Inhibition of PERK mediated UPR acts as a switch for reversal of residual senescence and as senolytic therapy in glioblastoma.

BACKGROUND: Glioblastoma due to recurrence is clinically challenging with 10-15months overall survival. Previously we showed therapy induced senescence (TIS) in glioblastoma reverses causing recurrence. Here, we aim to delineate TIS reversal mechanism for potential therapeutic intervention to prevent GBM recurrence. METHODS: Residual senescent (RS) and End of Residual Senescence (ERS) cells were captured from GBM patient-derived primary-cultures and cell lines mimicking clinical scenario. RNA-sequencing, transcript/protein validations, knock-down/inhibitor studies, ChIP RT-PCR, biochemical assays and IHCs were performed for mechanistics of TIS reversal. In vivo validations were conducted in GBM orthotopic mouse model. RESULTS: Transcriptome analysis showed co-expression of ER stress-UPR and senescence associated secretory phenotype (SASP) with TIS induction and reversal. Robust SASP production and secretion by RS cells could induce senescence, ROS, DNA damage and ER stress in paracrine fashion independent of radiation. Neutralization of most significantly enriched cytokine from RS-secretome IL1ß, suppressed SASP and delayed senescence reversal. Mechanistically, with SASP and massive protein accumulation in Endoplasmic reticulum, RS cells displayed stressed ER morphology, upregulated ER stress markers and PERK pathway activation via peIF2α-ATF4-CHOP which was spontaneously resolved in ERS. ChIP RT-PCR showed CHOP occupancy at CXCL8/IL8, CDKN1A/p21 and BCL2L1/BCLXL aiding survival. PERK knockdown/inhibition with GSK2606414 in combination with radiation led to sustained ER stress and senescence without SASP. PERKi in RS functioned as senolytic via apoptosis and prevented recurrence in vitro and in vivo ameliorating overall survival. CONCLUSION: We demonstrate that PERK mediated UPR regulates senescence reversal and its inhibition can be exploited as potential seno-therapeutic option in glioblastoma.

Utility of leukocyte-associated immunoglobulin-like receptor-1 (CD305) in flow cytometric detection of minimal bone marrow involvement by B-cell non-Hodgkin lymphoma.

Multicolor flow cytometry (MFC) is crucial in detecting occult or minimal bone marrow (BM) involvement by non-Hodgkin lymphomas (NHL), which may not be detected using trephine biopsy or imaging studies. Detection of low-level BM involvement can be challenging without definite immunophenotypic aberrancies. We studied the utility of CD305 in MFC detection of minimal BM involvement by B-NHL, especially in the absence of aberrancies by commonly used markers. The study included 1084 consecutive BM samples submitted for the staging of B-NHLs (excluding CLL) over two years. Samples were studied for morphological, immunophenotypic, and histopathological assessment. MFC studies were performed using 10-13 color MFC, including CD305-antibody (clone, DX26). Minimal BM involvement was defined with a cutoff of ≤10% lymphoma cells in viable cells on MFC assessment. Of 1084, 148 samples revealed overt morphological involvement by B-NHL and were excluded from analysis. BM samples of 172/936 patients were morphologically negative but revealed involvement using MFC independently. Corresponding trephine biopsy involvement was detected in only 79/172 (45.9%) patients. On MFC, 23/172 samples showed BM involvement with >10% lymphoma cells, and 149/172 (86.6%) samples revealed minimal involvement. In 54/149 (36.24%) samples, lymphoma cells were detected only with aberrant loss of CD305 expression. In 78 of the remaining 95 samples (82.1%), it provided an immunophenotypic aberrancy addition to other markers and supported the results. CD305 is a highly useful marker in the flow cytometric assessment of minimal BM involvement by B-NHL. MFC is a superior modality to trephine biopsy in detecting low-level BM involvement.

Risk-stratification for treatment de-intensification in WNT-pathway medulloblastoma: finding the optimal balance between survival and quality of survivorship.

INTRODUCTION: Advances in molecular biology have led to consensus classification of medulloblastoma into four broad molecular subgroups - wingless (WNT), sonic hedgehog (SHH), Group 3, and Group 4, respectively. Traditionally, children >3 years of age, with no/minimal residual tumor (<1.5 cm2) and lack of metastasis were classified as average-risk disease with >80% long-term survival. Younger age (<3 years), large residual disease (≥1.5 cm2), and leptomeningeal metastases either alone or in combination were considered high-risk features yielding much worse 5-year survival (30-60%). This clinico-radiological risk-stratification has been refined by incorporating molecular/genetic information. Contemporary multi-modality management for non-infantile medulloblastoma entails maximal safe resection followed by risk-stratified adjuvant radio(chemo)therapy. Aggressive multi-modality management achieves good survival but is associated with substantial dose-dependent treatment-related toxicity prompting conduct of subgroup-specific prospective clinical trials. AREAS COVERED: We conducted literature search on PubMed from 1969 till 2023 to identify putative prognostic factors and risk-stratification for medulloblastoma, including molecular subgrouping. Based on previously published data, including our own institutional experience, we discuss molecular risk-stratification focusing on WNT-pathway medulloblastoma to identify candidates suitable for treatment de-intensification to strike the optimal balance between survival and quality of survivorship. EXPERT OPINION: Prospective clinical trials and emerging biological information should further refine risk-stratification in WNT-pathway medulloblastoma.

Long-term clinical outcomes of combined modality therapy for advanced-stage Hodgkin lymphoma in the PET era: A retrospective study.

BACKGROUND OBJECTIVES: The role of consolidation radiation therapy (CRT) after complete metabolic response to chemotherapy in advanced-stage (stage III and IV) Hodgkin lymphoma (HL) is controversial. This study was undertaken to assess the clinical outcomes in terms of event free survival, local failure free survival and overall survival in individuals with advanced HL treated with chemotherapy and CRT. METHODS: A retrospective review was conducted to study the long-term clinical outcomes in individuals diagnosed with HL and treated with chemotherapy and CRT from 2012 to 2016 at a tertiary cancer care hospital in India. RESULTS: Data from 203 study participants with advanced-stage HL were analyzed. Positron emission tomography-computed tomography (PET-CT) was done at baseline and after 2 cycles for response assessment. The median age at presentation was 32 yr [interquartile range (IQR): 26-46]. Early metabolic response (after 2 cycles) and delayed metabolic response (after 4 or 6 cycles) were observed in 74.4 and 25.6 per cent of individuals, respectively. With a median follow up of 52 months (IQR: 40-67), the five-year event-free survival (EFS), local failure-free survival (LFFS) and overall survival (OS) were 83.2, 95.1 and 94.6 per cent, respectively. On univariate analysis, extranodal disease was associated with inferior EFS (P=0.043). Haemoglobin <10.5 g/dl (P=0.002) and Hasenclever index >3 (P=0.00047) were associated with poorer OS. Relapses were observed in 28/203 (13.8%) study participants with predominance at central nodal stations. The median time to relapse was 19.4 months (IQR: 13-33). Local relapse alone (at the irradiated site) was observed in 5/28 study participants, systemic (distant) relapse in 14/28 individuals, while both systemic and local relapse was observed in 9/28 participants. Extranodal disease (P=0.05), bulky disease (P=0.005) and haemoglobin concentration ≤10.5 g/dl (P=0.036) were significant predictors for disease relapse. INTERPRETATION CONCLUSIONS: Individuals with advanced-stage HL treated with anthracycline-based chemotherapy (anthracycline-based chemotherapy with doxorubicin, bleomycin, vinblastine and dacarbazine regimen) and CRT had excellent long-term outcomes. As isolated infield failures are uncommon, selective consolidation with conformal RT to high-risk sites improves final disease outcomes.

Epilepsia Partialis Continua as a Sequelae of Measles Infection in Children With Hematolymphoid Malignancies.

PURPOSE: To share our clinical experience with the diagnosis and management of children with hematolymphoid malignancies presenting with epilepsia partialis continua (EPC) as a sequelae of measles infection. MATERIALS AND METHODS: In December 2022, a series of children in our hemato-oncology unit presented with focal status epilepticus with no conclusive evidence pointing toward any underlying etiology. One such child had a typical measles rash a few weeks before the onset of this focal status epilepticus. After a series of cases with a similar presentation, a clinical pattern suspicious for measles became evident. cerebrospinal fluid polymerase chain reaction was positive for measles virus with measles immunoglobin M detected in the serum. This led to the diagnosis of measles inclusion-body encephalitis in a series of children who presented with EPC over a period of 3 months. EPC is a rare manifestation of measles that is seen only in immunocompromised patients. RESULTS: Among the 18 children reported in this series, only 10 had a history of rashes. The rash was mostly transient and elicited only on retrospective history taking. Five of the 18 children who did not lose consciousness during the prolonged seizure episode survived the disease but had residual neurologic sequelae. Among the 18 children, two were unimmunized and immunization status could not be confirmed in three other children. CONCLUSION: This case series highlights the threats posed by measles infection in children with cancer who are immunosuppressed because of the underlying disease and ongoing chemotherapy. Loss of herd immunity because of declining measles immunization rates secondary to vaccine hesitancy and COVID-19 lockdown pose a greater risk of measles infection and its complications for patients with deficient immune systems.

Clinico-Radiological Outcomes in WNT-Subgroup Medulloblastoma.

Medulloblastoma (MB) comprises four broad molecular subgroups, namely wingless (WNT), sonic hedgehog (SHH), Group 3, and Group 4, respectively, with subgroup-specific developmental origins, unique genetic profiles, distinct clinico-demographic characteristics, and diverse clinical outcomes. This is a retrospective audit of clinical outcomes in molecularly confirmed WNT-MB patients treated with maximal safe resection followed by postoperative standard-of-care risk-stratified adjuvant radio(chemo)therapy at a tertiary-care comprehensive cancer centre. Of the 74 WNT-MB patients registered in a neuro-oncology unit between 2004 to 2020, 7 patients accrued on a prospective clinical trial of treatment deintensification were excluded, leaving 67 patients that constitute the present study cohort. The median age at presentation was 12 years, with a male preponderance (2:1). The survival analysis was restricted to 61 patients and excluded 6 patients (1 postoperative mortality plus 5 without adequate details of treatment or outcomes). At a median follow-up of 72 months, Kaplan-Meier estimates of 5-year progression-free survival and overall survival were 87.7% and 91.2%, respectively. Traditional high-risk features, large residual tumour (≥1.5 cm2), and leptomeningeal metastases (M+) did not significantly impact upon survival in this molecularly characterized WNT-MB cohort treated with risk-stratified contemporary multimodality therapy. The lack of a prognostic impact of conventional high-risk features suggests the need for refined risk stratification and potential deintensification of therapy.

CDKN2A/B deletion in IDH-mutant astrocytomas: An evaluation by Fluorescence in-situ hybridization.

INTRODUCTION: CDKN2A/B homozygous deletion is one of the defining features of grade 4 in IDH-mutant astrocytic tumours. AIM: To evaluate CDKN2A/B-deletion in IDH-mutant astrocytic tumours and its clinicopathological impact. MATERIALS AND METHODS: CDKN2A/B-deletion was evaluated by Fluorescence in-situ hybridisation (FISH) and interpreted by two recently accepted methods. RESULTS: Eighty-three out of 94 cases (histologically-grade 2: 3, grade 3: 46, grade 4: 34) were interpretable on FISH. Concordant CDKN2A/B-deletion was observed in 71% (27/38) of lower-grade tumours (n = 49) and 90% (27/30) of histological grade 4 tumours (n = 34). Both the interpretation methods showed good agreement (Kappa = 0.75). CDKN2A/B-deletion showed an inverse correlation for < 10% MIB-1 labeling index (p = 0.01) while that by method-2 showed a significant correlation for grade 4 (p = 0.02). No significant correlation was observed for any other clinicopathological parameters. Twenty-four patients showed progression/recurrence (including deaths), and no significant difference in frequency of CDKN2A/B deletion was observed among cases with disease progression across different histological grades. CONCLUSIONS: CDKN2A/B-deletion was observed across all the histological grades of IDH-mutant astrocytic tumours, expectedly more in the higher grade. FISH, as a method, can be used for the detection of CDKN2A/B homozygous deletion, when there is concordant interpretation.

BRAF V600E Mutations and Beyond: A Molecular Perspective of Melanoma from a Tertiary Cancer Referral Center of India.

Vaibhavi VengurlekarObjectives Malignant melanoma demonstrates frequently occurring mutations of genes in the serine/threonine kinase pathway, namely BRAF, NRAS, and neurofibromin 1. There is rare documentation of a detailed analysis of these mutations in cases of melanoma among Indian patients. We present molecular features in cases of malignant melanoma, diagnosed at a tertiary cancer referral center in India, over a period of 8 years (2011-2018). Materials and Methods This study was performed on formalin fixed paraffin embedded tissues of 88 histologically confirmed cases of malignant melanoma. BRAF gene alterations were studied by both Sanger sequencing and real-time polymerase chain reaction techniques ( n = 74). Molecular testing for BRAF and NRAS gene alterations was accomplished in 74/88 cases (80%). Molecular test results were correlated with clinicopathological features using IBM SPSS Statistical software 25.0. Results The age ranged from 13 to 79 years (median = 57), with a M:F ratio of 1.4:1. BRAF mutations were observed in 12/74 (16.21%) patients, including V600E ( n = 7), A594T ( n = 1), T599 = ( n = 2), V600K ( n = 1), and Q612P ( n = 1), while NRAS mutations were observed in 6/38 (15.7%) patients. Among various subtypes, nodular melanoma was the most frequent subtype (33%) among cutaneous malignant melanomas. Among non-cutaneous melanomas, mucosal melanomas were observed in 37.5% of cases. Conclusion This constitutes one of the few reports on comprehensive analysis of molecular alterations underlying melanomas in Indian patients. A larger sample size, with more extensive molecular markers, would yield additional information on the disease manifestation.