Stiffness-dependent MSC homing and differentiation into CAFs - implications for breast cancer invasion.

Cellular heterogeneity and extracellular matrix (ECM) stiffening have been shown to be drivers of breast cancer invasiveness. Here, we examine how stiffness-dependent crosstalk between cancer cells and mesenchymal stem cells (MSCs) within an evolving tumor microenvironment regulates cancer invasion. By analyzing previously published single-cell RNA sequencing datasets, we establish the existence of a subpopulation of cells in primary tumors, secondary sites and circulatory tumor cell clusters of highly aggressive triple-negative breast cancer (TNBC) that co-express MSC and cancer-associated fibroblast (CAF) markers. By using hydrogels with stiffnesses of 0.5, 2 and 5 kPa to mimic different stages of ECM stiffening, we show that conditioned medium from MDA-MB-231 TNBC cells cultured on 2 kPa gels, which mimic the pre-metastatic stroma, drives efficient MSC chemotaxis and induces stable differentiation of MSC-derived CAFs in a TGFß (TGFB1)- and contractility-dependent manner. In addition to enhancing cancer cell proliferation, MSC-derived CAFs on 2 kPa gels maximally boost local invasion and confer resistance to flow-induced shear stresses. Collectively, our results suggest that homing of MSCs at the pre-metastatic stage and their differentiation into CAFs actively drives breast cancer invasion and metastasis in TNBC.

Microfluidics-based EGFR mutation detection and its implication in the resource-limited clinical setting.

Management of lung cancer today obligates a mutational analysis of the epidermal growth factor receptor (EGFR) gene particularly when Tyrosine Kinase Inhibitor (TKI) therapy is being considered as part of prognostic stratification. This study evaluates the performance of automated microfluidics-based EGFR mutation detection and its significance in clinical diagnostic settings. Formalin-fixed, paraffin-embedded (FFPE) samples from NSCLC patients (n = 174) were included in a two-phase study. Phase I: Validation of the platform by comparing the results with conventional real-time PCR and next-generation sequencing (NGS) platform. Phase II: EGFR mutation detection on microfluidics-based platform as part of routine diagnostics workup. The microfluidics-based platform demonstrates 96.5% and 89.2% concordance with conventional real-time PCR and NGS, respectively. The system efficiently detects mutations across the EGFR gene with 88.23% sensitivity and 100% specificity. Out of 144 samples analysed in phase II, the platform generated valid results in 94% with mutation detected in 41% of samples. This microfluidics-based platform can detect as low as 5% mutant allele fractions from the FFPE samples. Therefore the microfluidics-based platform is a rapid, complete walkaway, with minimum tissue requirement (two sections of 5 µ thickness) and technical skill requirement. The method can detect clinically actionable EGFR mutations efficiently and can be considered a reliable diagnostic platform in resource-limited settings. From receiving samples to reporting the results this platform provides accurate data without much manual intervention. The study helped to devise an algorithm that emphasizes effective screening of the NSCLC cases for EGFR mutations with varying tumour content. Thus it helps in triaging the cases judiciously before proceeding with multigene testing.

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.

Clinicopathological features of five cases of CIC::DUX4 positive sarcomas, including literature review.

According to the recent World Health Organization (WHO) classification, CIC-rearranged sarcomas, including CIC::DUX4-positive sarcomas constitute an aggressive subtype of undifferentiated round cell sarcomas. There is a single study on these tumors from our subcontinent. We present clinicopathological features of 5 additional cases of this tumor entity, including literature review. Thirty-nine undifferentiated round cell sarcomas, excluding Ewing sarcomas (ES), were tested for CIC::DUX4 fusion, including Type I (165 base pair size) and II (230 bp) by reverse transcription-polymerase chain reaction. Twenty-five of those tumors were tested for EWSR1 gene rearrangement, 5 for SS18 and 4 for SS18::SSX fusion, and were negative for those tests. Five tumors (12.8 %) were positive for CIC::DUX4(Type II) fusion. Five CIC:: DUX4-positive sarcomas occurred in 4 males and one female; of 25-43 years of age, in soft tissues, including thigh (n = 2), chest wall (n = 1), iliac region (n = 1) and foot (n = 1). Tumor size varied from 2.2 to 19 cm. Microscopically, the tumors were predominantly composed of nodules and sheets of malignant round to epithelioid cells, including "rhabdoid-like" (n = 2) and spindle-shaped (n = 2) with eosinophilic to vacuolated cytoplasm (4/5), distinct nucleoli (4/5), brisk mitoses, focal myxoid to hyalinised stroma (4/5) and necrosis (5/5). Immunohistochemically, tumor cells were positive for WT1 (5/5), calretinin (3/4), pan-keratin (1/4), CD99/MIC2 ("dot-like" to cytoplasmic membranous) (4/4), while negative for desmin (0/4), S100P (0/4), and NKX2.2 (0/5). INI1/SMARCB1 was retained (3/3). All patients underwent excision with adjuvant radiotherapy and chemotherapy (Ewing sarcoma regimen). A single patient developed recurrence, and 2 developed pulmonary metastasis, including one with brain metastasis. CIC:: DUX4-positive sarcomas are ultra-rare tumors, that mainly occur in the soft tissues and in young adult patients. Histopathologically, these tumors display a wide spectrum, including round to epithelioid cells, variable amount of cytoplasmic vacuolization and myxoid stroma with necrosis. Immunohistochemically, these tumors express WT1 and calretinin. Despite adjuvant therapies, these tumors have dismal outcomes, especially in large-sized tumors. CIC::DUX4-positive sarcomas need to be differentiated from their histopathological mimics, including ES, in view of significant treatment-related implications.

Incidence of SARS-CoV-2 infection among asymptomatic patients undergoing preoperative COVID testing prior to cancer surgery: ASPECT study.

BACKGROUND AND OBJECTIVES: The COVID-19 pandemic, with high rate of asymptomatic infections and increased perioperative complications, prompted widespread adoption of screening methods. We analyzed the incidence of asymptomatic infection and perioperative outcomes in patients undergoing cancer surgery. We also studied the impact on subsequent cancer treatment in those with COVID-19. METHODS: All patients who underwent elective and emergency cancer surgery from April to September 2020 were included. After screening for symptoms, a preoperative test was performed from nasopharyngeal and oropharyngeal swabs before the procedure. Patients were followed up for 30 days postoperatively and complications were noted. RESULTS: 2108 asymptomatic patients were tested, of which 200 (9.5%) tested positive. Of those who tested positive, 140 (70%) underwent the planned surgery at a median of 30 days from testing positive, and 20 (14.3%) had ≥ Grade III complications. Forty (20%) patients did not receive the intended treatment; 110 patients were retested in the Postoperative period, and 41 (37.3%) tested positive and 9(22%) patients died of COVID-related complications. CONCLUSION: Routine preoperative testing for COVID-19 helps to segregate patients with asymptomatic infection. Higher complications occur in those who develop COVID-19 in postoperative period. Prolonged delay in surgery after COVID infection may influence planned treatment.

A unique case of adamantinoma-like Ewing sarcoma in the calcaneus, exhibiting prominent squamous differentiation and displaying EWSR1 gene rearrangement.

Adamantinoma-like Ewing sarcoma is uncommonly reported in the skeletal sites, including small bones of the feet.A 15-year-old girl presented with pain and swelling in her left foot, leading to difficulty in walking for 8 months. Plain radiograph revealed an ill-defined, lytic-sclerotic lesion without significant periosteal reaction in her left calcaneus. Magnetic resonance imaging (MRI) revealed an expansile lesion involving the anterior calcaneus, which was hypointense on T1 and heterogeneously hyperintense on T2-weighted sequences, infiltrating the adjacent bones and soft tissues. On imaging, the differential diagnoses considered were a giant cell tumor and other primary bone tumors.Histopathological examination revealed a tumor composed of small round cells, with interspersed keratin pearls. Immunohistochemically, the tumor cells were positive for CD99/MIC2, pan-cytokeratin (AE1/AE3), p40, p63, NKX2.2, and synaptophysin. Diagnosis of adamantinoma-like Ewing sarcoma was offered on the initial biopsy. Furthermore, the tumor cells revealed EWSR1 gene rearrangement by fluorescence in situ hybridization, confirming this diagnosis. The patient underwent neoadjuvant chemotherapy, had a poor response, and finally underwent below-knee amputation.This constitutes a rare case of adamantinoma-like Ewing sarcoma in the calcaneus. Ewing sarcoma may be considered as a differential diagnosis for intraosseous lytic-sclerotic lesions, even without significant periosteal reaction, at unusual sites, such as the bones of the foot. Awareness of this entity and application of ancillary techniques is recommended for its exact diagnosis and in differentiating this rare variant from its diagnostic mimics. This case also indicates a poor chemotherapy response in this unusual variant of Ewing sarcoma, occurring in the calcaneus.

Primary intracranial Ewing sarcoma/ peripheral primitive neuroectodermal tumor, an entity of unacquaintance: a series of 8 cases.

PURPOSE: The purpose is to highlight the primary intracranial (meningeal-based) occurrence of Ewing sarcoma/primitive neuroectodermal tumor (ES/PNET). METHODS: This report is a collation of clinicopathological features of eight cases of molecularly and clinicoradiologically confirmed primary (non-metastatic) intracranial (non-osseous) meningeal ES/PNET. RESULTS: The age range was 1 to 33 years with a median age of 9 years. Male to female ratio was 0.6:1. All patients were diagnosed on the debulking surgical material (gross total resection, 2 cases; subtotal resection, 6 cases) and showed primitive embryonal histomorphology with diffuse membranous CD99 immunoexpression and EWSR1 gene rearrangement by fluorescence in situ hybridization. Seven of them showed a typical FISH pattern of split signals with break-apart probe, while one showed an unusual signal pattern of loss of green signals. EFT-2001 adjuvant protocol was followed along with focal radiotherapy (RT) in all cases (except case 8, full course of chemotherapy could not be completed). Two cases had local recurrence-one of them died of disease recurrence before the administration of further treatment. CONCLUSION: This series adds non-osseous intracranial site to the list of uncommon sites of occurrence for ES/PNET and more importantly emphasizes the need to be considered in a differential list of primary intracranial primitive embryonal tumors before embarking as primary central nervous system (CNS) embryonal tumor, NOS.

Clinicopathologic, immunohistochemical, molecular cytogenetic profile with treatment and outcomes of 34 cases of Ewing sarcoma with epithelial differentiation, including 6 cases with "Adamantinoma-like" features, diagnosed at a single institution, India.

To analyze clinicopathological features, including treatment profile of 34 cases of Ewing sarcomas with epithelial differentiation, including 6 cases with adamantinoma-like features. EWSR1 gene rearrangement was tested by fluorescence in-situ hybridization. Thirty-four tumors occurred in 19 males and 15 females (M:F = 1.26:1), with age ranging from 7 to 61 years (average = 24.2); in extremities (17), pelvis (5), paraspinal region (6), head and neck region (3), abdomen (2) and lung (1). Prior to molecular testing, 20/34(58.8%) cases were unequivocally diagnosed as Ewing sarcomas. Histopathologically, the most commonly observed pattern was nesting-type, comprising malignant round cells, including adamantinoma-like features, seen in 6 tumors. Immunohistochemically, tumor cells were diffusely positive (cytoplasmic membranous staining) for CD99/MIC2 (34/34), Fli1 (30/30); focally for synaptophysin (4/16) (25%); variably positive for AE1/AE3(31/32)(96.8%), including diffuse immunoexpression in 4 cases; EMA(6/8) and p40(3/8). All 34 (100%) tumors, tested for EWSR1 rearrangement, displayed positive results. According to the treatment details (available in 24/34 cases, 70.5%), most patients (13/24)(54.1%) were treated with surgical resection and a specific chemotherapy(CT) regimen (neoadjuvant or adjuvant settings), including 7 patients, who received adjuvant radiotherapy. During follow-up (16 cases, 47%), 5 patients developed recurrences and 8 developed metastasis, including a single, who developed recurrence. Finally, 10 patients were alive-with-disease (2-22 months); 6 free-of-disease (5-36 months). This constitutes one of the largest documentation of these rare tumors from our subcontinent, which are diagnostically challenging; require molecular confirmation and associated with treatment implications.

A Rare Case of a Vaginal Solitary Fibrous Tumor, Presenting as a Cystic Mass, Showing NAB2ex4-STAT6ex2 Fusion and STAT6 Immunostaining.

Solitary fibrous tumor (SFT) has been rarely documented in the female genital tract. Until now, only 5 such cases have been reported in the vagina. A 40-yr-old woman was referred to us with a history of antitubercular treatment and intra-abdominal adhesions; for which she previously underwent laparoscopic lysis of adhesions. Subsequently, she developed a vaginal mass, which appeared as a complex cyst on ultrasonogram. Histopathologic examination of the excised mass revealed a cellular spindle cell tumor with collagenous deposition in the stroma. The tumor cells were composed of oval to spindle shaped nuclei and were arranged in a diffuse, as well as in a focally, hemangiopercytomatous pattern. There were no significant mitotic figures or tumor necrosis. By immunohistochemistry, the tumor cells showed patchy positivity for CD34 and diffuse intranuclear positivity for STAT6, along with cytoplasmic positivity for MIC2 and BCL2. Few cells were estrogen receptor positive. MIB1/Ki67 highlighted 2% to 3% tumor nuclei (low). Diagnosis of a SFT was rendered. Subsequently, the tumor was subjected to molecular analysis, by reverse transcriptase-polymerase chain reaction and sequencing, which revealed presence of NAB2ex4-STAT6ex2 fusion transcript. This case constitutes the first case of a vaginal SFT confirmed by STAT6 immunostaining and NAB2-STAT6 fusion. It exemplifies the role of specific immunohistochemical markers, such as STAT6 in differentiating a SFT from various other spindle cell tumors occurring in this location. Literature review of similar reported cases and treatment implications in such cases are discussed herein.

Clinicopathologic features of undifferentiated round cell sarcomas of bone & soft tissues: An attempt to unravel the BCOR-CCNB3- & CIC-DUX4-positive sarcomas.

Background & objectives: Certain genetically defined undifferentiated round cell sarcomas, namely BCOR-CCNB3 and CIC-DUX4 positive, have been described. Here we present detailed clinicopathologic features and molecular results in such cases. Methods: Fifty one cases of undifferentiated round cell sarcomas, including 32 cases, tested for BCOR-CCNB3 and CIC-DUX4 fusions, by reverse transcription polymerase chain reaction technique and 44 tumours, for CCNB3 immunostaining, were analyzed. Results: Twenty seven (52.9%) tumours occurred in males and 24 (47%) in females; in soft tissues (38; 74.5%), commonly, trunk and extremities and bones (13; 25.4%), frequently, femur and tibia. Five of 32 (15.6%) tested cases were positive for BCOR-CCNB3 fusion and seven (21.8%) for CIC-DUX4 fusions. Histopathologically, CIC-DUX4-positive sarcomas comprised nodular aggregates of round to polygonal cells, containing hyperchromatic nuclei, prominent nucleoli and moderate cytoplasm, with focal myxoid stroma and variable necrosis, in certain cases. BCOR-CCNB3- positive sarcomas mostly comprised diffusely arranged, round to oval to short spindly cells with angulated nuclei, vesicular chromatin, inconspicuous nucleoli and interspersed vessels. Immunohistochemically, tumour cells were positive for MIC2 in 24 of 49 (48.9%) and CCNB3 in 12 of 44 (27.2%) cases. Four of five BCOR-CCNB3-positive sarcomas showed CCNB3 immunostaining and 6 of 7 CIC-DUX4-positive sarcomas displayed WT1 immunostaining. Most patients (27/37) (72.9%) underwent surgical resection and chemotherapy. Median overall survival was 12 months, and disease-free survival was seven months. Interpretation & conclusions: Undifferentiated round cell sarcomas are rare; mostly occur in soft tissues of extremities, with CIC-DUX4 positive, as these are relatively more frequent than BCOR-CCNB3 positive sarcomas. CCNB3 and WT1 are useful immunostains for triaging such cases for BCOR-CCNB3 and CIC-DUX4 fusion testing, respectively. Overall, these are relatively aggressive tumours, especially CIC-DUX4-positive sarcomas.

Pilocytic astrocytomas: BRAFV600E and BRAF fusion expression patterns in pediatric and adult age groups.

PURPOSE: Pilocytic astrocytomas (PCAs) are characterized by two dominant molecular alterations of the BRAF gene, i.e., BRAFV600E mutation and KIAA1549-BRAF fusions which show a differential pattern of frequency across different age-groups. METHODS: Formalin-fixed paraffin-embedded tissues of 358 (pediatric 276 and adult 82) consecutive PCAs were evaluated for BRAFV600E mutation by Sanger sequencing and KIAA1549:BRAF fusion transcripts (KIAA1549:BRAF 16-9, KIAA1549:BRAF 15-9, and KIAA1549:BRAF 16-11) by reverse transcriptase polymerase chain reaction, which were correlated with different clinicopathological features. RESULTS: BRAFV600E mutation was detected in 8.9% pediatric and 9.75% adult PCAs, whereas 41.1% and 25.7% of pediatric and adult cases showed KIAA1549-BRAF fusions respectively. BRAFV600E did not show any statistically significant correlation with any of the clinical parameters (age, location, and gender). KIAA1549:BRAF fusions showed a significant statistical association with the pediatric age group and cerebellar location. KIAA1549-BRAF 16-9 was the commonest variant and was predominantly associated with cerebellar location than non-cerebellar whereas fusion variant 15-9 negatively correlated with cerebellar locations. CONCLUSIONS: The present study showed overall frequency of 53.5% and 37.3% BRAF alterations in pediatric and adult PCA cases respectively. BRAF fusion in PCA cases showed a different distribution pattern across age groups and locations; while no such differential pattern was observed for BRAFV600E.

Five rare cases of Ewing sarcoma, including with epithelial differentiation, involving the female genital tract, displaying EWSR1 rearrangement: Diagnostic challenge and treatment implications.

We present clinicopathological and molecular cytogenetic features of five rare cases of Ewing sarcomas, occurring in the female genital tract. A 40 year-old lady presented with a 5.4 cm-sized vaginal mass of 3 months duration, which was histopathologically diagnosed as ES. She defaulted chemotherapy and 8 months later, presented with a recurrence. She underwent chemotherapy and radiotherapy. A 45 year-old lady presented with recurrent vaginal bleeding, for which she underwent total abdominal hysterectomy (TAH) and unilateral salpingo-oophorectomy (USO), 2 and 1/2 years back. Subsequent vaginal biopsy was reported inconclusively, elsewhere. Thereafter, a 5 cm-sized, residual cervicovaginal mass was reported as ES. She completed induction chemotherapy with a significant response. A 35 year-old-lady was referred with a 4 cm-sized cervical mass, for which she underwent TAH-USO with pelvic and para-aortic lymphadenectomy. A 39 year-old-lady presented with a right labial lesion, which recurred. She underwent initial excision, chemotherapy, wide excision and brachytherapy. A year later, she developed multiple metastases; received palliative radiotherapy and died-of-disease. A 16 year-old girl presented with perineal swelling of 4 months duration. She underwent surgical excision of a recurrent right-sided labial cyst, followed by chemotherapy. On histopathological review, all 5 cases were malignant round cell tumors. Immunohistochemically, tumor cells displayed MIC2/CD99 and Fli1 positivity, along with focal positivity for pan cytokeratin (AE1/AE3) (cases 1 and 2) and p63 (case 2). Furthermore, tumor cells in the 1st, 2nd, 3rd and 5th cases displayed EWSR1 rearrangement. Five uncommon cases of ES involving the female genital tract are presented with diagnostic challenges and therapeutic implications.

Correction to: HDAC6 deacetylates alpha tubulin in sperm and modulates sperm motility in Holtzman rat.

The published online version contains mistake. The chimeric peptide should read as 'DPSVLYVSLHRYGGYMNEGELRV'. It was inadvertently written as 'DPSVLYVSLYVSLHRYGGYMNEGELR' a mistake which we missed during proof reading.

Outcomes and complications of surgery in patients with intermediate-risk neuroblastoma: experience from an Indian tertiary Cancer Centre.

PURPOSE: The treatment of intermediate risk (IR) neuroblastoma has evolved with the focus now on reducing the drugs, dosage, and duration of chemotherapy. The aim of this study is to present the outcomes of treatment and the complications of surgery in patients with IR neuroblastoma treated at a tertiary cancer center in India. METHODS: All eligible patients with IR neuroblastoma treated between April 2005 and August 2016 were identified. The presence and number of image-defined risk factors (IDRF) before and after neoadjuvant chemotherapy were retrospectively analyzed as were the extent of surgery, complications, and outcomes. RESULTS: Of 282 neuroblastoma patients treated during the study period, 54 had IR neuroblastoma. Complete excision was achieved in 25 patients. There were 26 surgical complications in 22 patients with a similar incidence in patients with complete (n = 13) or incomplete (n = 13) resection (p = 0.78). After a median follow-up of 47 months, the 4-year overall and event-free survival was 91.5% and 75%, respectively. There was no difference in survival between patients who underwent complete resection versus those with incomplete resection (p = 0.9). CONCLUSION: Outcomes of IR neuroblastoma are favorable. The extent of resection does not affect the survival and complications can occur even when the resection is incomplete.

MGMT gene promoter methylation and its correlation with clinicopathological parameters in glioblastomas.

BACKGROUND: MGMT (O6-methyl guanine DNA methyl transferase) promoter hypermethylation is a prognostic and predictive biomarker for glioblastomas (GBM). AIMS: To evaluate the frequency of MGMT methylation status in a single institute series of 134 GBMs and correlate it with clinical (age, sex, location, survival) and other molecular parameters [such as p53 expression, alpha thalassemia/mental retardation syndrome X-linked (ATRX) expression, isocitrate dehydrogenase (IDH) 1R132H mutation, and epidermal growth factor receptor (EGFR) gene amplification]. RESULTS: One hundred and thirty-four GBMs were evaluated by methylation-specific polymerase chain reaction (MSP) for MGMT promoter methylation status. The results were correlated with the above mentioned clinicopathological parameters. MGMT gene promoter methylation was identified in 49.2% (66/134) GBMs, and was significantly associated with IDH1R132H mutation (14/66; 21%; P - value, 0.01) and ATRX loss (15/66; 23%; P - value, 0.01). Confluent necrosis was found to be significantly associated with MGMT unmethylation status (P - value: 0.002). Multivariable logistic regression analysis showed confluent necrosis as a single independent predictor (odds ratio [OR], 2.5; confidence interval [CI], 1.0-5.8; P - value, 0.04) of MGMT unmethylation status among all the parameters studied. CONCLUSIONS: The frequency of MGMT promoter methylation in GBMs was 49.2%, which was significantly associated with IDHR132H mutation and ATRX loss. In addition, the presence of confluent necrosis was significantly associated with MGMT unmethylation and was found to be an independent predictor of the same.

Diffuse glioma - Rare homozygous IDH point mutation, is it an oncogenetic mechanism?

Isocitrate dehydrogenase (IDH1/IDH2) mutations in gliomas of WHO grade II/III and secondary glioblastoma are almost always heterozygous missense mutations. Here, we report an extremely rare case of homozygous IDH1R132H mutation in a recurrent WHO grade III anaplastic astrocytoma. The authors here also review the relevant literature for the possible metabolic impact of homozygous IDH1/2 mutations in the gliomagenesis.