their migratory and tumorigenic properties. In addition to providing evidence for an autonomous vicious cycle driven by PKCε, our studies identified a compelling rationale for targeting the CXCL13-CXCR5 axis for prostate cancer treatment.

Chronic lymphocytic leukemia (CLL), the most common leukemia among adults in the western world, is characterized by a progressive accumulation of relatively mature CD5+ B cells in peripheral blood, lymph nodes and bone marrow. Despite much recent advancement in therapy, CLL is still incurable. Lymph nodes and bone marrow represent sanctuary sites preserving leukemic cells from spontaneous or drug-induced apoptosis, and infiltration of leukemic cells in these districts correlates with clinical stages and prognosis. The central role played by the microenvironment in the disease has become increasingly clear. Different chemokines (CXCL12, CXCL13, CCL19, CCL21) may in fact participate in attracting CLL cells into bone marrow and lymph nodes, where various factors, such as IL-15 and CXCL12, enhance leukemic cells survival. Recently, we have suggested that hepatocyte growth factor (HGF), produced by microenvironmental stromal cells, can contribute to CLL pathogenesis. We have demonstrated that HGF exerts a double effect on CLL B cells through the interaction with its receptor c- MET; HGF, infact, protects CLL B cells, which are c-MET+, from apoptosis, and also polarizes mono/macrophages towards the M2 phenotype, thus facilitating the evasion of the CLL clone from immune control. This double effect appears mediated by the activation of two major signaling pathways: STAT3TYR705 and AKT. The aim of this review is to summarize data on HGF and c-MET expression in normal B cells and in B cell malignancies, with a particular emphasis on our results obtained in CLL. Altogether, the observations described here suggest that the HGF/c-MET axis may have a prominent role in malignancy progression further indicating novel potential therapeutic options aimed to block HGF-induced signaling pathways in B lymphoproliferative disorders.

Follicular dendritic cell (FDC)-sarcoma is a rare neoplasm with morphologic and phenotypic features of FDCs. It shows an extremely heterogeneous morphology, therefore, its diagnosis relys on the phenotype of tumor cells. Aim of the present study was the identification of new specific markers for FDC-sarcoma by whole transcriptome sequencing (WTS). Candidate markers were selected based on gene expression level and biological function. Immunohistochemistry was performed on reactive tonsils, on 22 cases of FDC-sarcomas and 214 control cases including 114 carcinomas, 87 soft tissue tumors, 5 melanomas, 5 thymomas and 3 interdigitating dendritic cell sarcomas. FDC secreted protein (FDCSP) and Serglycin (SRGN) proved to be specific markers of FDC and related tumor. They showed better specificity and sensitivity values than some well known markers used in FDC sarcoma diagnosis (specificity: 98.6%, and 100%, respectively; sensitivity: 72.73% and 68.18%, respectively). In our cohorts CXCL13, CD21, CD35, FDCSP and SRGN were the best markers for FDC-sarcoma diagnosis and could discriminate 21/22 FDC sarcomas from other mesenchymal tumors by linear discriminant analysis. In summary, by WTS we identified two novel FDC markers and by the analysis of a wide cohort of cases and controls we propose an efficient marker panel for the diagnosis of this rare and enigmatic tumor.

In the era of primary vaccination against HPV and at the beginning of the low prevalence of cervical lesions, introduction of screening methods that can distinguish between low- and high-grade lesions is necessary in order to maintain the positive predictive value of screening. This case-control study included 562 women who attended cervical screening or were referred for colposcopy and 140 disease free controls, confirmed by histology and/or cytology. The cases were stratified by age. Using routine exfoliated liquid based cytological samples RT-PCR measurements of biomarker genes, high-risk HPV testing and liquid based cytology were performed and used to evaluate different testing protocols including sets of genes/tests with different test cut-offs for the diagnostic panels. Three new panels of cellular biomarkers for improved triage of hrHPV positive women (diagnostic panel) and for prognostic assessment of CIN lesions were proposed. The diagnostic panel (PIK3AP1, TP63 and DSG3) has the potential to distinguish cytologically normal hrHPV+ women from hrHPV+ women with CIN2+. The prognostic gene panels (KRT78, MUC5AC, BPIFB1 and CXCL13, TP63, DSG3) have the ability to differentiate hrHPV+ CIN1 and carcinoma cases. The diagnostic triage panel showed good likelihood ratios for all age groups. The panel showed age-unrelated performance and even better diagnostic value under age 30, a unique feature among the established cervical triage tests. The prognostic gene-panels demonstrated good discriminatory power and oncogenic, anti-oncogenic grouping of genes. The study highlights the potential for the gene expression panels to be used for diagnostic triage and lesion prognostics in cervical cancer screening.

PURPOSE: The tertiary lymphoid structure (TLS) is an important source of tumor-infiltrating lymphocytes (TILs), which have a strong prognostic and predictive value in triple-negative breast cancer (TNBC). A previous study reported that the levels of
MATERIALS AND METHODS: A total of 108 TNBC patients treated with neoadjuvant chemotherapy (NAC) were studied. The amounts of TILs and TLSs were measured histopathologically using hematoxylin and eosin-stained slides. The HEV densities and TIL subpopulations were measured by immunohistochemistry for MECA79, CD3, CD8, and CD20.
RESULTS: The mean number of HEVs in pre-NAC biopsies was 12 (range, 0 to 72). The amounts of TILs and TLSs, HEV density, and
CONCLUSION: MECA79-positive HEV density in pre-NAC biopsies is an objective and quantitative surrogate marker of TLS and might be a valuable tool for predicting pCR of TNBC in routine pathology practice.

Cancers often evade immune surveillance by adopting peripheral tissue- tolerance mechanisms, such as the expression of programmed cell death ligand 1 (PD-L1), the inhibition of which results in potent antitumor immunity. Here, we show that cyclin-dependent kinase 5 (Cdk5), a serine-threonine kinase that is highly active in postmitotic neurons and in many cancers, allows medulloblastoma (MB) to evade immune elimination. Interferon-γ (IFN-γ)-induced PD-L1 up-regulation on MB requires Cdk5, and disruption of Cdk5 expression in a mouse model of MB results in potent CD4(+) T cell-mediated tumor rejection. Loss of Cdk5 results in persistent expression of the PD-L1 transcriptional repressors, the interferon regulatory factors IRF2 and IRF2BP2, which likely leads to reduced PD-L1 expression on tumors. Our finding highlights a central role for Cdk5 in immune checkpoint regulation by tumor cells.

Whole-genome sequencing has identified highly prevalent somatic mutations including MYD88, CXCR4, and ARID1A in Waldenström macroglobulinemia (WM). The impact of these and other somatic mutations on transcriptional regulation in WM remains to be clarified. We performed next-generation transcriptional profiling in 57 WM patients and compared findings to healthy donor B cells. Compared with healthy donors, WM patient samples showed greatly enhanced expression of the VDJ recombination genes DNTT, RAG1, and RAG2, but not AICDA Genes related to CXCR4 signaling were also upregulated and included CXCR4, CXCL12, and VCAM1 regardless of CXCR4 mutation status, indicating a potential role for CXCR4 signaling in all WM patients. The WM transcriptional profile was equally dissimilar to healthy memory B cells and circulating B cells likely due increased differentiation rather than cellular origin. The profile for CXCR4 mutations corresponded to diminished B-cell differentiation and suppression of tumor suppressors upregulated by MYD88 mutations in a manner associated with the suppression of TLR4 signaling relative to those mutated for MYD88 alone. Promoter methylation studies of top findings failed to explain this suppressive effect but identified aberrant methylation patterns in MYD88 wild-type patients. CXCR4 and MYD88 transcription were negatively correlated, demonstrated allele-specific transcription bias, and, along with CXCL13, were associated with bone marrow disease involvement. Distinct gene expression profiles for patients with wild-type MYD88, mutated ARID1A, familial predisposition to WM, chr6q deletions, chr3q amplifications, and trisomy 4 are also described. The findings provide novel insights into the molecular pathogenesis and opportunities for targeted therapeutic strategies for WM.

We have previously shown that regulatory T cells (Tregs) infiltrating follicular lymphoma lymph nodes are quantitatively and qualitatively different than those infiltrating normal and reactive nodes. To gain insight into how such Treg populations differ, we performed RNA sequence (RNAseq) analyses on flow sorted Tregs from all three sources. We identify several molecules that could contribute to the observed increased suppressive capacity of follicular lymphoma nodal tregs, including upregulation of CTLA-4, IL-10, and GITR, all confirmed by protein expression. In addition, we identify, and confirm functionally, a novel mechanism by which Tregs target to and accumulate within a human tumor microenvironment, through the down regulation of S1PR1, SELL (L-selectin) and CCR7, potentially resulting in greater lymph node retention. In addition we identify and confirm functionally the upregulation of the chemokine receptor CXCR5 as well as the secretion of the chemokines CXCL13 and IL-16 demonstrating the unique ability of the follicular derived Tregs to localize and accumulate within not only the malignant lymph node, but also localize and accumulate within the malignant B cell follicle itself. Such findings offer significant new insights into how follicular lymphoma nodal Tregs may contribute to the biology of follicular lymphoma and identify several novel therapeutic targets.

Angioimmunoblastic T-cell lymphoma (AITL) is an infrequent subtype of peripheral T-cell lymphoma derived from follicular helper T cells. Recently, a somatic G17V RHOA gene mutation has been reported. In this article, we examined the RHOA G17V mutation in 18 cases of AITL by 3 different techniques of Sanger sequencing, fully automated SNP genotyping, and deep sequencing, using routine diagnostic formalin-fixed paraffin-embedded tissue. The RHOA G17V mutation was detected in 10 cases (56%). Among the 10 mutated cases, 8 cases were detected by all 3 methods. The status of RHOA mutation was subsequently compared with the clinicopathologic characteristics of AITL. RHOA-mutated AITL (10 cases) was clinically characterized by high serum IL-2R and a poor ECOG performance status. By immunohistochemistry, expression of CD10, PD-1, CXCL13, and CCR4 and a wide distribution of CD21(+) follicular dendritic cells were observed in RHOA-mutated cases. Among these, CCR4 expression and the CD21(+) network in RHOA-mutated AITL cases were more extensive than in the RHOA mutation-negative AITL cases (P<0.05). Thus, RHOA-mutated AITL cases are more characteristic of follicular helper T cells, and the presence of such a mutation is an important marker for AITL.

OBJECTIVE: To evaluate treatment response, survival, and the associations between KRAS mutation status and tumour expression levels of BRCA1, TYMS and SRC retrospectively in a cohort of patients with non-small cell lung cancer (NSCLC), treated exclusively with conjunctive platinum-based doublet chemotherapy.
METHODS: KRAS mutation status was determined via amplification refractory mutation and multiple quantitative polymerase chain reaction (PCR) analysis. Tumour expression levels of BRCA1, TYMS and SRC were determined via real time quantitative PCR.
RESULTS: Patients with KRAS mutations (n = 3) had significantly shorter survival duration than patients with wild type KRAS (n = 42). Tumour expression levels of BRCA1 and TYMS, but not SRC, were significantly lower in patients with, than in those without, KRAS mutations. Tumour expression level of BRCA1 was positively correlated with survival duration.
CONCLUSIONS: KRAS mutation status and BRCA1 tumour expression are potential biomarkers for tailoring chemotherapy and predicting clinical outcome.

UNLABELLED: Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer-related deaths worldwide, with an exceedingly low 5-year survival rate. PDAC tumors are characterized by an extensive desmoplastic stromal response and hypovascularity, suggesting that tumor hypoxia could regulate PDAC initiation and/or progression. Using a well-defined, autochthonous Kras(G12D)-driven murine model, as well as human tumors, we demonstrate that hypoxia and stabilization of hypoxia-inducible factor 1α (HIF1α), a principal mediator of hypoxic adaptation, emerge early during preinvasive stages of PDAC. Surprisingly, pancreas-specific Hif1a deletion drastically accelerated Kras(G12D)-driven pancreatic neoplasia and was accompanied by significant increases in intrapancreatic B lymphocytes, featuring prominent influx of a rare "B1b" B-cell subtype. Finally, treatment of HIF1α-deficient mice with B cell-depleting αCD20 monoclonal antibodies inhibited progression of pancreatic intraepithelial neoplasia (PanIN). Our data reveal a previously unrecognized role for B cells in promoting pancreatic tumorigenesis and implicate HIF1α as a critical regulator of PDAC development.
SIGNIFICANCE: We show here that pancreas-specific Hif1a deletion promotes PDAC initiation, coincident with increased intrapancreatic accumulation of B cells, and that B-cell depletion suppresses pancreatic tumorigenesis. We therefore demonstrate a protective role for HIF1α in pancreatic cancer initiation and uncover a previously unrecognized function of B cells.

Chemokines are a family of small, structurally related cytokines with chemoattractant and activation properties. In breast cancer, both epithelial cancer cells and cells within the microenvironment secrete chemokines with either tumor-promoting or anti-malignant potential. The equilibrium between these two chemokine activities plays a key role in the biology of the developing tumor, including its ability to metastasize. Here we evaluated the expression of chemokines in breast tumors and the plasma of breast cancer patients before treatment in order to identify a blood-based signature that could distinguish between malignant and non-malignant processes. We screened the mRNA expression of chemokine genes using cDNA microarray on homogenous, laser-capture microdissected breast cancer specimens. Further, using a protein array approach, we determined the levels of selected chemokines in the plasma of patients with breast cancer, benign breast tumors and healthy women. Finally, we analyzed the association between the levels of chemokines in breast and blood samples with the pathological characteristics of the disease. At mRNA level, 27 chemokines and 11 chemokine receptors were differentially expressed in cancers when compared with normal breast tissue. When compared to benign tumors, the only chemokine significantly upregulated in cancers was CXCL10. At protein level, with the exception of CXCL13, nine out of the ten selected chemokines (CCL2, CCL7, CCL18, CCL22, CXCL8, CXCL9, CXCL10, CXCL11 and osteoprotegerin) were significantly overexpressed in the plasma of breast cancers patients compared to healthy controls. After grouping, CXCL8, CXCL9 and CCL22 proved to be significant predictors for breast cancers as compared to healthy controls in a model of logistic regression. We found upregulation of CXCL8, CXCL11 and CXCL9 in triple negative carcinomas, CXCL9 in low proliferative carcinomas, and CXCL10, CCL7 and osteoprotegerin in poorly differentiated carcinomas. Furthermore, CXCL9 was overexpressed in lymph node negative tumors, whereas CXCL8 and CCL18 were higher in advanced stage carcinomas. We identified a panel of chemokines dysregulated in breast cancer that could be further investigated as prospective novel diagnostic markers or for therapeutic and prognostic applications.

BACKGROUND: Sensitizing effects of poly-ADP-ribose polymerase inhibitors have been studied in several preclinical models, but a clear understanding of predictive biomarkers is lacking. In this study, in vivo efficacy of veliparib combined with temozolomide (TMZ) was evaluated in a large panel of glioblastoma multiforme (GBM) patient-derived xenografts (PDX) and potential biomarkers were analyzed.
METHODS: The efficacy of TMZ alone vs TMZ/veliparib was compared in a panel of 28 GBM PDX lines grown as orthotopic xenografts (8-10 mice per group); all tests of statistical significance were two-sided. DNA damage was analyzed by γH2AX immunostaining and promoter methylation of DNA repair gene O6-methylguanine-DNA-methyltransferase (MGMT) by Clinical Laboratory Improvement Amendments-approved methylation-specific polymerase chain reaction.
RESULTS: The combination of TMZ/veliparib statistically significantly extended survival of GBM models (P < .05 by log-rank) compared with TMZ alone in five of 20 MGMT-hypermethylated lines (average extension in median survival = 87 days, range = 20-150 days), while the combination was ineffective in six MGMT-unmethylated lines. In the MGMT promoter-hypermethylated GBM12 line (median survival with TMZ+veliparib = 189 days, 95% confidence interval [CI] = 59 to 289 days, vs TMZ alone = 98 days, 95% CI = 49 to 210 days, P = .04), the profound TMZ-sensitizing effect of veliparib was lost when MGMT was overexpressed (median survival with TMZ+veliparib = 36 days, 95% CI = 28 to 38 days, vs TMZ alone = 35 days, 95% CI = 32 to 37 days, P = .87), and a similar association was observed in two nearly isogenic GBM28 sublines with an intact vs deleted MGMT locus. In comparing DNA damage signaling after dosing with veliparib/TMZ or TMZ alone, increased phosphorylation of damage-responsive proteins (KAP1, Chk1, Chk2, and H2AX) was observed only in MGMT promoter-hypermethylated lines.
CONCLUSION: Veliparib statistically significantly enhances (P < .001) the efficacy of TMZ in tumors with MGMT promoter hypermethylation. Based on these data, MGMT promoter hypermethylation is being used as an eligibility criterion for A071102 (NCT02152982), the phase II/III clinical trial evaluating TMZ/veliparib combination in patients with GBM.

To explore the mechanisms of MDSC trafficking and accumulation during tumor progression. In this study, we report significant CD40 upregulation in tumor-infiltrating MDSC when compared with splenic MDSC. Microarray analyses comparing CD40(high) and CD40l(ow) MDSC revealed 1872 differentially expressed genes, including CD83, CXCR5, BTLA, CXCL9, TLR1, FLT3, NOD2 and CXCL10. In vivo experiments comparing wild-type (WT) and CD40 knockout (KO) mice demonstrated that CD40 critically regulates CXCR5 expression. Consistently, the transwell analysis confirmed the essential role of CXCR5-CXCL13 crosstalk in the migration of CD40+ MDSC toward gastric cancer. Furthermore, more MDSC accumulated in the gastric cancers of WT mice when compared with KO mice, and the WT tumors mostly contained CD40+ cells. Functionally, tumors grew faster in WT than KO mice. In conclusion, we demonstrate that CD40 expression upregulates the chemokine receptor CXCR5 and promotes MDSC migration toward and accumulation within cancer. Therefore, this study provides preliminary evidence that CD40 may stimulate tumor growth by enabling immune evasion via MDSC recruitment and inhibition of T cell expansion.

The prognostic significance of tumor-infiltrating lymphocytes and immune signals has been described previously in triple-negative breast cancer (TNBC). Furthermore, recent studies have shown that immunologic parameters are relevant for the response to neoadjuvant chemotherapy (NAC) in breast cancer as well as for outcomes after adjuvant chemotherapy. However, immune signals are variable, and which signals are important is largely unknown. We, therefore, evaluated the expression of immune-related genes in TNBC treated with NAC. We retrospectively evaluated biopsy tissue from 55 patients with primary TNBC treated with NAC (anthracycline, cyclophosphamide, and docetaxel) against the NanoString nCounter GX Human Immunology Panel (579 immune-related genes). Higher expression of cytotoxic molecules, T cell receptor signaling pathway components, cytokines related to T helper cell type 1 (Th1), and B cell markers was associated with a pathologic complete response (pCR). Higher expression of NFKB1, MAPK1, TRAF1, CXCL13, GZMK, and IL7R was significantly associated with pCR, higher Miller-Payne grade, and lower residual cancer burden class. Expression of NFKB1, TRAF1, and CXCL13genes, in particular, was significantly correlated with a longer disease-free survival rate. Conversely, patients those who failed to achieve a pCR showed increased expression of genes related to neutrophils. Higher expression of cytotoxic molecules, T cell receptor signaling pathway components, Th1-related cytokines, and B cell markers is correlated with pCR and survival in TNBC patients treated with NAC. Our results suggest that the activation status of neutrophils may provide additional predictive information for TNBC patients treated with NAC.