Oncolytic DNX-2401 Virus for Pediatric Diffuse Intrinsic Pontine Glioma.

BACKGROUND: Pediatric patients with diffuse intrinsic pontine glioma (DIPG) have a poor prognosis, with a median survival of less than 1 year. Oncolytic viral therapy has been evaluated in patients with pediatric gliomas elsewhere in the brain, but data regarding oncolytic viral therapy in patients with DIPG are lacking. METHODS: We conducted a single-center, dose-escalation study of DNX-2401, an oncolytic adenovirus that selectively replicates in tumor cells, in patients with newly diagnosed DIPG. The patients received a single virus infusion through a catheter placed in the cerebellar peduncle, followed by radiotherapy. The primary objective was to assess the safety and adverse-event profile of DNX-2401. The secondary objectives were to evaluate the effect of DNX-2401 on overall survival and quality of life, to determine the percentage of patients who have an objective response, and to collect tumor-biopsy and peripheral-blood samples for correlative studies of the molecular features of DIPG and antitumor immune responses. RESULTS: A total of 12 patients, 3 to 18 years of age, with newly diagnosed DIPG received 1×1010 (the first 4 patients) or 5×1010 (the subsequent 8 patients) viral particles of DNX-2401, and 11 received subsequent radiotherapy. Adverse events among the patients included headache, nausea, vomiting, and fatigue. Hemiparesis and tetraparesis developed in 1 patient each. Over a median follow-up of 17.8 months (range, 5.9 to 33.5), a reduction in tumor size, as assessed on magnetic resonance imaging, was reported in 9 patients, a partial response in 3 patients, and stable disease in 8 patients. The median survival was 17.8 months. Two patients were alive at the time of preparation of the current report, 1 of whom was free of tumor progression at 38 months. Examination of a tumor sample obtained during autopsy from 1 patient and peripheral-blood studies revealed alteration of the tumor microenvironment and T-cell repertoire. CONCLUSIONS: Intratumoral infusion of oncolytic virus DNX-2401 followed by radiotherapy in pediatric patients with DIPG resulted in changes in T-cell activity and a reduction in or stabilization of tumor size in some patients but was associated with adverse events. (Funded by the European Research Council under the European Union's Horizon 2020 Research and Innovation Program and others; EudraCT number, 2016-001577-33; ClinicalTrials.gov number, NCT03178032.).

Next-generation sequencing of uveal melanoma with clinical and histological correlations: Prognostic value of new mutations in the PI3K/AKT/mTOR pathway.

BACKGROUND: Uveal melanoma (UM) is the eye's most common primary malignancy and there are no effective therapies for disseminated disease. It is important to try to know the patient's prognosis. The aim of this study was to reflect genetic variants, studied using NGS, of a series of 69 cases of UM and its correlation with histopathology and clinical progression. METHODS: We performed targeted NGS using a 519-gene panel. RESULTS: There were selected 28 different mutated genes, showing a total of 231 genetic variants that affected the function of the protein. The most common secondary mutations occurred in SF3B1 (in 26%), followed by BAP1 (in 23%), LRP1B (22%) and FGFR4 (20%). BAP1 mutation was associated with a greater likelihood of metastases and with greater presence of epithelioid cells. LRP1B was also associated with presence of epithelioid cells SF3B1 mutation was significantly associated with a spindle morphology. We found variants in the RAD51B, TOP2A, PTPRD, TSC2, DHX9, PDK1 and MTOR that have not been previously reported in consulted databases. The presence of a mutation in: CHEK2, DHX9 and PDK1 was associated with metastases. CONCLUSIONS: BAP1 is the most solid biomarker of a poor prognosis in UM and mutations can be detected using NGS. SF3B1 is associated with the spindle cell subtype of UM, which gives it probably a favourable prognostic value. Our study suggests that mutations in DHX9 and PDK1 can have prognostic value. These potential biomarkers are related to the PI3K/AKT/mTOR pathway and makes them candidates for developing new directed therapies.

Machine Learning Quantification of Intraepithelial Tumor-Infiltrating Lymphocytes as a Significant Prognostic Factor in High-Grade Serous Ovarian Carcinomas.

The prognostic and predictive role of tumor-infiltrating lymphocytes (TILs) has been demonstrated in various neoplasms. The few publications that have addressed this topic in high-grade serous ovarian carcinoma (HGSOC) have approached TIL quantification from a semiquantitative standpoint. Clinical correlation studies, therefore, need to be conducted based on more accurate TIL quantification. We created a machine learning system based on H&E-stained sections using 76 molecularly and clinically well-characterized advanced HGSOC. This system enabled immune cell classification. These immune parameters were subsequently correlated with overall survival (OS) and progression-free survival (PFI). An intense colonization of the tumor cords by TILs was associated with a better prognosis. Moreover, the multivariate analysis showed that the intraephitelial (ie) TILs concentration was an independent and favorable prognostic factor both for OS (p = 0.02) and PFI (p = 0.001). A synergistic effect between complete surgical cytoreduction and high levels of ieTILs was evidenced, both in terms of OS (p = 0.0005) and PFI (p = 0.0008). We consider that digital analysis with machine learning provided a more accurate TIL quantification in HGSOC. It has been demonstrated that ieTILs quantification in H&E-stained slides is an independent prognostic parameter. It is possible that intraepithelial TIL quantification could help identify candidate patients for immunotherapy.

Morphological and Molecular Study of Hybrid Oncocytic/Chromophobe Tumor of the Kidney Associated with Sporadic Renal Oncocytosis and Chronic B-Cell Lymphocytic Leukemia: The Possible Contribution of Lymphoma to Renal Oncocytosis.

Hybrid oncocytic/chromophobe tumor (HOCT) of the kidney arising from a precursor oncocytosis not associated with the Birt-Hogg-Dubé (BHD) syndrome is an unusual and highly interesting neoplasm. Immunohistochemical and molecular findings suggest that HOCT is an entity distinct from both oncocytoma and chromophobe carcinoma. Although uncertainty persists regarding the factors predisposing to the development of HOCT, experimental findings suggest that it may arise due to the effect of toxins or in association with chronic kidney failure. The potential role of prior renal lymphoma in the development of oncocytosis has not hitherto been examined. We present a morphological, immunohistochemical, and molecular analysis of an HOCT arising from renal oncocytosis in conjunction with CLL affecting the kidney. The findings suggest that this tumor belongs to a family of similar neoplasms including oncocytoma, the eosinophilic variant of chromophobe renal-cell carcinoma (CRCC), and low-grade oncocytic tumor, even though these neoplasms may arise from different precursor lesions. HOCT and oncocytosis revealed the same immunohistochemical profile consistent on positivity for epithelial membrane antigen (EMA), cytokeratin 7 (Ck7), E-cadherin, CAM 5.2 and negativity for Pax-8, vimentin, renal-cell carcinoma (RCC) antigen, CD117, racemase, progesterone receptor, and CD10. The Ki-67 proliferation index was <1%. Molecular analysis of the tumor revealed the AKT3 gene mutation variant, classified as probably pathogenic, together with FOS1 gene amplification and no copy number variations (CNVs). Finally, we present a case of HOCT arising from a nonhereditary renal oncocytosis in conjunction with B lymphoma that raises interesting questions regarding pathogenesis.

Diverse immune environments in human lung tuberculosis granulomas assessed by quantitative multiplexed immunofluorescence.

The precise nature of the local immune responses in lung tuberculosis (TB) granulomas requires a comprehensive understanding of their environmental complexities. At its most basic level, a granuloma is a compact, organized immune aggregate of macrophages surrounded by myeloid, B and T cells. We established two complementary multiplex immunolabeling panels to simultaneously evaluate the myeloid and lymphocytic contexture of 14 human lung TB granulomas in formalin-fixed paraffin-embedded tissue samples. We observed diverse CD3+ and CD8+ T-cell and CD20+ B lymphocyte compositions of the granuloma immune environment and a relatively homogeneous distribution of all myeloid cells. We also found significant associations between CD8+ T-cell densities and the myeloid marker CD11b and phagocytic cell marker CD68. In addition, significantly more CD68+ macrophages and CD8+ T cells were found in Mycobacterium tuberculosis-infected granulomas, as detected by Ziehl-Neelsen staining. FOXP3 expression was predominately found in a small subset of CD4+ T cells in different granulomas. As the success or failure of each granuloma is determined by the immune response within that granuloma at a local and not a systemic level, we attempted to identify the presence of reactive T cells based on expression of the T-cell activation marker CD137 (4-1BB) and programmed cell death-1 (PD-1). Only a small fraction of the CD4+ and CD8+ T cells expressed PD-1. CD137 expression was found only in a very small fraction of the CD4+ T cells in two granulomas. Our results also showed that multinucleated giant cells showed strong PD-L1 but not CTLA-4 membrane staining. This study offers new insights into the heterogeneity of immune cell infiltration in lung TB granulomas, suggesting that each TB granuloma represents a unique immune environment that might be independently influenced by the local adaptive immune response, bacterial state, and overall host disease status.

A case of unilateral linear capillaritis.

Unilateral linear capillaritis is a rare variant of the pigmented purpuric dermatoses that can be misdiagnosed due to confusion with other cutaneous diseases that follow a linear distribution. We present the case of an 8-year-old boy with hyperpigmented patches linearly distributed on the right arm, initially diagnosed with segmental neurofibromatosis.

5 protein-based signature for resectable lung squamous cell carcinoma improves the prognostic performance of the TNM staging.

INTRODUCTION: Prognostic biomarkers have been very elusive in the lung squamous cell carcinoma (SCC) and none is currently being used in the clinical setting. We aimed to identify and validate the clinical utility of a protein-based prognostic signature to stratify patients with early lung SCC according to their risk of recurrence or death. METHODS: Patients were staged following the new International Association for the Study of Lung Cancer (IASLC) staging criteria (eighth edition, 2018). Three independent retrospective cohorts of 117, 96 and 105 patients with lung SCC were analysed to develop and validate a prognostic signature based on immunohistochemistry for five proteins. RESULTS: We identified a five protein-based signature whose prognostic index (PI) was an independent and significant predictor of disease-free survival (DFS) (p<0.001; HR=4.06, 95% CI 2.18 to 7.56) and overall survival (OS) (p=0.004; HR=2.38, 95% CI 1.32 to 4.31). The prognostic capability of PI was confirmed in an external multi-institutional cohort for DFS (p=0.042; HR=2.01, 95% CI 1.03 to 3.94) and for OS (p=0.031; HR=2.29, 95% CI 1.08 to 4.86). Moreover, PI added complementary information to the newly established IASLC TNM 8th edition staging system. A combined prognostic model including both molecular and anatomical (TNM) criteria improved the risk stratification in both cohorts (p<0.05). CONCLUSION: We have identified and validated a clinically feasible protein-based prognostic model that complements the updated TNM system allowing more accurate risk stratification. This signature may be used as an advantageous tool to improve the clinical management of the patients, allowing the reduction of lung SCC mortality through a more accurate knowledge of the patient's potential outcome.

Recalcitrant granulomatous periorificial dermatitis with good response to low-dose oral isotretinoin.

Granulomatous periorificial dermatitis is a clinical variant of periorificial dermatitis. We present the case of an 18-year-old girl with several reddish papular lesions in the perioral, perinasal, and periorbital regions unresponsive to conventional therapy. After 6 months of therapy with low-dose oral isotretinoin, the lesions fully remitted.

A phase II trial of autologous dendritic cell vaccination and radiochemotherapy following fluorescence-guided surgery in newly diagnosed glioblastoma patients.

BACKGROUND: Prognosis of patients with glioblastoma multiforme (GBM) remains dismal, with median overall survival (OS) of about 15 months. It is therefore crucial to search alternative strategies that improve these results obtained with conventional treatments. In this context, immunotherapy seems to be a promising therapeutic option. We hypothesized that the addition of tumor lysate-pulsed autologous dendritic cells (DCs) vaccination to maximal safe resection followed by radiotherapy and concomitant and adjuvant temozolomide could improve patients' survival. METHODS: We conducted a phase-II clinical trial of autologous DCs vaccination in patients with newly diagnosed patients GBM who were candidates to complete or near complete resection. Candidates were finally included if residual tumor volume was lower than 1 cc on postoperative radiological examination. Autologous DCs were generated from peripheral blood monocytes and pulsed with autologous whole tumor lysate. The vaccination calendar started before radiotherapy and was continued during adjuvant chemotherapy. Progression free survival (PFS) and OS were analyzed with the Kaplan-Meier method. Immune response were assessed in blood samples obtained before each vaccines. RESULTS: Thirty-two consecutive patients were screened, one of which was a screening failure due to insufficient resection. Median age was 61 years (range 42-70). Karnofsky performance score (KPS) was 90-100 in 29%, 80 in 35.5% and 60-70 in 35.5% of cases. MGMT (O6-methylguanine-DNA-methyltransferase) promoter was methylated in 45.2% of patients. No severe adverse effects related to immunotherapy were registered. Median PFS was 12.7 months (CI 95% 7-16) and median OS was 23.4 months (95% CI 16-33.1). Increase in post-vaccination tumor specific immune response after vaccines (proliferation or cytokine production) was detected in 11/27 evaluated patients. No correlation between immune response and survival was found. CONCLUSIONS: Our results suggest that the addition of tumor lysate-pulsed autologous DCs vaccination to tumor resection and combined radio-chemotherapy is feasible and safe. A multicenter randomized clinical trial is warranted to evaluate the potential survival benefit of this therapeutic approach. Trial registration This phase-II trial was registered as EudraCT: 2009-009879-35 and ClinicalTrials.gov Identifier: NCT01006044 retrospectively registered.

MRI fused with prone FDG PET/CT improves the primary tumour staging of patients with breast cancer.

OBJECTIVE: Our aim was to evaluate the diagnostic accuracy of magnetic resonance imaging (MRI) fused with prone 2-[fluorine-18]-fluoro-2-deoxy-D-glucose (FDG) positron emission tomography (PET) in primary tumour staging of patients with breast cancer. METHODS: This retrospective study evaluated 45 women with 49 pathologically proven breast carcinomas. MRI and prone PET-CT scans with time-of-flight and point-spread-function reconstruction were performed with the same dedicated breast coil. The studies were assessed by a radiologist and a nuclear medicine physician, and evaluation of fused images was made by consensus. The final diagnosis was based on pathology (90 lesions) or follow-up ≥ 24 months (17 lesions). RESULTS: The study assessed 72 malignant and 35 benign lesions with a median size of 1.8 cm (range 0.3-8.4 cm): 31 focal, nine multifocal and nine multicentric cases. In lesion-by-lesion analysis, sensitivity, specificity, positive and negative predictive values were 97%, 80%, 91% and 93% for MRI, 96%, 71%, 87%, and 89% for prone PET, and 97%. 94%, 97% and 94% for MRI fused with PET. Areas under the curve (AUC) were 0.953, 0.850, and 0.983, respectively (p < 0.01). CONCLUSIONS: MRI fused with FDG-PET is more accurate than FDG-PET in primary tumour staging of breast cancer patients and increases the specificity of MRI. KEY POINTS: • FDG PET-CT may improve the specificity of MRI in breast cancer staging. • MRI fused with prone 2-[fluorine-18]-fluoro-2-deoxy-D-glucose PET-CT has better overall diagnostic performance than MRI. • The clinical role of fused PET-MRI has not yet been established.

Management of a pregnant woman with thyrotropinoma: a case report and review of the literature.

INTRODUCTION: Pituitary disorders during pregnancy are uncommon. The approach should include a close follow-up in order to reduce maternal and fetal risks associated with physiological changes during pregnancy or treatment side effects. MATERIALS AND METHODS: We report a 21-year-old woman with a thyroid-stimulating hormone-secreting pituitary macroadenoma and positive antithyroid antibodies. She was initially treated using transsphenoidal pituitary surgery. The patient relapsed 17-month post-surgery. Somatostatin analog therapy was started which rapidly controlled the hyperthyroidism. Eleven months later, while receiving octreotide, the patient reported to be pregnant and the medication was stopped. Gestation and delivery went well with a healthy full-term newborn. The patient developed a postpartum thyroiditis 15 weeks after giving birth. Twenty-eight months postpartum the patient remains euthyroid without medication. CONCLUSIONS: The overall positive outcomes of the four cases reported in literature, including this new case, suggest that pregnancy should not be absolutely contraindicated in women with thyrotropinomas. We emphasize the effectiveness of octreotide to control hyperthyroidism, as well as stopping medication when a patient is found to be pregnant. In our case, close observation following octreotide cessation had a positive outcome.

Aberrant mitochondria in a Bethlem myopathy patient with a homozygous amino acid substitution that destabilizes the collagen VI α2(VI) chain.

Bethlem myopathy and Ullrich congenital muscular dystrophy (UCMD) sit at opposite ends of a clinical spectrum caused by mutations in the extracellular matrix protein collagen VI. Bethlem myopathy is relatively mild, and patients remain ambulant in adulthood while many UCMD patients lose ambulation by their teenage years and require respiratory interventions. Dominant and recessive mutations are found across the entire clinical spectrum; however, recessive Bethlem myopathy is rare, and our understanding of the molecular pathology is limited. We studied a patient with Bethlem myopathy. Electron microscopy of his muscle biopsy revealed abnormal mitochondria. We identified a homozygous COL6A2 p.D871N amino acid substitution in the C-terminal C2 A-domain. Mutant α2(VI) chains are unable to associate with α1(VI) and α3(VI) and are degraded by the proteasomal pathway. Some collagen VI is assembled, albeit more slowly than normal, and is secreted. These molecules contain the minor α2(VI) C2a splice form that has an alternative C terminus that does include the mutation. Collagen VI tetramers containing the α2(VI) C2a chain do not assemble efficiently into microfibrils and there is a severe collagen VI deficiency in the extracellular matrix. We expressed wild-type and mutant α2(VI) C2 domains in mammalian cells and showed that while wild-type C2 domains are efficiently secreted, the mutant p.D871N domain is retained in the cell. These studies shed new light on the protein domains important for intracellular and extracellular collagen VI assembly and emphasize the importance of molecular investigations for families with collagen VI disorders to ensure accurate diagnosis and genetic counseling.

Role of HHV-8 and mTOR pathway in post-transplant Kaposi sarcoma staging.

Kaposi's sarcoma (KS) is one of the most frequent transplant related tumors. Several pathways are involved; however, the impact of the molecular phenotype associated to the tumor stage and the behavior-depending resultant therapy is still unknown. The aim of our study was to analyze the role of HHV-8 and mTOR pathway in tumor stages of skin KS after renal transplantation. Twelve renal transplant recipients with cutaneous KS from five transplant centers (1980-2007) under reduction of immunosuppression or conversion to mTOR inhibitor were included. The expression of HHV-8, PTEN, TGFß, VEGF, phospho-mTOR, and phospho-P70S6K in tumoral tissue was analyzed. KS lesions were classified as patch, plaque, and nodule state. HHV-8 infection was found in all tissue samples. KS lesions showed high activation of VEGF, p-mTOR and p-P70S6K, low PTEN, and null TGFß expression. The only pathway activated in a staging-dependent manner was mTOR with higher p-mTOR and p-P70S6K expression in nodule versus patch stage. KS lesions disappeared after 5.24 months in all converted patients without any recurrence in 14.05 years of mean follow-up. The activation of mTOR pathway according to KS stages supports the rational of the mTOR inhibitor in post-transplant Kaposi.

Clinical Neuropathology Views - 2/2016: Digital networking in European neuropathology: An initiative to facilitate truly interactive consultations.

Digital technology is progressively changing our vision of the practice of neuropathology. There are a number of facts that support the introduction of digital neuropathology. With the development of wholeslide imaging (WSI) systems the difficulties involved in implementing a neuropathology network have been solved. A relevant difficulty has been image standardization, but an open digital image communication protocol defined by the Digital Imaging and Communications in Medicine (DICOM) standard is already a reality. The neuropathology network should be established in Europe because it is the expected geographic context for relationships among European neuropathologists. There are several limitations in the implementation of a digital neuropathology consultancy network such as financial support, operational costs, legal issues, and technical assistance of clients. All of these items have been considered and should be solved before implementing the proposal. Finally, the authors conclude that a European digital neuropathology network should be created for patients' benefit.

Biological and clinical significance of the intratumour heterogeneity of PTEN protein expression and the corresponding molecular abnormalities of the PTEN gene in glioblastomas.

AIMS: Glioblastomas display marked phenotypic and molecular heterogeneity. The expression of the PTEN protein in glioblastomas also shows great intratumour heterogeneity, but the significance of this heterogeneity has so far received little attention. METHODS: We conducted a comparative study on paraffin and frozen samples from 60 glioblastomas. Based on PTEN immunostaining, paraffin glioblastomas were divided into positive (homogeneous staining) and both positive and negative (heterogeneous staining) tumours. DNA was extracted from manually microdissected samples from representative areas, and from frozen samples taken randomly from the same tumours. Loss of heterozygosity (LOH) of 10q23 and hypermethylation status of the PTEN promoter were studied, and the molecular findings were correlated with overall survival. RESULTS: PTEN protein was present heterogeneously in 42 cases and homogeneously in 18 cases. In homogeneous glioblastomas, no correlation was found between PTEN protein expression and the LOH of the gene. Surprisingly, in the heterogeneous glioblastomas, LOH was found significantly more frequently (P < 0.001) in PTEN-positive areas (81%) than in PTEN-negative ones (35.7%). In general, molecular results of frozen tissue were representative of the tumour. Only two cases of methylation of the PTEN promoter were identified. A significant difference was found for overall survival for LOH10q23 status (P = 0.005) and for homogeneous vs. heterogeneous tumours (P = 0.014). CONCLUSION: The expression of PTEN protein does not correlate with the abnormalities of the LOH of the gene. Interestingly, patients with glioblastomas presenting either LOH of 10q23 or heterogeneous PTEN expression have a poorer prognosis.

Fox Fordyce disease as a secondary effect of laser hair removal.

Fox Fordyce disease (FFD) has been recently described as an adverse effect of laser hair removal. It is an apocrine gland disorder characterized by pruritus and a folliculocentric papular eruption in apocrine sweat gland areas. Different etiologies have been proposed to be the cause of this entity. It has been suggested that a fisical factor could contribute to FFD phatogenesis. We report a new case of FFD after laser hair removal.