The alternative RelB NF-κB subunit is a novel critical player in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most frequent lymphoid malignancy affecting adults. The NF-κB transcription factor family is activated by 2 main pathways, the canonical and the alternative NF-κB activation pathway, with different functions. The alternative NF-κB pathway leads to activation of the transcriptionally active RelB NF-κB subunit. Alternative NF-κB activation status and its role in DLBCL pathogenesis remain undefined. Here, we reveal a frequent activation of RelB in a large cohort of DLBCL patients and cell lines, independently of their activated B-cell-like or germinal center B-cell-like subtype. RelB activity defines a new subset of patients with DLBCL and a peculiar gene expression profile and mutational pattern. Importantly, RelB activation does not correlate with the MCD genetic subtype, enriched for activated B-cell-like tumors carrying MYD88L265P and CD79B mutations that cooperatively activate canonical NF-κB, thus indicating that current genetic tools to evaluate NF-κB activity in DLBCL do not provide information on the alternative NF-κB activation. Furthermore, the newly defined RelB-positive subgroup of patients with DLBCL exhibits a dismal outcome after immunochemotherapy. Functional studies revealed that RelB confers DLBCL cell resistance to DNA damage-induced apoptosis in response to doxorubicin, a genotoxic agent used in the front-line treatment of DLBCL. We also show that RelB positivity is associated with high expression of cellular inhibitor of apoptosis protein 2 (cIAP2). Altogether, RelB activation can be used to refine the prognostic stratification of DLBCL and may contribute to subvert the therapeutic DNA damage response in a segment of patients with DLBCL.

Detection of Nine Oncogenes Amplification in Lung and Colorectal Cancer Formalin-Fixed Paraffin-Embedded Tissue Samples using Combined Next-Generation Sequencing-Based Script and Digital Droplet Polymerase Chain Reaction.

INTRODUCTION: Gene copy number variations have theranostic impact and require reliable methods for their identification. We aimed to evaluate the reliability of combined next-generation sequencing (NGS) and digital droplet PCR (ddPCR) method for gene amplification evaluation. METHODS: We conducted a retrospective multicentric observational study. MET/ERBB2 amplifications were assessed in patients with lung or colorectal carcinoma (cohort A), from 2016 to 2020, by fluorescence in situ hybridization (FISH)/immunohistochemistry (IHC), NGS and ddPCR. NGS-based script and ddPCR were then used to detect amplifications of 7 additional oncogenes (EGFR, KRAS, BRAF, FGFR1, FGFR2, FGFR3, PIK3CA) in a cohort of patients (cohort B). RESULTS: 55 patients (9 control, 25 ERBB2-amplified and 21 MET-amplified) out of 3779 patients tested were included in cohort A. Correlation coefficient between NGS-based script and FISH/IHC results were .88 for MET (P < .001) and .89 (P < .001) for ERBB2. Using a threshold ratio of 1.56 with the NGS-based script, the sensitivity was 100% for both genes and the specificity 69% for MET and 90% for ERBB2, respectively. With an alternative 1.76 threshold, sensitivity was 94% for MET and 96% for ERBB2, while specificity was 85% for MET and 90% for ERBB2. Correlation coefficient between FISH and ddPCR ratio was .90 for MET and .88 for ERBB2. In both cohorts, NGS-based script and ddPCR results were significantly correlated regarding all genes (P < .001). CONCLUSION: Combined NGS-based script and ddPCR method is reliable and easily feasible for the detection of gene amplifications, providing useful data for guided therapy in cancer.

The strategic roles of four enzymes in the interconnection between metabolism and oncogene activation in non-small cell lung cancer: Therapeutic implications.

NSCLC is the leading cause of cancer mortality and represents a major challenge in cancer therapy. Intrinsic and acquired anticancer drug resistance are promoted by hypoxia and HIF-1α. Moreover, chemoresistance is sustained by the activation of key signaling pathways (such as RAS and its well-known downstream targets PI3K/AKT and MAPK) and several mutated oncogenes (including KRAS and EGFR among others). In this review, we highlight how these oncogenic factors are interconnected with cell metabolism (aerobic glycolysis, glutaminolysis and lipid synthesis). Also, we stress the key role of four metabolic enzymes (PFK1, dimeric-PKM2, GLS1 and ACLY), which promote the activation of these oncogenic pathways in a positive feedback loop. These four tenors orchestrating the coordination of metabolism and oncogenic pathways could be key druggable targets for specific inhibition. Since PFK1 appears as the first tenor of this orchestra, its inhibition (and/or that of its main activator PFK2/PFKFB3) could be an efficacious strategy against NSCLC. Citrate is a potent physiologic inhibitor of both PFK1 and PFKFB3, and NSCLC cells seem to maintain a low citrate level to sustain aerobic glycolysis and the PFK1/PI3K/EGFR axis. Awaiting the development of specific non-toxic inhibitors of PFK1 and PFK2/PFKFB3, we propose to test strategies increasing citrate levels in NSCLC tumors to disrupt this interconnection. This could be attempted by evaluating inhibitors of the citrate-consuming enzyme ACLY and/or by direct administration of citrate at high doses. In preclinical models, this "citrate strategy" efficiently inhibits PFK1/PFK2, HIF-1α, and IGFR/PI3K/AKT axes. It also blocks tumor growth in RAS-driven lung cancer models, reversing dedifferentiation, promoting T lymphocytes tumor infiltration, and increasing sensitivity to cytotoxic drugs.

Discovery and validation of a transcriptional signature identifying homologous recombination-deficient breast, endometrial and ovarian cancers.

BACKGROUND: Molecular alterations leading to homologous recombination deficiency (HRD) are heterogeneous. We aimed to identify a transcriptional profile shared by endometrial (UCEC), breast (BRCA) and ovarian (OV) cancers with HRD. METHODS: Genes differentially expressed with HRD genomic score (continuous gHRD score) in UCEC/BRCA/OV were identified using edgeR, and used to train a RNAseq score (ridge-regression model) predictive of the gHRD score (PanCanAtlas, N = 1684 samples). The RNAseq score was applied in independent gynaecological datasets (CARPEM/CPTAC/SCAN/TCGA, N = 4038 samples). Validations used ROC curves, linear regressions and Pearson correlations. Overall survival (OS) analyses used Kaplan-Meier curves and Cox models. RESULTS: In total, 656 genes were commonly up/downregulated with gHRD score in UCEC/BRCA/OV. Upregulated genes were enriched for nuclear/chromatin/DNA-repair processes, while downregulated genes for cytoskeleton (gene ontologies). The RNAseq score correlated with gHRD score in independent gynaecological cancers (R² = 0.4-0.7, Pearson correlation = 0.64-0.86, all P < 10-11), and was predictive of gHRD score >42 (RNAseq HRD profile; AUC = 0.95/0.92/0.78 in UCEC/BRCA/OV). RNAseq HRD profile was associated (i) with better OS in platinum-treated advanced TP53-mutated-UCEC (P < 0.001) and OV (P = 0.013), and (ii) with poorer OS (P < 0.001) and higher benefit of adjuvant chemotherapy in Stage I-III BRCA (interaction test, P < 0.001). CONCLUSIONS: UCEC/BRCA/OV with HRD-associated genomic scars share a common transcriptional profile. RNAseq signatures might be relevant for identifying HRD-gynaecological cancers, for prognostication and for therapeutic decision.

Highly Specific Droplet-Digital PCR Detection of Universally Methylated Circulating Tumor DNA in Endometrial Carcinoma.

BACKGROUND: No circulating biomarker is available for endometrial carcinoma (EC). We aimed to identify DNA positions universally hypermethylated in EC, and to develop a digital droplet PCR (ddPCR) assay for detection of hypermethylated circulating tumor DNA (meth-ctDNA) in plasma from patients with EC. METHODS: DNA positions hypermethylated in EC, and without unspecific hypermethylation in tissue/cell types releasing circulating cell-free DNA in plasma, were identified in silico from TCGA/Gene Expression Omnibus (GEO) data. A methylation-specific ddPCR (meth-ddPCR) assay following bisulfite conversion of DNA extracted from plasma was optimized for detection of meth-ctDNA according to dMIQE guidelines. Performances were validated on a retrospective cohort (n = 78 tumors, n = 30 tumor-adjacent tissues), a prospective pilot cohort (n = 33 stage I-IV patients), and 55 patients/donors without cancer. RESULTS: Hypermethylation of zinc finger and SCAN domain containing 12 (ZSCAN12) and/or oxytocin (OXT) classified EC samples from multiple noncancer samples with high diagnostic specificity/sensitivity [>97%; area under the curve (AUC) = 0.99; TCGA/GEO tissues/blood samples]. These results were confirmed in the independent retrospective cohort (AUC = 0.99). Meth-ddPCR showed a high analytical specificity (limit of blank = 2) and sensitivity (absolute lower threshold of detection = 50 pgmethDNA/mLplasma). In the pilot cohort, meth-ctDNA was detected in pretreatment plasma samples from 9/11 and 5/20 patients with advanced and non-advanced EC, respectively. 2 of 9 patients had ctDNA detected after macroscopic complete surgery and experienced progression within 6 months. No healthy donors had any copy of hypermethylated DNA detected in plasma. CONCLUSIONS: Meth-ddPCR of ZSCAN12/OXT allows a highly specific and sensitive detection of ctDNA in plasma from patients with EC and appears promising for personalized approaches for these patients.

Development and validation of a RNAseq signature for prognostic stratification in endometrial cancer.

BACKGROUND: Despite recent advances in endometrial carcinoma (EC) molecular characterization, its prognostication remains challenging. We aimed to assess whether RNAseq could stratify EC patient prognosis beyond current classification systems. METHODS: A prognostic signature was identified using a LASSO-penalized Cox model trained on TCGA (N = 543 patients). A clinically applicable polyA-RNAseq-based work-flow was developed for validation of the signature in a cohort of stage I-IV patients treated in two Hospitals [2010-2017]. Model performances were evaluated using time-dependent ROC curves (prediction of disease-specific-survival (DSS)). The additional value of the RNAseq signature was evaluated by multivariable Cox model, adjusted on high-risk prognostic group (2021 ESGO-ESTRO-ESP guidelines: non-endometrioid histology or stage III-IVA orTP53-mutated molecular subgroup). RESULTS: Among 209 patients included in the external validation cohort, 61 (30%), 10 (5%), 52 (25%), and 82 (40%), had mismatch repair-deficient, POLE-mutated, TP53-mutated tumors, and tumors with no specific molecular profile, respectively. The 38-genes signature accurately predicted DSS (AUC = 0.80). Most disease-related deaths occurred in high-risk patients (5-years DSS = 78% (95% CI = [68%-89%]) versus 99% [97%-100%] in patients without high-risk). A composite classifier accounting for the TP53-mutated subgroup and the RNAseq signature identified three classes independently associated with DSS: RNAseq-good prognosis (reference, 5-years DSS = 99%), non-TP53 tumors but with RNAseq-poor prognosis (adjusted-hazard ratio (aHR) = 5.75, 95% CI[1.14-29.0]), and TP53-mutated subgroup (aHR = 5.64 [1.12-28.3]). The model accounting for the high-risk group and the composite classifier predicted DSS with AUC = 0.84, versus AUC = 0.76 without (p = 0.01). CONCLUSION: RNA-seq profiling can provide an additional prognostic information to established classification systems, and warrants validation for potential RNAseq-based therapeutic strategies in EC.

Chemoresistant pleomorphic rhabdomyosarcoma: whole exome sequencing reveals underlying cancer predisposition and therapeutic options.

BACKGROUND: Rhabdomyosarcoma (RMS) is rare cancer affecting children and adults. Pleomorphic RMS histology is almost exclusive to adult patients and often resistant to chemotherapy. OBJECTIVE: We report the case of a 19-year-old patient who presented with a metastatic chemoresistant pleomorphic RMS. METHODS: Considering the poor prognosis and the few systemic therapeutic options, we decided to carry out a whole exome sequencing (WES) of the tumour and germline DNA. RESULTS: WES identified a germline variation (c.1863_1864insT) in the MLH1 gene corresponding to a pathogenic mutation: (p. Leu622Serfs*10), whereas the family history did not fit with classical criteria for Lynch syndrome. Loss-of-heterozygosity at MLH1 locus was found in the tumour. Immunohistochemistry showed loss of MLH1 and PMS2 nuclear expression in the tumour cells. In view of the mismatch repair defects and a high programmed cell death ligand 1 (PD-L1) expression (60% of tumour cells expressed PD-L1), we administrated an anti-PD-1 antibody to the patient. He achieved a rapid complete response of the lung metastases, which appears sustained after a 1-year follow-up. CONCLUSION: This observation of an RMS revealing an unexpected Lynch syndrome underlines the overlap between tumorous and germline molecular genetics and emphasises the major impact of cancer genomic medicine in clinical practice for guiding treatment decision.

Somatic IL4R mutations in primary mediastinal large B-cell lymphoma lead to constitutive JAK-STAT signaling activation.

Primary mediastinal large B-cell lymphoma (PMBCL) is a distinct subtype of diffuse large B-cell lymphoma thought to arise from thymic medullary B cells. Gene mutations underlying the molecular pathogenesis of the disease are incompletely characterized. Here, we describe novel somatic IL4R mutations in 15 of 62 primary cases of PMBCL (24.2%) and in all PMBCL-derived cell lines tested. The majority of mutations (11/21; 52%) were hotspot single nucleotide variants in exon 8, leading to an I242N amino acid change in the transmembrane domain. Functional analyses establish this mutation as gain of function leading to constitutive activation of the JAK-STAT pathway and upregulation of downstream cytokine expression profiles and B cell-specific antigens. Moreover, expression of I242N mutant IL4R in a mouse xenotransplantation model conferred growth advantage in vivo. The pattern of concurrent mutations within the JAK-STAT signaling pathway suggests additive/synergistic effects of these gene mutations contributing to lymphomagenesis. Our data establish IL4R mutations as novel driver alterations and provide a strong preclinical rationale for therapeutic targeting of JAK-STAT signaling in PMBCL.

Identification of TP53 mutated group using a molecular and immunohistochemical classification of endometrial carcinoma to improve prognostic evaluation for adjuvant treatments.

INTRODUCTION: Molecular classification of endometrial carcinoma has been proposed to predict survival. However, its role in patient management remains to be determined. We aimed to identify whether a molecular and immunohistochemical classification of endometrial carcinoma could improve decision-making for adjuvant therapy. METHODS: All consecutive patients treated for endometrial carcinoma between 2010 and 2017 at Cochin University Hospital were included. Clinical risk of relapse was based on European Society for Medical Oncology-European Society of Gynaecological Oncology-European SocieTy for Radiotherapy & Oncology (ESMO-ESGO-ESTRO) consensus. The clinical event of interest was event-free survival. Formalin-fixed paraffin-embedded tissue samples were processed for histopathological analysis and DNA extraction. The nuclear expression of mismatch repair and TP53 proteins was analyzed by immunohistochemistry. Next-generation sequencing of a panel of 15 genes including TP53 and POLE was performed using Ampliseq panels on Ion Torrent PGM (ThermoFisher). Tumors were allocated into four molecular groups using a sequential method based on next-generation sequencing and immunohistochemistry data: (1) POLE/ultramutated-like; (2) MSI/hypermutated-like (mismatch repair-deficient); (3) TP53-mutated (without POLE mutations or mismatch repair deficiency); (4) not otherwise specified (the remaining tumors). RESULTS: 159 patients were included; 125 tumors were available for molecular characterization and distributed as follows: (1) POLE/ultramutated-like: n=4 (3%); (2) MSI/hypermutated-like: n=35 (30%); (3) TP53-mutated: n=30 (25%); and (4) not otherwise specified: n=49 (42%). Assessing the TP53 status by immunohistochemistry only rather than next-generation sequencing would have misclassified 6 tumors (5%). TP53-mutated tumors were associated with poor prognosis, independently of International Federation of Gynecology and Obstetrics (FIGO) stage and histological grade (Cox-based adjusted hazard ratio (aHR) 5.54, 95% CI 2.30 to 13.4), and independently of clinical risk of relapse (aHR 3.92, 95% CI 1.59 to 9.64). Among patients with FIGO stage I-II tumors, 6 (38%) TP53-mutated tumors had low/intermediate clinical risk of relapse and did not receive adjuvant chemotherapy or radiotherapy. CONCLUSION: Endometrial carcinoma molecular classification identified potentially under-treated patients with poor molecular prognosis despite being at low/intermediate clinical risk of relapse. Consideration of molecular classification in adjuvant therapeutic decisions should be evaluated in prospective trials.

Dimethyl fumarate, a two-edged drug: Current status and future directions.

Dimethyl fumarate (DMF) is a fumaric acid ester registered for the treatment of relapsing-remitting multiple sclerosis (RRMS). It induces protein succination leading to inactivation of cysteine-rich proteins. It was first shown to possess cytoprotective and antioxidant effects in noncancer models, which appeared related to the induction of the nuclear factor erythroid 2 (NF-E2)-related factor 2 (NRF2) pathway. DMF also displays antitumor activity in several cellular and mice models. Recently, we showed that the anticancer mechanism of DMF is dose-dependent and is paradoxically related to the decrease in the nuclear translocation of NRF2. Some other studies performed indicate also the potential role of DMF in cancers, which are dependent on the NRF2 antioxidant and cellular detoxification program, such as KRAS-mutated lung adenocarcinoma. It, however, seems that DMF has multiple biological effects as it has been shown to also inhibit the transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), thus blocking downstream targets that may be involved in the development and progression of inflammatory cascades leading to various disease processes, including tumors, lymphomas, diabetic retinopathy, arthritis, and psoriasis. Herein, we present the current status and future directions of the use of DMF in various diseases models with particular emphases on its targeting of specific intracellular signal transduction cascades in cancer; to shed some light on its possible mode of action.

The tumor inflammation signature (TIS) is associated with anti-PD-1 treatment benefit in the CERTIM pan-cancer cohort.

BACKGROUND: The 18-gene tumor inflammation signature (TIS) is a clinical research assay that enriches for clinical benefit to immune checkpoint blockade. We evaluated its ability to predict clinical benefit of immunotherapy in cancer patients treated with PD-1 checkpoint inhibitors in routine clinical care. METHODS: The CERTIM cohort is a prospective cohort which includes patients receiving immune checkpoint inhibitors in Cochin University hospital. RNA extracted from 58 archival formalin fixed paraffin embedded tumor blocks (including 38 lung cancers, 5 melanomas, 10 renal carcinomas, 4 urothelial carcinomas and 1 colon carcinoma) was hybridized to a beta version of the NanoString® PanCancer IO360™ CodeSet using nCounter® technology. Gene expression signatures were correlated with tumor responses (by RECIST criteria) and overall survival. PD-L1 immunostaining on tumor cells was assessed in 37 non-small cell lung cancer (NSCLC) samples and tumor mutational burden (TMB) measured by whole exome sequencing in 19 of these. RESULTS: TIS scores were significantly associated with complete or partial response to anti-PD-1 treatment in the whole cohort (odds ratio = 2.64, 95% CI [1.4; 6.0], p = 0.008), as well as in the NSCLC population (odds ratio = 3.27, 95% CI [1.2; 11.6], p = 0.03). Patients whose tumor had a high TIS score (upper tertile) showed prolonged overall survival compared to patients whose tumor had lower TIS scores, both in the whole cohort (hazard ratio = 0.37, 95% CI [0.18, 0.76], p = 0.005) and in the NSCLC population (hazard ratio = 0.36, 95% CI [0.14, 0.90], p = 0.02). In the latter, the TIS score was independent from either PD-L1 staining on tumor cells (spearman coefficient 0.2) and TMB (spearman coefficient - 0.2). CONCLUSIONS: These results indicate that validated gene expression assay measuring the level of tumor microenvironment inflammation such as TIS, are accurate and independent predictive biomarkers and can be easily implemented in the clinical practice.

MYC-IG rearrangements are negative predictors of survival in DLBCL patients treated with immunochemotherapy: a GELA/LYSA study.

Diffuse large B-cell lymphoma (DLBCL) with MYC rearrangement (MYC-R) carries an unfavorable outcome. We explored the prognostic value of the MYC translocation partner gene in a series of MYC-R de novo DLBCL patients enrolled in first-line prospective clinical trials (Groupe d'Etudes des Lymphomes de l'Adulte/Lymphoma Study Association) and treated with rituximab-anthracycline-based chemotherapy. A total of 774 DLBCL cases characterized for cell of origin by the Hans classifier were analyzed using fluorescence in situ hybridization with BCL2, BCL6, MYC, immunoglobulin (IG)K, and IGL break-apart and IGH/MYC, IGK/MYC, and IGL/MYC fusion probes. MYC-R was observed in 51/574 (8.9%) evaluable DLBCL cases. MYC-R cases were predominantly of the germinal center B-cell-like subtype 37/51 (74%) with no distinctive morphologic and phenotypic features. Nineteen cases were MYC single-hit and 32 cases were MYC double-hit (MYC plus BCL2 and/or BCL6) DLBCL. MYC translocation partner was an IG gene in 24 cases (MYC-IG) and a non-IG gene (MYC-non-IG) in 26 of 50 evaluable cases. Noteworthy, MYC-IG patients had shorter overall survival (OS) (P = .0002) compared with MYC-negative patients, whereas no survival difference was observed between MYC-non-IG and MYC-negative patients. In multivariate analyses, MYC-IG predicted poor progression-free survival (P = .0051) and OS (P = .0006) independently from the International Prognostic Index and the Hans classifier. In conclusion, we show in this prospective randomized trial that the adverse prognostic impact of MYC-R is correlated to the MYC-IG translocation partner gene in DLBCL patients treated with immunochemotherapy. These results may have an important impact on the clinical management of DLBCL patients with MYC-R who should be routinely characterized according to MYC partner gene. These trials are individually registered at www.clinicaltrials.gov as #NCT00144807, #NCT01087424, #NCT00169143, #NCT00144755, #NCT00140660, #NCT00140595, and #NCT00135499.

Sarcopenic overweight is associated with early acute limiting toxicity of anti-PD1 checkpoint inhibitors in melanoma patients.

Little is known on factors predicting toxicity of anti-PD1 checkpoint inhibitors. Sarcopenic obesity is associated with increased acute toxicity of cytotoxic agents and targeted therapies. We explored whether body composition also influenced the occurrence of early acute limiting toxicity (ALT) of anti-PD1 in melanoma patients. This is a monocentric, retrospective study analyzing toxicity outcome in consecutive melanoma patients treated with nivolumab or pembrolizumab. Various parameters linked to the patient or the disease status have been analysed. Body mass index (BMI; kg/m2) and muscle mass using CT were measured prior to treatment initiation. Chi-squared test and Mann-Whitney's tests were used for the comparison of categorical and continuous variables respectively. Among 68 melanoma patients treated with anti-PD1 (47 pembrolizumab, 21 nivolumab), 38 (56%) patients had a BMI ≥ 25 kg/m2 and 11 (16%) a BMI ≥ 30, while 13 (19%) had both sarcopenia and a BMI ≥ 25 kg/m2. For the 11 (16%) patients who experienced early ALT, the mean BMI was higher (27.9 versus 24.7 kg/m2; p = 0.04). Among the 32 female patients, sarcopenic overweight patients had a 6.5-fold increased risk of ALT (50 versus 7.7%; p = 0.01). Sarcopenic overweight is associated with more early ALT of anti-PD1 in melanoma patients.

Whole exome sequencing of relapsed/refractory patients expands the repertoire of somatic mutations in diffuse large B-cell lymphoma.

Despite the many efforts already spent to enumerate somatic mutations in diffuse large B-cell lymphoma (DLBCL), previous whole-genome and whole-exome studies conducted on patients of mixed outcomes failed at characterizing the 30% of patients who will relapse or resist current immunochemotherapies. To address this issue, we performed whole-exome sequencing of normal/tumoral DNA pairs in 14 relapsed/refractory (R/R) patients subclassified by full-transcriptome arrays (six activated B-cell like, three germinal center B-cell like, and five primary mediastinal B-cell lymphomas), from the LNH-03 LYSA clinical trial program. Aside from well-known DLBCL features, gene and pathway level recurrence analyses proposed several interesting leads including TBL1XR1 and activating mutations in IRF4 or in the insulin regulation pathway. Sequencing-based copy number analysis defined 23 short recurrently altered regions involving genes such as REL, CDKN2A, HYAL2, and TP53. Moreover, it highlighted mutations in genes such as GNA13, CARD11, MFHAS1, and PCLO as associated with secondary variant allele amplification events. The five primary mediastinal B-cell lymphomas (PMBL), while unexpected in a R/R cohort, showed a significantly higher mutation rate (P = 0.003) and provided many insights on this classical Hodgkin lymphoma related subtype. Novel genes such as XPO1, MFHAS1, and ITPKB were found particularly mutated, along with various cytokine-based signaling pathways. Among these analyses, somatic events in the NF-κB pathway were found preponderant in the three DLBCL subtypes, confirming its major implication in DLBCL aggressiveness and pinpointing several new candidate genes.

Recurrent RAS and PIK3CA mutations in Erdheim-Chester disease.

Erdheim-Chester disease (ECD) is a rare histiocytic disorder that is challenging to diagnose and treat. We performed molecular analysis of BRAF in the largest cohort of ECD patients studied to date followed by N/KRAS, PIK3CA, and AKT1 mutational analysis in BRAF wild-type patients. Forty-six of 80 (57.5%) of patients were BRAFV600E-mutant. NRAS mutations were detected in 3 of 17 ECD BRAFV600E wild-type patients. PIK3CA mutations (p.E542K, p.E545K, p.A1046T, and p.H1047R) were detected in 7 of 55 patients, 4 of whom also had BRAF mutations. Mutant NRAS was present in peripheral blood CD14(+) cells, but not lymphoid cells, from an NRASQ61R mutant patient. Our results underscore the central role of RAS-RAF-MEK-ERK activation in ECD and identify an important role of activation of RAS-PI3K-AKT signaling in ECD. These results provide a rationale for targeting mutant RAS or PI3K/AKT/mTOR signaling in the subset of ECD patients with NRAS or PIK3CA mutations.

Recurrent mutations of the exportin 1 gene (XPO1) and their impact on selective inhibitor of nuclear export compounds sensitivity in primary mediastinal B-cell lymphoma.

Primary mediastinal B-cell lymphoma (PMBL) is an entity of B-cell lymphoma distinct from the other molecular subtypes of diffuse large B-cell lymphoma (DLBCL). We investigated the prevalence, specificity, and clinical relevance of mutations of XPO1, which encodes a member of the karyopherin-ß nuclear transporters, in a large cohort of PMBL. PMBL cases defined histologically or by gene expression profiling (GEP) were sequenced and the XPO1 mutational status was correlated to genetic and clinical characteristics. The XPO1 mutational status was also assessed in DLBCL, Hodgkin lymphoma (HL) and mediastinal gray-zone lymphoma (MGZL).The biological impact of the mutation on Selective Inhibitor of Nuclear Export (SINE) compounds (KPT-185/330) sensitivity was investigated in vitro. XPO1 mutations were present in 28/117 (24%) PMBL cases and in 5/19 (26%) HL cases but absent/rare in MGZL (0/20) or DLBCL (3/197). A higher prevalence (50%) of the recurrent codon 571 variant (p.E571K) was observed in GEP-defined PMBL and was associated with shorter PFS. Age, International Prognostic Index and bulky mass were similar in XPO1 mutant and wild-type cases. KPT-185 induced a dose-dependent decrease in cell proliferation and increased cell-death in PMBL cell lines harboring wild type or XPO1 E571K mutant alleles. Experiments in transfected U2OS cells further confirmed that the XPO1 E571K mutation does not have a drastic impact on KPT-330 binding. To conclude the XPO1 E571K mutation represents a genetic hallmark of the PMBL subtype and serves as a new relevant PMBL biomarker. SINE compounds appear active for both mutated and wild-type protein. Am. J. Hematol. 91:923-930, 2016. © 2016 Wiley Periodicals, Inc.

Neoadjuvant FOLFOX 4 versus FOLFOX 4 with Cetuximab versus immediate surgery for high-risk stage II and III colon cancers: a multicentre randomised controlled phase II trial--the PRODIGE 22--ECKINOXE trial.

BACKGROUND: In patients with high risk stage II and stage III colon cancer (CC), curative surgery followed by adjuvant FOLFOX-4 chemotherapy has become the standard of care. However, for 20 to 30% of these patients, the current curative treatment strategy of surgical excision followed by adjuvant chemotherapy fails either to clear locoregional spread or to eradicate distant micrometastases, leading to disease recurrence. Preoperative chemotherapy is an attractive concept for these CCs and has the potential to impact upon both of these causes of failure. Optimum systemic therapy at the earliest possible opportunity may be more effective at eradicating distant metastases than the same treatment given after the delay and immunological stress of surgery. Added to this, shrinking the primary tumor before surgery may reduce the risk of incomplete surgical excision, and the risk of tumor cell shedding during surgery. METHODS/DESIGN: PRODIGE 22--ECKINOXE is a multicenter randomized phase II trial designed to evaluate efficacy and feasibility of two chemotherapy regimens (FOLFOX-4 alone and FOLFOX-4 + Cetuximab) in a peri-operative strategy in patients with bulky CCs. Patients with CC deemed as high risk T3, T4 and/or N2 on initial abdominopelvic CT scan are randomized to either colectomy and adjuvant chemotherapy (control arm), or 4 cycles of neoadjuvant chemotherapy with FOLFOX-4 (for RAS mutated patients). In RAS wild-type patients a third arm testing FOLFOX+ cetuximab has been added prior to colectomy. Patients in the neoadjuvant chemotherapy arms will receive postoperative treatment for 4 months (8 cycles) to complete their therapeutic schedule. The primary endpoint of the study is the histological Tumor Regression Grade (TRG) as defined by Ryan. The secondary endpoints are: treatment strategy safety (toxicity, primary tumor related complications under chemotherapy, peri-operative morbidity), disease-free and recurrence free survivals at 3 years, quality of life, carcinologic quality and completeness of the surgery, initial radiological staging and radiological response to neoadjuvant chemotherapy, and the correlation between histopathological and radiological response. Taking into account a 50% prevalence of CC without RAS mutation, accrual of 165 patients is needed for this Phase II trial. TRIAL REGISTRATION: NCT01675999 (ClinicalTrials.gov).

DNA comparison between operative and biopsy specimens to investigate stage pT0 after radical prostatectomy.

PURPOSE: The aim was to eliminate, by DNA comparison, any identity mismatch between operative and biopsy specimens and to analyse the determinants of all pT0 prostate cancers occurred in a single institution. METHODS: All prostate pT0 cases in a single institution over 20 years were investigated. None of the patients had been diagnosed after a transurethral resection of the prostate nor had they received neoadjuvant hormonal treatment. The biopsies performed in other centres had been referred for a centralized pathologic re-analysis. DNA analysis was performed in samples from operative and biopsy specimens, and pairs of tissues were blindly constituted. Correct matching was verified in each pair and compared to the original database in order to comment on the occurrence of identity mismatches in the series. RESULTS: Nineteen patients (0.77 %) had been diagnosed as having pT0 prostate cancer among the 2,462 RP procedures performed over 19 years. The biopsy re-analysis invalidated the initial diagnosis of prostate cancer in one biopsy set performed elsewhere. Among 12 entirely processed cases, the biochemistry procedure evaluated as "very unlikely" the occurrence of an error in tissue identification in the biopsy setting, during the surgical procedure or the pathological analysis. No identification error of tissue samples was established in this first verified pT0 series. CONCLUSIONS: Although it must be suspected, specimen identification error was not a cause for pT0 prostate cancer. Only after a full pathological and DNA verification, the pT0 stage remains a sole entity, unexplained in most cases.

A Dysfunctional T-cell Gene Signature for Predicting Nonresponse to PD-1 Blockade in Non-small Cell Lung Cancer That Is Suitable for Routine Clinical Diagnostics.

PURPOSE: Because PD-1 blockade is only effective in a minority of patients with advanced-stage non-small cell lung cancer (NSCLC), biomarkers are needed to guide treatment decisions. Tumor infiltration by PD-1T tumor-infiltrating lymphocytes (TIL), a dysfunctional TIL pool with tumor-reactive capacity, can be detected by digital quantitative IHC and has been established as a novel predictive biomarker in NSCLC. To facilitate translation of this biomarker to the clinic, we aimed to develop a robust RNA signature reflecting a tumor's PD-1T TIL status. EXPERIMENTAL DESIGN: mRNA expression analysis using the NanoString nCounter platform was performed in baseline tumor samples from 41 patients with advanced-stage NSCLC treated with nivolumab that were selected on the basis of PD-1T TIL infiltration by IHC. Samples were included as a training cohort (n = 41) to develop a predictive gene signature. This signature was independently validated in a second cohort (n = 42). Primary outcome was disease control at 12 months (DC 12 m), and secondary outcome was progression-free and overall survival. RESULTS: Regularized regression analysis yielded a signature using 12 out of 56 differentially expressed genes between PD-1T IHC-high tumors from patients with DC 12 m and PD-1T IHC-low tumors from patients with progressive disease (PD). In the validation cohort, 6/6 (100%) patients with DC 12 m and 23/36 (64%) with PD were correctly classified with a negative predictive value (NPV) of 100% and a positive predictive value of 32%. CONCLUSIONS: The PD-1T mRNA signature showed a similar high sensitivity and high NPV as the digital IHC quantification of PD-1T TIL. This finding provides a straightforward approach allowing for easy implementation in a routine diagnostic clinical setting.

EBV-Positive Inflammatory Follicular Dendritic Cell Sarcoma of the Spleen: Report of an Aggressive Form With Molecular Characterization.

EBV-positive inflammatory follicular dendritic cell sarcoma (EBV+ inflammatory FDCS) is a rare neoplasm almost exclusively located in the spleen or liver. It is characterized by a proliferation of EBV-positive spindle-shaped cells bearing follicular dendritic cell markers, associated with an abundant lymphoplasmacytic infiltrate. EBV+ inflammatory FDCS is often asymptomatic or responsible for mild symptoms. It usually displays an indolent course and its prognosis is excellent after tumor removal, although relapsing and metastatic forms exist. Herein, we describe an aggressive form of splenic EBV+ inflammatory FDCS in a 79-year-old woman presenting with abdominal pain, deterioration of general health status, major inflammatory syndrome, and symptomatic hypercalcemia. A splenectomy was performed leading to a rapid improvement in her clinical condition and normalization of laboratory abnormalities. Unfortunately, her symptoms and laboratory abnormalities reappeared 4 months later. Computed tomography showed a mass in the splenectomy site and multiple liver and peritoneal nodules. Further analyses were performed on tumor tissue and showed positive phospho-ERK staining of tumoral cells indicating activation of MAPK pathway. Inactivating mutations were found on CDKN2A and NF1 genes. Subsequently, the patient's condition deteriorated rapidly. Since interleukin-6 levels were dramatically increased, tocilizumab was used but only had a transient effect on the patient's symptoms and inflammatory syndrome. Antitumor agent gemcitabine was initiated but her clinical condition continued to deteriorate and the patient died 2 weeks later. The management of aggressive forms of EBV+ inflammatory FDCS remains challenging. However, since these tumors seem to display genetic alterations, better characterization could lead to molecular targeted therapies.