Cell-type specific concentration regulation of the basal transcription factor TFIIH in XPBy/y mice model.

BACKGROUND: The basal transcription/repair factor TFIIH is a ten sub-unit complex essential for RNA polymerase II (RNAP2) transcription initiation and DNA repair. In both these processes TFIIH acts as a DNA helix opener, required for promoter escape of RNAP2 in transcription initiation, and to set the stage for strand incision within the nucleotide excision repair (NER) pathway. METHODS: We used a knock-in mouse model that we generated and that endogenously expresses a fluorescent version of XPB (XPB-YFP). Using different microscopy, cellular biology and biochemistry approaches we quantified the steady state levels of this protein in different cells, and cells imbedded in tissues. RESULTS: Here we demonstrate, via confocal imaging of ex vivo tissues and cells derived from this mouse model, that TFIIH steady state levels are tightly regulated at the single cell level, thus keeping nuclear TFIIH concentrations remarkably constant in a cell type dependent manner. Moreover, we show that individual cellular TFIIH levels are proportional to the speed of mRNA production, hence to a cell's transcriptional activity, which we can correlate to proliferation status. Importantly, cancer tissue presents a higher TFIIH than normal healthy tissues. CONCLUSION: This study shows that TFIIH cellular concentration can be used as a bona-fide quantitative marker of transcriptional activity and cellular proliferation.

Familial focal epilepsy with focal cortical dysplasia due to DEPDC5 mutations.

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.

Clinical, pathological, and molecular data on desmoplastic/nodular medulloblastoma: case studies and a review of the literature.

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 immunoprofile. 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 overallsurvival 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.

Rubinstein-Taybi syndrome predisposing to non-WNT, non-SHH, group 3 medulloblastoma.

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.

Cutaneous location of atypical teratoid/rhabdoid tumour.

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.

Comprehensive genome characterization of solitary fibrous tumors using high-resolution array-based comparative genomic hybridization.

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.

[National network of paediatric central nervous system tumours reviewing by the Groupe d'Étude de Neuropathologie Oncologique Pediatrique (GENOP)]. / Réseau de relecture des tumeurs pédiatriques du système nerveux central par le Groupe d'étude de neuropathologie oncologique pédiatrique (GENOP).

Diagnosis of paediatric tumours of the central nervous system is often difficult because WHO classification criteria are mainly defined for adults tumours and do not always apply to their paediatric counterparts. These tumours are rare (400 cases/year among more than 50 pathological subtypes per year in France). Pathological diagnosis may be a challenge for a general pathologist with a too low number of paediatric cases in his recruitment. Hence, a reference group of paediatric neuropathologists was formed (GENOP) on the behalf of the comité "Tumeurs Cérébrales" de la Société Française de lutte contre les Cancers de l'Enfant. This network is supported by the Institut National du Cancer (INCa). GENOP aim is to structure a centralised review of paediatric central nervous system tumours in order to harmonise neuropathological diagnosis at the national level and enhance patients care. Cases assessed during the last 3 years led GENOP to better identify tumours subtypes for which there is a diagnostic challenge. A set of immunohistochemical or molecular specialised techniques was developed, leading to an increased diagnostic accuracy. It allowed a better distinction between diffuse and circumscribed glioma, a better recognition of glioneuronal differentiation and a better subtyping of embryonal tumours such as medulloblastomas. Inter-observer agreement varied according to the tumour subtypes.

Histological Risk Factors for Local Recurrence of Phyllodes Tumors of the Breast.

BACKGROUND/AIM: The rate of local recurrence (LR) of phyllodes tumor (PT) varies from 4 to 18%. Several histological risk factors of LR of PT are known. The aim of this study was to estimate the LR rate of PT according to PT grade and to evaluate histological risk factors of PT LR in our retrospective cohort. PATIENTS AND METHODS: This was a two-center study, conducted from 1995 to 2019. All patients with PT diagnosed on surgical specimen were included. PT was diagnosed histologically according to the grade category defined by the 2012 World Health Organization classification as benign, borderline or malignant PT. Univariate analysis and then multivariate logistic regression analysis were performed to determine histological risk factors of LR of PT. RESULTS: A total of 224 patients with PT were included: 152 with benign, 49 with borderline and 23 with malignant PT. The median and standard deviation for the duration of follow-up was 136.60 ± 167.43 months, and 18 patients (8.04%) developed LR: 7 (4.61%), 7 and (14.29%) and 4 (17.39%) with benign, borderline and malignant PT, respectively. In univariate analysis, LR was statistically increased for histological size ≥45 mm (p=0.003), borderline/malignant TP (p=0.006) and dense stromal cellularity (p<0.001). In multivariate analysis, only histological size ≥45 mm and cellularity were statistically associated with LR (odds ratio=1.83, 95% confidence interval=1.06-9.83, p=0.04; and odds ratio=3.69, 95% confidence interval=1.11-12.28, p=0.03, respectively). CONCLUSION: Histological size ≥45 mm and dense stromal cellularity were demonstrated as histological risk factors of LR of PT. In our cohort, no association was found between LR and PT grade nor LR and surgical margins ≥10 mm.

Differentiation driven changes in the dynamic organization of Basal transcription initiation.

Studies based on cell-free systems and on in vitro-cultured living cells support the concept that many cellular processes, such as transcription initiation, are highly dynamic: individual proteins stochastically bind to their substrates and disassemble after reaction completion. This dynamic nature allows quick adaptation of transcription to changing conditions. However, it is unknown to what extent this dynamic transcription organization holds for postmitotic cells embedded in mammalian tissue. To allow analysis of transcription initiation dynamics directly into living mammalian tissues, we created a knock-in mouse model expressing fluorescently tagged TFIIH. Surprisingly and in contrast to what has been observed in cultured and proliferating cells, postmitotic murine cells embedded in their tissue exhibit a strong and long-lasting transcription-dependent immobilization of TFIIH. This immobilization is both differentiation driven and development dependent. Furthermore, although very statically bound, TFIIH can be remobilized to respond to new transcriptional needs. This divergent spatiotemporal transcriptional organization in different cells of the soma revisits the generally accepted highly dynamic concept of the kinetic framework of transcription and shows how basic processes, such as transcription, can be organized in a fundamentally different fashion in intact organisms as previously deduced from in vitro studies.

IDH1 mutation, a genetic alteration associated with adult gliomatosis cerebri.

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.

Negative Relationship between Post-Treatment Stromal Tumor-Infiltrating Lymphocyte (TIL) and Survival in Triple-Negative Breast Cancer Patients Treated with Dose-Dense Dose-Intense NeoAdjuvant Chemotherapy.

Background: Patients with triple-negative breast cancers (TNBC) have a poor prognosis unless a pathological complete response (pCR) is achieved after neoadjuvant chemotherapy (NAC). Few studies have analyzed changes in TIL levels following dose-dense dose-intense (dd-di) NAC. Patients and methods: From 2009 to 2018, 117 patients with TNBC received dd-di NAC at our institution. We aimed to identify factors associated with pre- and post-NAC TIL levels, and oncological outcomes relapse-free survival (RFS), and overall survival (OS). Results: Median pre-NAC and post-NAC TIL levels were 15% and 3%, respectively. Change in TIL levels with treatment was significantly correlated with metabolic response (SUV) and pCR. High post-NAC TIL levels were associated with a weak metabolic response after two cycles of NAC, with the presence of residual disease and nodal involvement at NAC completion. In multivariate analyses, high post-NAC TIL levels independently predicted poor RFS and poor OS (HR = 1.4 per 10% increment, 95%CI (1.1; 1.9) p = 0.014 and HR = 1.8 per 10% increment 95%CI (1.3−2.3), p < 0.0001, respectively). Conclusion: Our results suggest that TNBC patients with TIL enrichment after NAC are at higher risk of relapse. These patients are potential candidates for adjuvant treatment, such as immunotherapy, in clinical trials.

Medulloblastomas: update on a heterogeneous disease.

PURPOSE OF REVIEW: 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. RECENT FINDINGS: 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. SUMMARY: 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.

EGFR immunolabeling pattern may discriminate low-grade gliomas from gliosis.

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.

Expression of angiopoietin-like 4 fibrinogen-like domain (cANGPTL4) increases risk of brain metastases in women with breast cancer.

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. MATERIALS AND METHODS: From June 2015 to June 2016, serum samples from 113 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, the two cleaved fragments of ANGPTL4, in the occurrence of brain metastases. RESULTS: An ANGPTL4 serum concentration over 0.1 ng/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 < 0.05), while mice injected with cANGPTL4 had longer survival (p < 0.01). On tissue sections, compared to wild-type mice, mice injected with nANGPTL4 cells had significantly larger surface areas of lung metastases (p < 0.01), and mice injected with cANGPTL4 had significantly larger surface areas of brain metastases (p < 0.01). CONCLUSIONS: In this study, we showed that a higher expression of Angiopoietin-like 4 Fibrinogen-Like Domain (cANGPTL4) was associated with an increased risk of brain metastases in women with breast cancer.

Prognostic Impact of Stromal Immune Infiltration before and after Neoadjuvant Chemotherapy (NAC) in Triple Negative Inflammatory Breast Cancers (TNIBC) Treated with Dose-Dense Dose-Intense NAC.

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 1.13-3.74; p = 0.02), high post-NAC TIL (HR 1.81; CI 1.07-3.06; p = 0.03) and positive delta TIL (HR 2.20; CI 1.36-3.52; 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 1.05-3.35; 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.

Dynamic interaction of TTDA with TFIIH is stabilized by nucleotide excision repair in living cells.

Transcription/repair factor IIH (TFIIH) is essential for RNA polymerase II transcription and nucleotide excision repair (NER). This multi-subunit complex consists of ten polypeptides, including the recently identified small 8-kDa trichothiodystrophy group A (TTDA)/ hTFB5 protein. Patients belonging to the rare neurodevelopmental repair syndrome TTD-A carry inactivating mutations in the TTDA/hTFB5 gene. One of these mutations completely inactivates the protein, whereas other TFIIH genes only tolerate point mutations that do not compromise the essential role in transcription. Nevertheless, the severe NER-deficiency in TTD-A suggests that the TTDA protein is critical for repair. Using a fluorescently tagged and biologically active version of TTDA, we have investigated the involvement of TTDA in repair and transcription in living cells. Under non-challenging conditions, TTDA is present in two distinct kinetic pools: one bound to TFIIH, and a free fraction that shuttles between the cytoplasm and nucleus. After induction of NER-specific DNA lesions, the equilibrium between these two pools dramatically shifts towards a more stable association of TTDA to TFIIH. Modulating transcriptional activity in cells did not induce a similar shift in this equilibrium. Surprisingly, DNA conformations that only provoke an abortive-type of NER reaction do not result into a more stable incorporation of TTDA into TFIIH. These findings identify TTDA as the first TFIIH subunit with a primarily NER-dedicated role in vivo and indicate that its interaction with TFIIH reflects productive NER.

GFAPdelta immunostaining improves visualization of normal and pathologic astrocytic heterogeneity.

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.