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Background The EndoPredict (EP) test has been developed and validated for assessing recurrence risk in patients with estrogen receptor (ER)-positive HER2-negative breast cancer (BC). This test is based on the expression of 12 genes (molecular score), combined with clinicopathological criteria (i.e. tumor size and nodal status) (EPclin risk score). For node-positive patients, the weight given to the node status in the EPclin score is similar when considering pN1mi (]0.2-2mm]) or pN1 (>2mm, 1-3 positive lymph nodes). Our aim was to characterize the EPclin classification of the pN1mi subgroup in the French national registry for molecular signatures in ER+ BC. Methods Since April 2016, nine French laboratories using EP test in clinical practice prospectively implement a national registry for molecular prognostic signatures in ER+ BC. We analyzed the pN1mi subgroup with regards to their clinico-pathological characteristics, molecular EP score and EPclin risk score. Using the definition formula of the EPclin score [EPclin=0.35t + 0.64n + 0.28EP, with n=1 for pN0, 2 for pN1mi/pN1, 3 for pN2, 4 for pN3], we could calculate a hypothetical EPclin score if the pN1mi had been considered as pN0 [n factor=1]. Results By the end of 2017, the database included 1246 EP tests performed in routine practice, including 67 (5.4%) pN1mi. The pN1mi BC were ER+ HER2- (67/67, 100%), invasive carcinoma of no special type in 52/67 (78%), with a median tumor size of 18 mm (range 7-45; pT1c in 40/67). Among these, 22 were classified as EPclin low and 45 as EPclin high, with a median EP score of 6 (range 2-14), a median EPclin score of 3.70 (range 3-6), and a median relapse risk at 10-year of 15%(range 5-73). Most interestingly, 23/67 (34%; i.e. 1.8% of all EP tests performed) pN1mi BC displayed an EPclin score comprised between 3.4 and 4, just above the cut-off value of 3.3: these cases (and only these cases) would have been classified in a different risk category (i.e. EPclin low risk) if the node status would have been considered as negative. Conclusion With regards to EndoPredict test in pN1mi BC - and the persistent debate as to whether they should bear the same weight as node-positive (pN1) or negative (pN0) tumors - categorization in the EPclin class is not likely to be impacted by the node factor weight in the vast majority (>65%) of the pN1mi cases and >98% of all patients tested with EP. Only cases with an EPclin between 3.4 and 4 might be impacted. Citation Format: LaCroix-Triki M, Arnould L, MacGrogan G, Penault-Llorca F, Haudebourg J, Tas P, Garnier A, Vincent-Salomon A, Miquel C, Lehmann-Che J, Callens C, Quillien V, Cayre A, Lamy P-J, Rouleau E, De Cremoux P. EndoPredict prognostic signature in pN1mi, estrogen receptor-positive breast cancer: analysis of the French national registry for molecular signatures [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-08-23.

Inflammatory breast cancers are very aggressive, and among them, triple negative breast cancer (TNBC) has the worst prognosis. While many studies have investigated the association between tumor-infiltrating lymphocytes (TIL) before neoadjuvant chemotherapy (NAC) and outcome in TNBC, the impact of post-NAC TIL and TIL variation in triple negative inflammatory breast cancer (TNIBC) outcome is unknown. Between January 2010 to December 2018, all patients with TNIBC seen at the breast disease unit (Saint-Louis Hospital) were treated with dose-dense dose-intense NAC. The main objective of the study was to determine factors associated with event-free survival (EFS), particularly pathological complete response (pCR), pre- and post-NAC TIL, delta TIL and post-NAC lymphovascular invasion (LVI). After univariate analysis, post-NAC LVI (HR 2.06, CI 1.13&ndash, 3.74, p = 0.02), high post-NAC TIL (HR 1.81, CI 1.07&ndash, 3.06, p = 0.03) and positive delta TIL (HR 2.20, CI 1.36&ndash, 3.52, p = 0.001) were significantly associated with impaired EFS. After multivariate analysis, only a positive TIL variation remained negatively associated with EFS (HR 1.88, CI 1.05&ndash, 3.35, p = 0.01). TNIBC patients treated with intensive NAC who present TIL enrichment after NAC have a high risk of relapse, which could be used as a prognostic marker in TNIBC and could help to choose adjuvant post-NAC treatment.

International audience; An intraductal carcinoma, 55 mm across, was diagnosed on a total mastectomy in a 45-year-old woman. The 2 micro-invasive areas found were too small for reliable immunostainings for estrogen, progesterone, and HER2 receptors. In the sentinel lymph-node, a subcapsular tumor embole of about 50 cancer cells was identified on the extemporaneous cryo-cut section, but not on further sections after paraffinembedding of the sample. Considering this tumor metastatic potential, we decided to assess HER2 status on the metastatic embole using pathological and molecular micro-methods. We lasermicrodissected the tumor cells, extracted their DNA, and performed droplet-digital-PCR (ddPCR) for HER2 gene copy number variation. The HER2/RNaseP allele ratio was 5.2 in the laser-microdissected tumor cells, similar to the 5.3 ratio in the HER2overexpressing breast cancer cell line BT-474. We thus optimized the adjuvant treatment of our patient and she received a trastuzumab-based adjuvant chemotherapy.

Cancer cells often express differentiation programs unrelated to their tissue of origin, although the contribution of these aberrant phenotypes to malignancy is poorly understood. An aggressive subgroup of medulloblastoma, a malignant pediatric brain tumor of the cerebellum, expresses a photoreceptor differentiation program normally expressed in the retina. We establish that two photoreceptor-specific transcription factors, NRL and CRX, are master regulators of this program and are required for tumor maintenance in this subgroup. Beyond photoreceptor lineage genes, we identify BCL-XL as a key transcriptional target of NRL and provide evidence substantiating anti-BCL therapy as a rational treatment opportunity for select MB patients. Our results highlight the utility of studying aberrant differentiation programs in cancer and their potential as selective therapeutic vulnerabilities.

OBJECTIVEThe goal of this study was to provide insight into the influence of gliomas on gestational outcomes, the impact of pregnancy on gliomas, and the identification of patients at risk.METHODSIn this multiinstitutional retrospective study, the authors identified 52 pregnancies in 50 women diagnosed with a glioma.RESULTSFor gliomas known prior to pregnancy (n = 24), we found the following: 1) An increase in the quantified imaging growth rates occurred during pregnancy in 87% of cases. 2) Clinical deterioration occurred in 38% of cases, with seizures alone resolving after delivery in 57.2% of cases. 3) Oncological treatments were immediately performed after delivery in 25% of cases. For gliomas diagnosed during pregnancy (n = 28), we demonstrated the following: 1) The tumor was discovered during the second and third trimesters in 29% and 54% of cases, respectively, with seizures being the presenting symptom in 68% of cases. 2) The quantified imaging growth rates did not significantly decrease after delivery and before oncological treatment. 3) Clinical deterioration resolved after delivery in 21.4% of cases. 4) Oncological treatments were immediately performed after delivery in 70% of cases. Gliomas with a high grade of malignancy, negative immunoexpression of alpha-internexin, or positive immunoexpression for p53 were more likely to be associated with tumor progression during pregnancy. Deliveries were all uneventful (cesarean section in 54.5% of cases and vaginal delivery in 45.5%), and the infants were developmentally normal.CONCLUSIONSWhen a woman harboring a glioma envisions a pregnancy, or when a glioma is discovered in a pregnant patient, the authors suggest informing her and her partner that pregnancy may impact the evolution of the glioma clinically and radiologically. They strongly advise a multidisciplinary approach to management.■ CLASSIFICATION OF EVIDENCE Type of question: association; study design: case series; evidence: Class IV.

The 2016 WHO classification of tumours of the central nervous system represents the new paradigm among the specialists in the brain tumours and proposes a new approach combining histopathological and molecular features into diagnosis named 'integrated diagnosis'. The aim of this challenge is to overstep the interobserver variability of diagnosis based on previous classifications in order to ensure homogenous biological entities with a more accurate clinical significance. Over the last two decades, several molecular aberrations into gliomagenesis were highlighted and then confirmed as emerging biomarkers through prognostic stratification. In particular, IDH1/IDH2 genes mutations, 1p/19q codeletion and mutations in genes encoding histone H3 variants drastically changed the knowledge about diffuse gliomas inducing the WHO working group to consider the phenotype-genotype approach. In the present review, the historical development of the diagnosis of brain tumours from the 3D spatial configuration to the integration of multidisciplinary data up to recent molecular alterations is discussed. At the national level, the RENOCLIP network (supported by the National Cancer Institute) contributes to improve the standardization of histological diagnosis and the facilitation of access to molecular biology platforms for the detection of genetic aberrations necessary for integrated diagnosis. Importantly, the French POLA cohort allowed to test the clinical impact of the new criteria introduced by 2016 WHO classification of CNS tumours confirming the high accuracy in predicting clinical behaviour for diffuse gliomas.

Objective The DEPDC5 (DEP domain-containing protein 5) gene, encoding a repressor of the mTORC1 signaling pathway, has recently emerged as a major gene mutated in familial focal epilepsies. We aimed to further extend the role of DEPDC5 to focal cortical dysplasias (FCDs). Methods Seven patients from 4 families with DEPDC5 mutations and focal epilepsy associated with FCD were recruited and investigated at the clinical, neuroimaging, and histopathological levels. The DEPDC5 gene was sequenced from genomic blood and brain DNA. Results All patients had drug-resistant focal epilepsy, 5 of them underwent surgery, and 1 had a brain biopsy. Electroclinical phenotypes were compatible with FCD II, although magnetic resonance imaging (MRI) was typical in only 4 cases. Histopathology confirmed FCD IIa in 2 patients (including 1 MRI-negative case) and showed FCD I in 2 other patients, and remained inconclusive in the last 2 patients. Three patients were seizure-free postsurgically, and 1 had a worthwhile improvement. Sequencing of blood DNA revealed truncating DEPDC5 mutations in all 4 families; 1 mutation was found to be mosaic in an asymptomatic father. A brain somatic DEPDC5 mutation was identified in 1 patient in addition to the germline mutation. Interpretation Germline, germline mosaic, and brain somatic DEPDC5 mutations may cause epilepsy associated with FCD, reinforcing the link between mTORC1 pathway and FCDs. Similarly to other mTORopathies, a “2-hit” mutational model could be responsible for cortical lesions. Our study also indicates that epilepsy surgery is a valuable alternative in the treatment of drug-resistant DEPDC5-positive focal epilepsies, even if the MRI is unremarkable.

Medulloblastoma (MB) is the most common malignant brain tumor of childhood arising in the cerebellum. Although multimodal treatments have significantly increased the survival, 20–30% of patients remain incurable. Most of them belong to a poorly characterized MB subgroup, called group3, which expresses a retina photoreceptor-specific differentiation program. Cancer cells often express such differentiation programs unrelated to their tissue of origin. They likely result from cancer cell plasticity but are usually not thought to contribute to cancer progression. Accordingly, the functional relevance of this aberrant differentiation program to MB remains unknown. By analyzing group3 enhancers (Enh) and super-enhancers (SE), we identified key interconnected transcription factors of this subgroup, Among those, two lineage restricted transcription factors, NRL and CRX, are known to play key roles during retina development and in particular for photoreceptor specification. We identified a list of genes harboring a co-occurrence of NRL and CRX binding sites within Enh/SE, and displaying higher expression in group3. Interestingly, photoreceptors genes are enriched among this list. This analysis showed that NRL and CRX are important players in group3 MB and control the photoreceptor differentiation program. By loss-of-function experiments in cell lines and PDXs, we showed that NRL and CRX are absolutely required for MB growth in vitro and in vivo upon orthotopic grafting. We then confirmed that NRL controls the expression of photoreceptor genes in group3 MB and established that it protects MB from apoptosis. By ChIP experiments we identified one of its key target genes in this process: the anti-apoptotic BCL-XL gene. We confirmed its key role downstream of NRL by rescue experiments. Importantly, we established the potential of anti-BCL therapies in MB. In conclusion, we showed that the photoreceptor program is instrumental to MB through NRL and CRX and that the anti-BCL therapies could represent an alternative therapeutic strategy.

The aim of this study was to review and describe therapeutic approaches in children with choroid plexus tumor (CPT) based on a nationwide series. The World Health Organization classification subdivides these rare tumors into three histological subtypes corresponding to three grades of malignancy: low grade (grade I) choroid plexus papilloma (CPP), intermediate grade (grade II) atypical choroid plexus papilloma (aCPP) and high grade (grade III) choroid plexus carcinoma (CPC). This retrospective study included 102 French children younger than 18 years, treated from 2000 to 2012: 54 CPP, 26 aCPP and 22 CPC. The 5 year overall survival was 100% in CPP, 96.2% in aCPP and 64.7% in CPC. In patients with localized disease, complete surgical resection was achieved in 48/52 CPP, 20/26 aCPP and 7/14 CPC. In this group, patients with complete surgical resection had better event free survival than patients with partial resection (88.9 vs. 41.6%). 28 patients (1 CPP, 6 aCPP and 22 CPC) had adjuvant chemotherapy. 2 aCPP and 9 CPC had radiotherapy. We underlined the need for a central histological review to accurately analyze clinical data; we reported a much higher overall survival for CPC than in most previous CPT series probably including atypical teratoid rhabdoid tumors. In our series, the 5 years overall survival in CPC (64.7%) was higher than event free survival (25.2%) and could be interpreted as a clue for the efficiency of adjuvant/salvage therapy even if the heterogeneity of applied treatments in this retrospective series does not allow for meaningful statistical comparisons.

Little is known about the genomic basis of primary central nervous system lymphoma (PCNSL) tumorigenesis. To investigate the mutational profile of PCNSL, we analyzed nine paired tumor and germline DNA samples from PCNSL patients by high throughput exome sequencing. Eight genes of interest have been further investigated by focused resequencing in 28 additional PCNSL tumors to better estimate their incidence. Our study identified recurrent somatic mutations in 37 genes, some involved in key signaling pathways such as NFKB, B cell differentiation and cell cycle control. Focused resequencing in the larger cohort revealed high mutation rates for genes already described as mutated in PCNSL such as MYD88 (38%), CD79B (30%), PIM1 (22%) and TBL1XR1 (19%) and for genes not previously reported to be involved in PCNSL tumorigenesis such as ETV6 (16%), IRF4 (14%), IRF2BP2 (11%) and EBF1 (11%). Of note, only 3 somatically acquired SNVs were annotated in the COSMIC database. Our results demonstrate a high genetic heterogeneity of PCNSL and mutational pattern similarities with extracerebral diffuse large B cell lymphomas, particularly of the activated B-cell (ABC) subtype, suggesting shared underlying biological mechanisms. The present study provides new insights into the mutational profile of PCNSL and potential targets for therapeutic strategies.

Background Brain metastases challenge daily clinical practice, and the mechanisms by which cancer cells cross the blood-brain barrier remain largely undeciphered. Angiopoietin-like 4 (ANGPTL4) proteolytic fragments have controversial biological effects on endothelium permeability. Here, we studied the link between ANGPTL4 and the risk of brain metastasis in cancer patients. Methods From June 2014 to June 2015, serum samples from 86 cancer patients were prospectively collected, and ANGPTL4 concentrations were assessed. Using a murine model of brain metastases, we investigated the roles of nANGPTL4 and cANGPTL4 fragments in the occurrence of brain metastases. Results A positive ANGPTL4 serum concentration over 0.1ng/mL was associated with decreased overall-survival. Multivariate analyses found that only breast cancer brain metastases were significantly associated with elevated ANGPTL4 serum concentrations. 4T1 murine breast cancer cells were transfected with either nANGPTL4- or cANGPTL4-encoding cDNAs. Compared to mice injected with wild-type 4T1 cells, mice injected with nANGPTL4 cells had shorter median survival (p Conclusions This study opens the way for further translational research on the role of cANGPTL4 fragment in human breast cancer brain metastases.

Journal of Neuroradiology - In Press.Proof corrected by the author Available online since samedi 6 avril 2013

Medulloblastomas (MB) are classified in four subgroups: the well defined WNT and Sonic Hedgehog (SHH) subgroups, and the less defined groups 3 and 4. They occasionally occur in the context of a cancer predisposition syndrome. While germline APC mutations predispose to WNT MB, germline mutations in SUFU, PTCH1, and TP53 predispose to SHH tumors. We report on a child with a Rubinstein-Taybi syndrome (RTS) due to a germline deletion in CREBBP, who developed a MB. Biological profilings demonstrate that this tumor belongs to the group 3. RTS may therefore be the first predisposition syndrome identified for non-WNT/non-SHH MB.

As prognostic factors, MYC and MYCN amplifications are routinely assessed in medulloblastomas. Fluorescence in situ hybridization (FISH) is currently considered as the technique of reference. Recently, array comparative genomic hybridization (aCGH) has been developed as an alternative technique to evaluate genomic abnormalities in other tumor types; however, this technique has not been widely adopted as a replacement for FISH in medulloblastoma. In this study, 34 tumors were screened by both FISH and aCGH. In all cases showing amplification by FISH, aCGH also unambiguously revealed the abnormality. The aCGH technique was also performed on tumors showing no amplification by FISH, and the absence of amplification was confirmed in all cases. Interestingly, one tumor showed a subclonal MYC amplification by FISH. This subclonal amplification was observed in approximately 20% of tumor cells and was clearly evident on aCGH. In conclusion, our analysis confirms that aCGH is as safe as FISH for the detection of MYC/MYCN gene amplification. Given its cost efficiency in comparison to two FISH tests and the global genomic information additionally provided by an aCGH experiment, this reproducible technique can be safely retained as an alternative to FISH for routine investigation of medulloblastoma.

The aim of this study was to better define the clinical and biopathological features of patients with desmoplastic/nodular medulloblastoma (DNMB) and to further characterize this subgroup. 17 children aged < 5 years, with initial DNMB treated according to the HIT-SKK protocol, were evaluated. A retrospective central radiological review, a pathological and immunohistochemical study, and array-CGH and sequencing of germline SUFU and PTCH1 genes were performed. 15 histologically reviewed cases were confirmed as DNMB including three cases of medulloblastoma with extensive nodularity. Median age at diagnosis was 26 months. Radiology showed five cases with a vermis location and one with T2 hyperintensity. All cases showed a SHH immuno-profile. A 9q deletion was found in 6 cases, a MYCN–MYCL amplification in 1 case, and a SUFU germline mutation in 1 case (/9). The presence of SUFU and PTCH1 germline mutations agreed with previous reports. At 3 years, progression-free survival and overall-survival rates were 72 ± 15% and 85 ± 10%, respectively. The rate of recurrence was relatively high (4 patients). This may have been because chemotherapy was delayed in two cases. Age > 3 years, and residual tumor may also have been an explanation for recurrence.

Solitary fibrous tumors (SFTs) are rare spindle cell tumors with limited therapeutic options. Their molecular basis is poorly known. No consistent cytogenetic abnormality has been reported. We used high-resolution whole-genome array-based comparative genomic hybridization (Agilent 244K oligonucleotide chips) to profile 47 samples, meningeal in >75% of cases. Few copy number aberrations (CNAs) were observed. Sixty-eight percent of samples did not show any gene CNA after exclusion of probes located in regions with referenced copy number variation (CNV). Only low-level CNAs were observed. The genomic profiles were very homogeneous among samples. No molecular class was revealed by clustering of DNA copy numbers. All cases displayed a “simplex” profile. No recurrent CNA was identified. Imbalances occurring in >20%, such as the gain of 8p11.23–11.22 region, contained known CNVs. The 13q14.11–13q31.1 region (lost in 4% of cases) was the largest altered region and contained the lowest percentage of genes with referenced CNVs. A total of 425 genes without CNV showed copy number transition in at least one sample, but only but only 1 in at least 10% of samples. The genomic profiles of meningeal and extra-meningeal cases did not show any differences. © 2012 Wiley Periodicals, Inc.

To determine optimal resections in the 3 dysembryoplastic neuroepithelial tumor (DNT) histologic subtypes (simple, complex, and nonspecific) based on MRI features.In 78 consecutive epilepsy patients operated for DNT, MRI features were classified as follows: type 1 (cystic/polycystic-like, well-delineated, strongly hypointense T1), type 2 (nodular-like, heterogeneous), or type 3 (dysplastic-like, iso/hyposignal T1, poor delineation, gray-white matter blurring). Correlations between histology, neurophysiologic findings, and surgical outcome were established for each MRI subtype.Type 1 MRI (25 cases, in temporal and extratemporal areas) always corresponded to simple or complex DNTs. Type 2 MRI (25 cases, predominantly in neocortical areas) and type 3 MRI (28 cases, mainly in the mesial temporal lobe) corresponded to nonspecific forms. The epileptogenic zone (EZ) differed significantly according to the MRI subtype (p = 0.0029). It colocalized with the tumor in type 1 MRI, included perilesional cortex in type 2 MRI, and involved extensive areas in type 3 MRI. Cortical dysplasia was predominantly found in type 3 MRI (p0.0001). The main prognostic factors for seizure-free outcome (83%) were complete tumor (p0.0001) and EZ (p = 0.0115) removal. Other factors favorably influencing the outcome were a short epilepsy duration (p = 0.013) and absence of cortical-subcortical damage at the resection site (p = 0.053). Age at surgery was not related to outcome; however, cortical-subcortical damage was correlated with old age (p = 0.021). Treatment discontinuation was correlated with young age at surgery (p = 0.004) and short epilepsy duration (p = 0.001).We propose that resection might be restricted to the tumor in type 1 MRI and be more extensive in other MRI subtypes, especially in type 3 MRI. Early surgery and clean surgical margins are crucial for curing epilepsy.

Purpose: Our objective was to identify the genetic changes involved in primary central nervous system lymphoma (PCNSL) oncogenesis and evaluate their clinical relevance. Experimental Design: We investigated a series of 29 newly diagnosed, HIV-negative, PCNSL patients using high-resolution single-nucleotide polymorphism (SNP) arrays (n = 29) and whole-exome sequencing (n = 4) approaches. Recurrent homozygous deletions and somatic gene mutations found were validated by quantitative real-time PCR and Sanger sequencing, respectively. Molecular results were correlated with prognosis. Results: All PCNSLs were diffuse large B-cell lymphomas, and the patients received chemotherapy without radiotherapy as initial treatment. The SNP analysis revealed recurrent large and focal chromosome imbalances that target candidate genes in PCNSL oncogenesis. The most frequent genomic abnormalities were (i) 6p21.32 loss (HLA locus), (ii) 6q loss, (iii) CDKN2A homozygous deletions, (iv) 12q12-q22, and (v) chromosome 7q21 and 7q31 gains. Homozygous deletions of PRMD1, TOX, and DOCK5 and the amplification of HDAC9 were also detected. Sequencing of matched tumor and blood DNA samples identified novel somatic mutations in MYD88 and TBL1XR1 in 38% and 14% of the cases, respectively. The correlation of genetic abnormalities with clinical outcomes using multivariate analysis showed that 6q22 loss (P = 0.006 and P = 0.01) and CDKN2A homozygous deletion (P = 0.02 and P = 0.01) were significantly associated with shorter progression-free survival and overall survival. Conclusions: Our study provides new insights into the molecular tumorigenesis of PCNSL and identifies novel genetic alterations in this disease, especially MYD88 and TBL1XR1 mutations activating the NF-κB signaling pathway, which may be promising targets for future therapeutic strategies. Clin Cancer Res; 18(19); 5203–11. ©2012 AACR.

M. Fevre-Montange, A. Vasiljevic, D. Frappaz, J. Champier, A. Szathmari, M.-H. Aubriot Lorton, F. Chapon, A. Coulon, I. Quintin Roue, M.-B. Delisle, D. Figarella-Branger, A. Laquerriere, C. Miquel, J.-F. Michiels, M. Peoch, M. Polivka, F. Fauchon and A. Jouvet (2012) Neuropathology and Applied Neurobiology38, 87–94 Utility of Ki67 immunostaining in the grading of pineal parenchymal tumours: a multicentre study Aims: Pineal parenchymal tumours (PPTs) are rare neoplasms that are divided into pineocytoma (PC), pineoblastoma (PB) and PPT of intermediate differentiation (PPTID). Factors affecting the survival of patients with PPTs are morphological subtype and histological grading according to mitotic index and neurofilament immunostaining. Grading criteria to distinguish PPTIDs are difficult to define, particularly when using small specimens. The Ki67 labelling index (LI) might be helpful in distinguishing between grade II and III PPTIDs. Our study was performed to assess the predictive value of the Ki67 LI in a large cooperative series of PPTs and to evaluate whether inclusion of this data would improve and refine the World Health Organization classification. Methods: A retrospective analysis of 33 PPTs was performed. The histological features of the tumours were reviewed and Ki67 LI scoring was evaluated by immunohistochemistry. Data were correlated with the patients' survival. Results: The mean Ki67 LI was significantly different for tumour grades (0 in PC, 5.2 ± 0.4 in PPTID grade II, 11.2 ± 2.0 in PPTID grade III, 36.4 ± 6.2 in PB; P

Recently, mutations in IDH1 and IDH2 have been reported as an early and common genetic alteration in diffuse gliomas, being possibly followed by 1p/19q loss in oligodendrogliomas and TP53 mutations in astrocytomas. Lately, IDH1 mutations have also been identified in adult gliomatosis cerebri (GC). The aim of our study was to test the status of IDH1/2, p53 and of chromosomes 1 and 19 in a series of 12 adult and three pediatric GC. For all tumors, clinico-radiologic characteristics, histopathologic features, status of IDH1/2, p53 and of chromosomes 1 and 19 were evaluated. IDH1 mutations were detected only in GC of adult patients (5/12). They all corresponded to R132H. Additional 1p/19q losses were observed in two of them with histological features of oligodendroglial lineage. Other copy number alterations of chromosomes 1 and 19 were also noticed. The median overall survival in adults was 10.5 months in non-mutated GC and 43.5 months in mutated GC. IDH1 mutations were present in GC of adult patients, but not in those of children. There was a trend toward longer overall survival in mutated GC when compared to non-mutated ones. Concomitant 1p/19q loss was observed in IDH1-mutated GC with oligodendroglial phenotype. These observations contribute toward establishing a stronger link between GC and diffuse glioma. In addition, these results also emphasize the importance of testing for IDH1/2 mutations and 1p/19q deletions in GC to classify them better and to allow the development of targeted therapy.

The clinical use of intravenous immunoglobulin (IVIg) based on its immunomodulatory and anti-inflammatory potential remains an ongoing challenge. Fcγ receptor-mediated effects of IVIg, although well elucidated in certain pathologies, cannot entirely account for its proven benefit in several autoimmune disorders mediated by autoreactive T cells. In this study, we show that prophylactic infusion of IVIg prevents the development of experimental autoimmune encephalomyelitis (EAE), an accepted animal model for multiple sclerosis (MS). The protection was associated with peripheral increase in CD4+CD25+Foxp3+ regulatory T cell (Treg) numbers and function. The protection was Treg-mediated because IVIg failed to protect against EAE in mice that were depleted of the Treg population. Rather than inducing de novo generation from conventional T cells, IVIg had a direct effect on proliferation of natural Treg. In conclusion, our results highlight a novel mechanism of action of IVIg and provide a rationale to test the use of IVIg as an immunomodulatory tool to enhance Treg in early onset MS and other autoimmune and inflammatory conditions.

Empirical radiotherapy is the current treatment for children with diffuse pontine lesions that have imaging characteristics of an infiltrative malignant astrocytoma. The use of chemotherapeutic agents is, however, currently under investigation in the treatment of these tumors. To be included into a trial, patients need a definitive histological diagnosis. The authors present their prospective study of the stereotactic biopsy of these lesions during a 4-year period.A suboccipital, transcerebellar approach was used to obtain biopsy samples in 24 children.Two patients suffered deficits. Both had a transient (2 months) new cranial nerve palsy; one of these patients also experienced an exacerbation of a preoperative hemiparesis. No patient died during the perioperative period. A histological diagnosis was made in all 24 patients as follows: 22 had a malignant infiltrative astrocytoma, one had a low-grade astrocytoma, and one had a pilocytic astrocytoma. The diagnosis of the latter two patients affected the initial treatment after the biopsy.The findings of this study imply that stereotactic biopsy sampling of a diffuse pontine tumor is a safe procedure, is associated with minimal morbidity, and has a high diagnostic yield. A nonmalignant tumor was identified in two of the 24 patients in whom the imaging findings were characteristic of a malignant infiltrative astrocytoma. With the advent of new treatment protocols, stereotactic biopsy sampling, which would allow specific tumor characterization of diffuse pontine lesions, may become standard.

CERVOXY COLL; International audience

Objective: Defects in the DNA mismatch repair result in microsatellite instability (MSI), which characterise most tumours related to the hereditary non-polyposis colorectal cancer syndrome and some sporadic tumours. Several studies have reported the occurrence of MSI in urothelial cell carcinoma (UCC) of the bladder with a particularly high incidence in tumours from young patients. In this study, we have evaluated the occurrence of MSI in primary bladder UCC arising in seventeen young patients selected for being below 45 years of age at diagnosis. Methods: Microsatellite analysis has been performed using the panel of five quasimonomorphic mononucleotide repeats (BAT-25, BAT-26, NR-21, NR-24, NR-27) recently recommended to detect MSI tumours. The original Bethesda panel including BAT-25, BAT-26 and three dinucleotide repeats (D2S123, D5S346, D17S250) has further been studied in 10 UCC samples. Results: MSI has been observed in only one of the 17 bladder UCC studied. Using the original Bethesda panel, identical results were obtained, indicating that the panel of five mononucleotide markers adequately detected MSI in UCC tumours. Conclusions: Our data indicate that classical MSI affecting mono- or di-nucleotides are rarely involved in bladder UCC developing in young patients. Further studies using gold standard criteria would help clarifying the involvement of MSI in the pathogenesis of bladder UCC.

A subset of upper urinary tract urothelial cell carcinomas (UUC), arising sporadically or as a manifestation of hereditary non-polyposis colorectal cancer, displays microsatellite instability (MSI). MSI tumours are characterized by defective mismatch repair and accumulation of frameshift mutations in numerous genes harbouring repeats in their coding sequences. We have evaluated the incidence of MSI in UUC and the intratumoral distribution of mutations in 13 candidate target genes. A total of 58 unselected UUC were screened for MSI using the panel of five mononucleotide markers recently recommended by the National Cancer Institute for a precise MSI assessment. Four tumours displayed MSI (7%), among which at least three had alterations in the genes MSH3, BAX, MRE11, RAD50. Mutations in genes involved in key cellular pathways (ATR, DNA-PKcs, MBD4, TCF-4, MSH6, and BLM) were further detected. BAX and MRE11 mutations tend to present homogeneously within the three MSI UUC. Immunohistochemistry (MLH1, MSH2, MSH6) showed that loss of mismatch repair protein expression occurred in all MSI UUC defining the gene defect and that MRE11 and RAD50 mutations were associated with their concomitant loss expression. In conclusion, MSI UUC represent a small proportion of UUC in which BAX and MRE11 mutations are frequent and may play a role early in UUC tumorigenesis.

Resume En France, deux systemes de classifications sont principalement utilises pour etablir le diagnostic et le grading de malignite des oligodendrogliomes (oligo): la classification de l’OMS et celle de l’Hopital Sainte-Anne (SA). Ces classifications reposent sur une approche diagnostique tres differente. L’OMS n’utilise que des criteres histologiques et demeure basee sur le type cellulaire predominant (oligodendroglial versus astrocytaire). La classification SA repose sur l’analyse de la structure histologique tumorale: tissu tumoral solide versus cellules tumorales isolees, et integre les donnees cliniques et de l’imagerie. En consequence: 1- la classification SA integre, dans la categorie des oligo, les tumeurs classees selon l’OMS en astrocytomes infiltrants diffus fibrillaires ou gemistocytiques, et en partie les astrocytomes anaplasiques ; 2- selon l’OMS, le grade de malignite de ces tumeurs (grade II versus grade III) est defini en fonction de multiples criteres histologiques (sous-type tumoral, densite cellulaire, atypies nucleaires, mitose, necrose, proliferation microvasculaire), tandis que le grading SA (grade A versus grade B) n’utilise que deux criteres, l’un histologique: presence ou absence d’hyperplasie endotheliale, l’autre radiologique: presence ou absence de prise de contraste. Ce dernier permet de pallier les problemes lies a la representativite des prelevements. Les difficultes et divergences concernant le diagnostic des oligo sont en partie dues a l’absence de marqueur de l’oligodendrocyte tumoral. L’apport potentiel des nouveaux marqueurs des precurseurs oligodendrogliaux sera discute.

Purpose: The aim of our study was to assess whether the polymorphism of the nucleotide excision repair enzyme, excision repair cross-complementing rodent repair deficiency, complementation group 1 (ERCC1), had an effect on the tumor response in patients treated with standard chemotherapy regimens for a metastatic colorectal cancer. We have studied the synonymous polymorphism that causes a single nucleotide change C to T at codon 118 converting a codon of common usage (AAC) to a less used codon (AAT), both coding asparagine. This change results in a decreased ERCC1 gene expression, which impairs repair activity. Experimental Design: Ninety-one patients with a median age of 55.1 years treated for a metastatic colorectal cancer were included in our retrospective study. The ERCC1 polymorphism was analyzed in the normal tissue of all patients. Results: Twenty (22%) were homozygous for AAC codon (C/C genotype), 30 were (33%) homozygous for AAT codon (T/T genotype), and 41 (45%) were heterozygous (C/T genotype). The objective response rate to oxaliplatin in combination with 5-fluorouracil (5-FU) was significantly higher in the T/T genotype group compared with the C/T and the C/C genotype groups (61.9%, 42.3%, and 21.4%, respectively; P = 0.018). By contrast, no significant difference was observed when patients were treated with either 5-FU alone (45%, 29.2%, and 33.3%, respectively; P = 0.407) or in combination with irinotecan (46.1%, 25.0%, and 27.3%, respectively; P = 0.305). Conclusions: Our observations allowed us to define the first useful predictive criterion for oxaliplatin/5-FU response in patients with metastatic colorectal cancer.

Resume Objectif La constitution de groupes homogenes de patients est indispensable pour que les resultats therapeutiques des oligodendrogliomes puissent etre valablement apprecies et compares d’une institution a l’autre. Des discordances croissantes sont cependant observees dans le diagnostic histologique des oligodendrogliomes ou oligo-astrocytomes. La presente etude a eu pour objectif principal d’apprecier si, pour les etudes retrospectives, l’IRM peut servir de base commune et de « trait d’union » entre les diagnostics etablis selon la classification de l’Organisation Mondiale de la Sante (OMS) ou selon celle l’hopital Sainte-Anne (classification SA). Materiel et methodes Cette etude a porte sur une serie de 251 patients adultes porteurs d’un oligodendrogliome ou oligo-astrocytome de grade A ou B selon SA et qui ont ete pris en charge dans notre Hopital durant la periode 1984-2003. Les preparations histologiques de routine et l’IRM ou le scanner preoperatoires effectues apres injection de produit de contraste ont ete simultanement revus afin d’apprecier l’influence sur la survie des parametres suivants : presence ou absence d’une composante astrocytaire polymorphe ou gemistocytique, de necrose, d’une prise de contraste (PC) ; hyperplasie endotheliale : absente, discrete (HE+), ou conforme au seuil defini selon le grading SA (HE++). Les tumeurs ont ete classees en grade A : absence de PC et d’HE, en grade B : PC et/ou HE++, ou en grade « A/B » : HE+, absence de PC. Resultats 70,1 % des tumeurs ont ete classees en « oligodendrogliome pur », 19,5 % en « oligo-astrocytome polymorphe », et 10,3 % en « oligo-astrocytome gemistocytique ». Dans les tumeurs de grade A, A/B ou B, la presence d’une composante astrocytaire polymorphe ou gemistocytique n’avait pas d’influence significative sur la survie, mais respectivement 53 % et 65 % de ces tumeurs versus 32 % des oligodendrogliomes « purs » etaient de grade B lors du diagnostic. Dans ces trois sous-types histologiques, la survie n’etait egalement pas significativement differente selon que l’hyperplasie endotheliale etait absente ou discrete (HE+). Apres regroupement des sous-types histologiques, d’une part, et des tumeurs avec HE+ ou absente, la serie etait composee de 153 oligodendrogliomes de grade A et de 98 oligodendrogliomes de grade B ; la survie correspondante etait de 142 mois versus 52 mois (p Conclusions Sur la base de ces resultats et ceux de nos etudes precedentes montrant que, parmi les formes communes de gliomes classees selon SA, seuls les oligodendrogliomes ou oligo-astrocytomes peuvent ne pas prendre le contraste, nous proposons que, pour les etudes cliniques retrospectives : 1) les oligodendrogliomes et les oligodendrogliomes selon la classification SA soient regroupes en une categorie unique, 2) les gliomes sans PC, quels que soient leur type histologique et leur grade de malignite selon l’OMS, soient regroupes avec les oligodendrogliomes A selon la classification SA, 3) en ce qui concerne les gliomes avec PC : les tumeurs classees, selon l’OMS, en oligodendrogliomes ou oligo-astrocytomes de grade II ou III ainsi que les glioblastomes secondaires ou avec composante oligodendrogliale soient regroupees avec les oligodendrogliomes de grade B selon la classification SA. Cependant, les tumeurs qui presentent une PC en anneau et un œdeme « en doigt de gant » doivent etre exclus ou analyses separement.

Half of colorectal tumors with microsatellite instability contain frameshift mutations in the (G)8 tract of bax, a major apoptosis effector, but their functional significance remains unclear. We studied the role of bax mutations on bax expression and apoptosis in 59 primary colorectal cancers of which 41 were microsatellite unstable. Tumors were screened for bax(G)8 mutations and evaluated immunohistochemically for bax, bcl-2, and p53 protein expression and apoptotic (M30 cytoDEATH) and proliferative (Ki-67) indexes. We identified bax(G)8 mutations in 20 (49%) of 41 unstable tumors; the mutations were associated significantly with proximal, poorly differentiated, or mucinous adenocarcinomas. Most bax-mutated cases displayed a bax-immunonegative zone in all or part of the tumor that was proved to correspond to biallelic bax(G)8 mutations by microdissection and to confer growth advantage to the tumor by decreasing apoptosis compared with adjacent bax-immunopositive tumor. Biallelic bax(G)8 mutations are subject to positive selection pressure and might disable apoptosis in colorectal cancer. There is substantial evidence that apoptosis has a role in the development of human cancer and that it might be a major mechanism in the action of current anticancer drugs. 1 Members of the bcl-2 family of proteins that regulate apoptotic signaling through mitochondria are key players in the determination of cell survival and death. 2,3

Gastrointestinal carcinoid tumors are often associated with other tumors, particularly colon adenocarcinomas; but the association between carcinoid tumors and hereditary nonpolyposis colorectal cancer (HNPCC) syndrome has not yet been explored. We report an unusual case of a 28-year-old woman with HNPCC who underwent surgery for a transverse colon adenocarcinoma in whom an appendix carcinoid tumor was incidentally found. To assess whether the carcinoid tumor displayed the characteristic molecular features of HNPCC tumors, we investigated the expression of mismatch-repair (MMR) proteins and microsatellite instability (MSI) status in both tumors. Both tumors demonstrated normal expression of the MMR proteins hMLH1, hMSH2, hMSH6, and hPMS2. Interestingly, the adenocarcinoma exhibited an MSI phenotype but the carcinoid tumor did not, indicating that these 2 tumors arose through different molecular pathways.

Atypical teratoid/rhabdoid tumour is a rare and highly malignant tumour of the posterior fossae nervous system that occurs in children especially in the first few years of life. Cutaneous location is not previously reported. A newborn boy was referred for both aqueductal stenosis detected antenatally and skin tags mimicking hamartoma. The cerebral tumour increased in size during a few months leading to both skin and cerebral biopsies. Integrase Interactor-1 (INI-1) immunostaining and tumoural and leukocytes INI-1 gene sequencing confirmed the atypical teratoid/rhabdoid tumour nature of the cerebral tumour. INI-1 immunostaining in skin biopsy confirmed the dermal location of rhabdoid tumour. Thus, unusual cutaneous lesions may be part of atypical teratoid/rhabdoid tumour. The loss of Integrase INI-1 on immunohistochemical staining is characteristic.

International audience; BackgroundSolitary Fibrous Tumours (SFT) and haemangiopericytomas (HPC) are rare meningeal tumours that have to be distinguished from meningiomas and more rarely from synovial sarcomas. We recently found that ALDH1A1 was overexpressed in SFT and HPC as compared to soft tissue sarcomas. Using whole-genome DNA microarrays, we defined the gene expression profiles of 16 SFT/HPC (9 HPC and 7 SFT). Expression profiles were compared to publicly available expression profiles of additional SFT or HPC, meningiomas and synovial sarcomas. We also performed an immunohistochemical (IHC) study with anti-ALDH1 and anti-CD34 antibodies on Tissue Micro-Arrays including 38 SFT (25 meningeal and 13 extrameningeal), 55 meningeal haemangiopericytomas (24 grade II, 31 grade III), 163 meningiomas (86 grade I, 62 grade II, 15 grade III) and 98 genetically confirmed synovial sarcomas.Results ALDH1A1 gene was overexpressed in SFT/HPC, as compared to meningiomas and synovial sarcomas. These findings were confirmed at the protein level. 84% of the SFT and 85.4% of the HPC were positive with anti-ALDH1 antibody, while only 7.1% of synovial sarcomas and 1.2% of meningiomas showed consistent expression. Positivity was usually more diffuse in SFT/HPC compared to other tumours with more than 50% of tumour cells immunostained in 32% of SFT and 50.8% of HPC. ALDH1 was a sensitive and specific marker for the diagnosis of SFT (SE = 84%, SP = 98.8%) and HPC (SE = 84.5%, SP = 98.7%) of the meninges. In association with CD34, ALDH1 expression had a specificity and positive predictive value of 100%.ConclusionWe show that ALDH1, a stem cell marker, is an accurate diagnostic marker for SFT and HPC, which improves the diagnostic value of CD34. ALDH1 could also be a new therapeutic target for these tumours which are not sensitive to conventional chemotherapy.

International audience; Background: Solitary fibrous tumors (SFTs) are rare spindle-cell tumors. Their cell-of-origin and molecular basis are poorly known. They raise several clinical problems. Differential diagnosis may be difficult, prognosis is poorly apprehended by histoclinical features, and no effective therapy exists for advanced stages.Methods: We profiled 16 SFT samples using whole-genome DNA microarrays and analyzed their expression profiles with publicly available profiles of 36 additional SFTs and 212 soft tissue sarcomas (STSs). Immunohistochemistry was applied to validate the expression of some discriminating genes.Results: SFTs displayed whole-genome expression profiles more homogeneous and different from STSs, but closer to genetically-simple than genetically-complex STSs. The SFTs/STSs comparison identified a high percentage (∼30%) of genes as differentially expressed, most of them without any DNA copy number alteration. One of the genes most overexpressed in SFTs encoded the ALDH1 stem cell marker. Several upregulated genes and associated ontologies were also related to progenitor/stem cells. SFTs also overexpressed genes encoding therapeutic targets such as kinases (EGFR, ERBB2, FGFR1, JAK2), histone deacetylases, or retinoic acid receptors. Their overexpression was found in all SFTs, regardless the anatomical location. Finally, we identified a 31-gene signature associated with the mitotic count, containing many genes related to cell cycle/mitosis, including AURKA.Conclusion: We established a robust repertoire of genes differentially expressed in SFTs. Certain overexpressed genes could provide new diagnostic (ALDH1A1), prognostic (AURKA) and/or therapeutic targets.

Small ArfGAP1 (stromal membrane-associated protein 1, SMAP1), a GTPase-activating protein specific for ADP-ribosylation factor 6 (Arf6), which is a small GTPase acting on membrane trafficking and actin remodeling, is frequently mutated in various tumors displaying microsatellite instability (MSI), notably in MSI colorectal cancers (CRC). Genotyping of 93 MSI CRCs (40 stage II, 32 stage III and 21 stage IV) allowed us to underscore that SMAP1 mutation frequency was inversely correlated with disease stage (P=0.01). Analysis of 46 cancer cell lines showed that SMAP1 mutations occurred only in MSI tumors, and consisted exclusively in short insertion or deletion in the coding 10-adenine repeat, generating a premature termination codon located downstream the ArfGAP domain. SMAP1 transcript levels were significant decreased (P=0.006), and truncated SMAP1 protein could not be detected in cells displaying biallelic SMAP1 mutations, owing to its sensitivity to proteasome degradation. To investigate the role of SMAP1 mutations, we used the SMAP1-null HCT116 cell line and we established three isogenic SMAP1-complemented clones. Cell proliferation was first assessed in vivo using subcutaneous xenografts into immunodeficient mice. Tumors developed in all animals regardless of the cell line injected, but tumor volumes were significantly smaller for both SMAP1-complemented clones compared with HCT116 (P

Neuroepithelial papillary tumor of the pineal region (PTPR) has been defined as a distinct entity that is increasingly being recognized, with 96 cases now reported. This tumor shares morphologic features with both ependymomas and choroid plexus tumors. PTPR is characterized by an epithelial-like growth pattern in which the vessels are covered by layers of tumor cells forming perivascular pseudorosettes. These tumors exhibit various combinations of papillary and solid architecture, making the differential diagnosis of PTPR difficult to establish. We report the detailed description of the histopathologic features of a large series of PTPRs from 20 different centers and distinguish 2 subgroups of tumors with either a striking papillary growth pattern or a papillary and solid growth pattern. We highlight the findings that PTPRs have unusual vessels with multiple lumina and frequently show detachment of the border of the tumoral cells from the vascular wall. The 2 PTPR subgroups present similar clinical characteristics and immunophenotypes. We confirmed and extended the results of previous ultrastructural studies on the presence of intercellular junctions at the apical part of tumoral cells. The expression of the tight junction proteins claudin-1, claudin-2, and claudin-3 was investigated by immunohistochemistry. Claudin-1 and claudin-3, but not claudin-2, were expressed in PTPRs and in the fetal subcommissural organ, potentially the origin of this tumor. In contrast, all 3 claudins were expressed in choroid plexus papillomas. Claudin expression may help in the diagnosis of PTPRs and can be used in combination with other markers, such as CK18, NCAM, E-cadherin, MAP-2, and Kir 7.1.

Medulloblastoma is the main primitive neuroectodermal tumour of the posterior fossa in childhood. The classical therapeutic approach consists of surgical resection, followed by craniospinal irradiation. Because of the good overall survival (75%), the main recent research efforts focus on refining the most relevant prognostic stratification and in decreasing the long-term sequelae.Thanks to the better understanding of the heterogeneity of medulloblastomas, clinical, histological and biological markers have been clearly identified and allow risk-adapted strategies. A subset of tumours of early childhood (3-5 years), frequently associated with a Sonic Hedgehog signalling, might be cured without irradiation. In older children, several trials have demonstrated the safety of reduced craniospinal irradiation in standard risk tumours. Furthermore, the evidence of an excellent prognosis associated with a subset of tumours characterized by an activation of the WNT pathway leads to forthcoming de-escalating strategies. Reducing long-term sequelae also relies on new surgical approaches aiming at reducing the cerebellar injuries. Tremendous efforts have also been made in defining the most adapted irradiation doses and fields. Intensity-modulated radiotherapy and proton beam therapy might also influence the long-term neurological and endocrine defects of the patients.Histological and biological characteristics clearly define various prognostic groups within medulloblastomas; confirming the overall good outcome and reducing long-term sequelae are the main focus of current clinical trials.

BACKGROUND: The diagnostic accuracy and reproducibility for glioma histological diagnosis are suboptimal. OBJECTIVE: To characterize radiological and histological features in pediatric malignant gliomas and to determine whether they had an impact on survival. METHODS: We retrospectively reviewed a series of 96 pediatric malignant gliomas. All histological samples were blindly and independently reviewed and classified according to World Health Organization 2007 and Sainte-Anne classifications. Radiological features were reviewed independently. Statistical analyses were performed to investigate the relationship between clinical, radiological, and histological features and survival. RESULTS: Cohort median age was 7.8 years; median follow-up was 4.8 years. Tumors involved cerebral hemispheres or basal ganglia in 82% of cases and brainstem in the remaining 18%. After histopathological review, low-grade gliomas and nonglial tumors were excluded (n = 27). The World Health Organization classification was not able to demonstrate differences between groups and patients survival. The Sainte-Anne classification identified a 3-year survival rate difference between the histological subgroups (oligodendroglioma A, oligodendroglioma B, malignant glioneuronal tumors, and glioblastomas; P = .02). The malignant glioneuronal tumor was the only glioma subtype with specific radiological features. Tumor location was significantly associated with 3-year survival rate (P = .005). Meningeal attachment was the only radiological criteria associated with longer survival (P = .02). CONCLUSION: The Sainte-Anne classification was better able to distinguish pediatric malignant gliomas in terms of survival compared with the World Health Organization classification. In this series, neither of these 2 histological classifications provided a prognostic stratification of the patients.

International audience; BACKGROUND: Primitive brain tumors are the leading cause of cancer-related death in children. Tumor cells with stem-like properties (TSCs), thought to account for tumorigenesis and therapeutic resistance, have been isolated from high-grade gliomas in adults. Whether TSCs are a common component of pediatric brain tumors and are of clinical relevance remains to be determined. METHODOLOGY/PRINCIPAL FINDINGS: Tumor cells with self-renewal properties were isolated with cell biology techniques from a majority of 55 pediatric brain tumors samples, regardless of their histopathologies and grades of malignancy (57% of embryonal tumors, 57% of low-grade gliomas and neuro-glial tumors, 70% of ependymomas, 91% of high-grade gliomas). Most high-grade glioma-derived oncospheres (10/12) sustained long-term self-renewal akin to neural stem cells (>7 self-renewals), whereas cells with limited renewing abilities akin to neural progenitors dominated in all other tumors. Regardless of tumor entities, the young age group was associated with self-renewal properties akin to neural stem cells (P = 0.05, chi-square test). Survival analysis of the cohort showed an association between isolation of cells with long-term self-renewal abilities and a higher patient mortality rate (P = 0.013, log-rank test). Sampling of low- and high-grade glioma cultures showed that self-renewing cells forming oncospheres shared a molecular profile comprising embryonic and neural stem cell markers. Further characterization performed on subsets of high-grade gliomas and one low-grade glioma culture showed combination of this profile with mesenchymal markers, the radio-chemoresistance of the cells and the formation of aggressive tumors after intracerebral grafting. CONCLUSIONS/SIGNIFICANCE: In brain tumors affecting adult patients, TSCs have been isolated only from high-grade gliomas. In contrast, our data show that tumor cells with stem cell-like or progenitor-like properties can be isolated from a wide range of histological sub-types and grades of pediatric brain tumors. They suggest that cellular mechanisms fueling tumor development differ between adult and pediatric brain tumors.

Neuropathological analysis of cellular mechanisms underlying gliosis and brain tumors is slowed by the lack of markers allowing to distinguish glial subpopulations in normal or pathological human brains. We therefore evaluated GFAPdelta immunostaining in a wide panel of astrogliosis and gliomas, and compared these with GFAP and vimentin. In normal tissue, gliosis and gliomas, GFAPdelta immunostaining was observed in astrocytes with relatively high GFAP levels. GFAPdelta immunostaining was most conspicuous in glia limitans astrocytes. In Chaslin's gliosis accompanying chronic epilepsy, GFAPdelta immunostaining evidenced the glia limitans reactive astrocytes as the source of the dense fibrillary meshwork typical of Chaslin's gliosis. Interestingly GFAPdelta and vimentin immunostainings coincided in normal tissues and gliosis, but not in gliomas. Altogether these results show that combined GFAP, GFAPdelta and vimentin labelling reveals fine gliofilament regulation in normal and pathological brain.

Overexpression of epidermal growth factor receptor (EGFR) is common in gliomas. Gliomas are infiltrating tumors in which neoplastic glial cells can be intermingled with reactive glial cells, particularly in diffuse low-grade gliomas. As overexpression of EGFR has also been described in gliosis, it can be difficult to evaluate EGFR immunolabeling in diffuse low-grade gliomas because of this cell mix. We compared EGFR immunolabeling between gliosis and low-grade gliomas in order to identify distinctive criteria. We studied EGFR expression in 28 cases of gliosis and 39 diffuse low-grade gliomas (23 astrocytomas and 16 oligodendrogliomas). EGFR immunohistochemistry staining was performed on paraffin-embedded sections with a mouse monoclonal antibody (clone 2-18C9; Dako). Co-expression of EGFR with Olig2, Mib-1, and p53 was assessed in seven cases of low-grade gliomas using double immunolabeling. Then, EGFR immunostaining was blindly tested on 22 small specimens of indeterminate glial lesions provided by a reference neuropathological center. Two pathologists of our local center were asked to classify the lesions into diffuse low-grade glioma or gliosis according to the pattern of EGFR expression. Weak expression of EGFR was commonly detected in gliosis (23/28 cases). Strongly-stained cells were absent. Positive cells had reactive glial cell morphology. EGFR expression in gliomas was characterized by constant strongly-stained cells (39/39 cases). All strongly-stained cells had a high nucleus-to-cytoplasm ratio, with minimal to moderate nuclear atypia. Most of the strongly EGFR-positive cells were Olig2-positive. All the cases displayed cells co-expressing EGFR and Mib-1. In three p53-positive tumors, many p53-positive cells were strongly EGFR-positive. On the basis of EGFR expression, 14 out of the 22 indeterminate cases were classified as gliomas and eight as gliosis by both pathologists. Concordance with the initial diagnosis established by the reference center and concordance between the pathologists were 100%. Our results confirm that weak EGFR expression can be detected by immunohistochemistry in gliosis. They show that strong EGFR expression may be specific for neoplastic glial cells. As all low-grade gliomas contained strongly-stained cells in our study, we believe that EGFR immunohistochemistry could be a useful tool for detection of neoplastic glial cells in case of indeterminate glial lesions.

Accumulation of frameshift mutations at genes containing coding mononucleotide repeats is thought to be the major molecular mechanism by which mismatch repair-deficient cells accumulate functional alterations. These mutations resulting from microsatellite instability (MSI) can affect genes involved in pathways with a putative oncogenic role, but may also arise in genes without any expected role in MSI carcinogenesis because of the high mutation background of these tumours. We here screened 39 MSI colorectal tumours for the presence of mutations in 25 genes involved in DNA damage signalling and repair pathways. Using a maximum likelihood statistical method, these genes were divided into two different groups that differed significantly in their mutation frequencies, and likely represent mutations that do or do not provide selective pressure during MSI tumour progression. Interestingly, the so-called real-target mutational events were found to be distributed among genes involved in different functional pathways of the DNA metabolism, for example, DNA damage signalling (DNA-PKcs, ATR), double-strand break (DSB) repair (DNA-PKcs, RAD50), mismatch repair (MSH3, MSH6, MBD4) and replication (POLD3). In particular, mutations in MRE11 and/or RAD50 were observed in the vast majority of the tumours and resulted in the concomitant loss of immunohistochemical expression of both proteins. These data might explain why MSI colorectal cancers (CRC) behave differently in response to a wide variety of chemotherapeutic agents, notably those targeting DNA. More generally, they give further insights into how MSI leads to functional changes with synergistic effects in oncogenic pathways.

Primary central nervous system lymphoma (PCNSL) is a rare extranodal diffuse large B-cell lymphoma (DLBCL) restricted to the CNS and associated with a poor prognosis. Despite a notable effort contributing to better characterize the genomic basis of these tumors, few molecular abnormalities were proposed as predictive or prognostic biomarkers. Recently, two hotspot mutations, C228T and C250T, and the polymorphism rs2853669, located within TERT core promoter region have known a growing interest due to their prognostic value in a wide range of tumors, including melanoma, hepatocellular carcinoma or glioblastoma. Forty-one PCNSL tumor samples of newly diagnosed PCNSL were sequenced for C228T and C250T TERT somatic mutations. TERT promoter mutations status was correlated with clinical outcome and rs2853669 genotype. We identified C228T and C250T mutations and rs2853669 genotype in 20%, 9.4% and 50%, respectively. No case with co-occurrence of C228T and C250T mutations was identified. C228T mutations, but not C250T, were significantly associated with shorter overall survival (OS) (median OS = 4.5 months vs 33 months for non-mutated, p = 0.0048) and progression free survival (PFS) (median PFS = 3 months vs 11.5 months for non-mutated, p = 0.0260). We found no association between TERT promoter mutations and the rs2853669 genotype. To our knowledge, we showed the presence of TERT promoter mutations in PCNSL for the first time. In addition, C228T promoter mutation was associated with a poor outcome. Considering that these mutations are known to confer enhanced TERT promoter activity, we speculate that an increased telomerase activity could play a role in PCNSL aggressiveness. This work was granted by the LOC study group network (réseau national de centres experts des lymphomes primitifs du SNC, INCa), the Ligue contre le Cancer (Comité du Val d’Oise), the Fondation pour la Recherche Médicale (FDT20140930968), the APHP-CRIC 2013 (assistance publique - hôpitaux de Paris), and the Association pour la Recherche sur les Tumeurs Cérébrales. Citation Format: Aurélie Bruno, Mailys Daniau, Karima Mokhtari, Amithys Rahimian, Marc Polivka, Anne Jouvet, Clovis Adam, Dominique Figarella-Branger, Catherine Miquel, Sandrine Eimer, Caroline Houillier, Carole Soussain, Agusti Alentorn, Khê Hoang-Xuan. Recurrent TERT promoter mutations in primary central nervous system lymphoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3896. doi:10.1158/1538-7445.AM2015-3896

Hereditary nonpolyposis colon cancer (HNPCC) syndrome is the most frequent hereditary cancer syndrome predisposing to cancers of various locations, especially colon, endometrium, stomach, and upper urinary tract. Carcinomas of the kidney parenchyma are not considered as an HNPCC-related tumor. HNPCC tumors are characterized by microsatellite instability (MSI) due to a defect in mismatch repair (MMR) and carry somatic frameshift mutations in mononucleotide repeats within the coding regions of key genes. We report the first case of a papillary carcinoma of the kidney in an HNPCC patient who developed carcinomas of the upper urinary tract, endocervix, and colon. Whereas the HNPCC-related tumors demonstrated MSI phenotype, loss of MSH2 protein expression, and frameshift mutations in several of the 13 target genes analyzed, the kidney cancer displayed MSS phenotype, normal MMR protein expression, and no frameshift mutation in target genes. Our observations do not support the possibility that papillary carcinomas are part of HNPCC syndrome.

Loss of a functional mismatch repair (MMR) system in colorectal cancer (CRC) cells is associated with microsatellite instability and increased sensitivity to topoisomerase inhibitors. In this study, we have investigated whether a defect in double-strand break (DSB) repair by non-homologous end-joining (NHEJ) could explain why MMR-deficient CRC cells are hypersensitive to camptothecin (CPT), a topoisomerase I inhibitor. To evaluate the efficiency and the fidelity of DSB repair, we have transiently transfected plasmids containing cohesive or non-complementary ends in cells with various MMR defects. We have observed that the repair efficiency of DSB with cohesive and non-complementary ends is comparable in all cell lines. In contrast to the MMR-proficient cell line HT29, the MMR-deficient cell lines were highly accurate in repairing DSB with cohesive ends, but this characteristic could not be directly assigned to the primary MMR deficiency. Furthermore, CPT treatment had no detectable effect on the repair of cohesive ends but significantly decreased the repair efficiency of non-complementary DSB. In conclusion, although our observations show that DSB repair efficiency by NHEJ decreases upon treatment with CPT, which possibly contributes to its cytotoxicity, it is quite unlikely that it accounts for the hypersensitivity of MMR-deficient cells to topoisomerase inhibitors.

Objective To demonstrate that malignant glioneuronal tumors comprise a large spectrum of neoplasms, without mature ganglion-like cells, that may histologically resemble any malignant glioma (World Health Organization Grade III or IV) but have a distinct biological behavior. Methods This series includes all tumors diagnosed as malignant glioneuronal tumors (MGNTs) in our routine practice during a 2-year period during which neurofilament protein (NFP) immunostaining was performed in any case of suspected malignant glioma with unusual clinical, radiographic, and/or histological features. Immunostaining using neuronal markers (NFP, NeuN, synaptophysin, and chromogranin) and glial fibrillary acidic protein was done on paraffin sections after antigen retrieval. The presence of NFP-positive tumor cells, including those in mitosis, was used as a hallmark diagnostic criterion of MGNT. Results All tumors coexpressed glial fibrillary acidic protein and NFP. Other neuronal markers tested were inconstantly expressed. No recurrence was observed at the primary site in 36.4% of patients who underwent gross total resection. Twelve patients (33.3%) developed intra-axial and/or systemic metastases, and 4 were free of disease at 39 to 184 months. Univariate analysis revealed that gross total surgical resection was the most important prognostic factor predicting survival (44 versus 15 mo; P 1 yr; P = 0.005), young age at symptom onset (children versus adults; P = 0.045), and absence of necrosis (P = 0.02). Gross total surgical resection (P = 0.001) and a long duration of symptoms (symptoms > 1 yr; P = 0.013) proved to be independent and statistically significant prognostic factors in the multivariate analysis. Conclusion NFP immunostaining is required to identify MGNTs accurately. Their distinction from malignant gliomas is of paramount clinical importance, particularly for neurosurgeons, because gross total surgical resection may be curative in some cases. Finally, MGNTs may account for the long-term survival and/or occurrence of metastases demonstrated in a subset of malignant gliomas.

Background: Primary central nervous system lymphoma (PCNSL) is a rare extranodal diffuse large B-cell lymphoma (DLBCL). Little is known about the genomic basis of tumorigenesis in PCNSL partly explained by the rarity of biological material available for research studies. PCNSL patients display poor outcomes compared to systemic DLBCL and it is still unclear whether this is linked to the organ-specific microenvironment or reflects a specific, aggressive, intrinsic biological behavior. To our knowledge, the mutational landscape of PCNSL has never been investigated Experimental design: We analyzed 9 paired tumor and germline DNA samples from PCNSL patients by high throughput exome sequencing. Then, we performed focused sequencing in 28 additional PCNSL tumors to validate somatic genomic alterations and better estimate their incidence. Mutational data were correlated with copy number variations (CNV) identified by SNP and CGH arrays and clinical data. Results: We identified recurrent somatic mutations in genes involved in key signalling pathways such as NFκB (i.e. MYD88, CD79B, TBL1XR1), B cell differentiation (i.e. ETV6, EBF1, IRF4) and cell cycle control (i.e. BTG1, PIM1). We found somatic mutations on MYD88 (38%), CD79B (30%), TBL1XR1 (22%) or ETV6 (16%) genes and described bi-allelic inactivation of TBL1XR1 and ETV6 genes. Interestingly, the correlation with the outcome showed a significant negative impact of TBL1XR1 mutation on survival in a multivariate analysis. Conclusions: The present study showed that PCNSLs have high genetic heterogeneity and that their mutational patterns display similarities with extracerebral DLBCL, particularly of the ABC subtype, suggesting shared underlying biological mechanisms. These results provide new insights into the mutational landscape of PCNSL and potential targets for therapeutic strategies. This work benefited from the LOC study group network (réseau national de centres experts des lymphomes primitifs du SNC, INCa). Citation Format: Aurélie Bruno, Blandine Boisselier, Karim Labreche, Yannick Marie, Marc Polivka, Anne Jouvet, Clovis Adam, Dominique Figarella-Branger, Catherine Miquel, Anne Vital, Caroline Houillier, Carole Soussain, Karima Mokhtari, Romain Daveau, Khê Hoang-Xuan. Mutational analysis of primary central nervous system lymphoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5192. doi:10.1158/1538-7445.AM2014-5192