Overexpression of laminin-5 gamma-2 promotes tumorigenesis of pancreatic ductal adenocarcinoma through EGFR/ERK1/2/AKT/mTOR cascade.

Pancreatic ductal adenocarcinoma (PDAC) is correlated with poor outcomes because of limited therapeutic options. Laminin-5 gamma-2 (LAMC2) plays a critical role in key biological processes. However, the detailed molecular mechanism and potential roles of LAMC2 in PDAC stay unexplored. The present study examines the essential role and molecular mechanisms of LAMC2 in the tumorigenesis of PDAC. Here, we identified that LAMC2 is significantly upregulated in microarray cohorts and TCGA RNA sequencing data of PDAC patients compared to non-cancerous/normal tissues. Patients with higher transcript levels of LAMC2 were correlated with clinical stages; dismal overall, as well as, disease-free survival. Additionally, we confirmed significant upregulation of LAMC2 in a panel of PDAC cell lines and PDAC tumor specimens in contrast to normal pancreatic tissues and cells. Inhibition of LAMC2 significantly decreased cell growth, clonogenic ability, migration and invasion of PDAC cells, and tumor growth in the PDAC xenograft model. Mechanistically, silencing of LAMC2 suppressed expression of ZEB1, SNAIL, N-cadherin (CDH2), vimentin (VIM), and induced E-cadherin (CDH1) expression leading to a reversal of mesenchymal to an epithelial phenotype. Interestingly, co-immunoprecipitation experiments demonstrated LAMC2 interaction with epidermal growth factor receptor (EGFR). Further, stable knockdown of LAMC2 inhibited phosphorylation of EGFR, ERK1/2, AKT, mTOR, and P70S6 kinase signaling cascade in PDAC cells. Altogether, our findings suggest that silencing of LAMC2 inhibited PDAC tumorigenesis and metastasis through repression of epithelial-mesenchymal transition and modulation of EGFR/ERK1/2/AKT/mTOR axis and could be a potential diagnostic, prognostic, and therapeutic target for PDAC.

ALDH1A1+ ovarian cancer stem cells co-expressing surface markers CD24, EPHA1 and CD9 form tumours in vivo.

One of the reasons for recurrence following treatment of high grade serous ovarian carcinoma (HGSOC) is the persistence of residual cancer stem cells (CSCs). There has been variability between laboratories in the identification of CSC markers for HGSOC. We have identified new surface markers (CD24, CD9 and EPHA1) in addition to those previously known (CD44, CD117 and CD133) using a bioinformatics approach. The expression of these surface markers was evaluated in ovarian cancer cell lines, primary malignant cells (PMCs), normal ovary and HGSOC. There was no preferential expression of any of the markers or a combination. All the markers were expressed at variable levels in ovarian cancer cell lines and PMCs. Only CD117 and CD9 were expressed in the normal ovarian surface epithelium and fallopian tube. Both ALDEFLUOR (ALDH1A1) and side population assays identified a small proportion of cells (<3%) separately that did not overlap with little variability in cell lines and PMCs. All surface markers were co-expressed in ALDH1A1+ cells without preference for one combination. The cell cycle analysis of ALDH1A1+ cells alone revealed that majority of them reside in G0/G1 phase of cell cycle. Further separation of G0 and G1 phases showed that ALDH1A1+ cells reside in G1 phase of the cell cycle. Xenograft assays showed that the combinations of ALDH1A1 + cells co-expressing CD9, CD24 or EPHA1 were more tumorigenic and aggressive with respect to ALDH1A1-cells. These data suggest that a combined approach could be more useful in identifying CSCs in HGSOC.

Intravenous fosaprepitant for the prevention of chemotherapy-induced vomiting in children: A double-blind, placebo-controlled, phase III randomized trial.

BACKGROUND: Fosaprepitant is a neurokinin-1 receptor antagonist, approved for the prevention of chemotherapy-induced nausea and vomiting. The data on the use of fosaprepitant in children are limited and therefore we conducted a phase III randomized controlled trial. PROCEDURE: Children aged 1-12 years scheduled to receive moderately or highly emetogenic chemotherapy were randomly assigned to arm-A (fosaprepitant) or arm-B (placebo). Children recruited to arm-A received intravenous ondansetron plus dexamethasone followed by fosaprepitant infusion. Children recruited to arm-B received the same drugs as those given to children in arm-A, except that fosaprepitant was substituted with a placebo. Ondansetron and dexamethasone were continued for 48 hours after completion of chemotherapy. The primary end point of the study was to determine the proportion of patients who achieved a complete response (CR), defined as no vomiting, no retching, and no use of rescue medication, during the 24-120 hours (delayed phase) after administration of the last dose of chemotherapy. Secondary end points were the proportion of patients who achieved a CR during the acute phase (0-24 hours) and overall after administration of the last dose of chemotherapy. RESULTS: One-hundred-sixty-three patients were analyzed (81 in the fosaprepitant arm and 82 in the placebo arm). CR rates were significantly higher in the fosaprepitant arm compared to those in the placebo arm during the acute phase (86% vs 60%, P < 0.001), delayed phase (79% vs 51%, P < 0.001), and overall phase (70% vs 41%, P < 0.001). Three (4%) patients in the fosaprepitant arm and sixteen (20%) in the placebo arm required rescue anti-emetics (P = 0.0017). CONCLUSION: Addition of fosaprepitant to ondansetron and dexamethasone improved chemotherapy-induced vomiting control in children treated with moderately or highly emetogenic chemotherapy.

Long non-coding RNAs: Fundamental regulators and emerging targets of cancer stem cells.

Cancer is one of the leading causes of death worldwide, primarily due to the dearth of efficient therapies that result in long-lasting remission. This is especially true in cases of metastatic cancer where drug resistance causes the disease to recur after treatment. One of the factors contributing to drug resistance, metastasis, and aggressiveness of the cancer is cancer stem cells (CSCs) or tumor-initiating cells. As a result, CSCs have emerged as a potential target for drug development. In the present review, we have examined and highlighted the lncRNAs with their regulatory functions specific to CSCs. Moreover, we have discussed the difficulties and various methods involved in identifying lncRNAs that can play a particular role in regulating and maintaining CSCs. Interestingly, this review only focuses on those lncRNAs with strong functional evidence for CSC specificity and the mechanistic role that allows them to be CSC regulators and be the focus of CSC-specific drug development.

Outcomes with chemotherapy in carcinoma penis: Experience from a tertiary cancer center.

Background: Carcinoma penis is more common in India compared to the West. The role of chemotherapy in carcinoma penis is ambiguous. We analyzed the profile and outcomes of patients with carcinoma penis treated with chemotherapy. Methods: We analyzed the details of all patients with carcinoma penis treated at our institute between 2012 and 2015. We collected particulars regarding demography, clinical presentation, treatment details, toxicities, and outcomes of these patients. Event-free and overall (OS) survival were calculated from the time of diagnosis until documentation of disease relapse/progression or death for the patients with advanced carcinoma penis who were eligible for chemotherapy. Results: There were 171 patients with carcinoma penis treated at our institute during the study period including 54 (31.6%) patients with stage I, 49 (28.7%) patients with stage II, 24 (14.0%) patients with stage III, 25 (14.6%) patients with stage IV, and 19 (11.1%) patients with recurrent disease at presentation. The present study included 68 patients with advanced carcinoma penis (stages III and IV) who were eligible for chemotherapy, with a median age of 55 years (range: 27-79 years). Sixteen patients received paclitaxel and carboplatin (PC) and 26 patients cisplatin and 5-FluoroUracil (CF). Neoadjuvant chemotherapy (NACT) was given to four patients with stage III and nine patients with stage IV disease. Of the 13 patients given NACT, we observed a partial response in five (38.5%), stable disease in two (15.4%), and progressive disease in five (38.5%) evaluable patients. Six (46%) patients underwent surgery after NACT. Only 28/54 (52%) patients received adjuvant chemotherapy. After a median follow-up of 17.2 months, the 2-year OS rates were 95.8, 89, 62.7, 51.9, and 28.6% for stages I, II, III, IV, and recurrent disease, respectively. The 2-year OS of patients who were given chemotherapy versus those who were not given chemotherapy were 52.7 and 63.2%, respectively (P = 0.762). Conclusions: We report the real-world outcomes of two chemotherapeutic regimens used in consecutive patients with advanced carcinoma penis. Both PC and CF seemed effective and safe. However, approximately half of patients with advanced carcinoma penis do not receive the planned/indicated chemotherapy. We need further prospective trials regarding the sequencing, protocols and indications of chemotherapy in this malignancy.

Neutropenic versus regular diet for acute leukaemia induction chemotherapy: randomised controlled trial.

OBJECTIVES: Restriction of raw fruits and vegetables (neutropenic diet) is advised for patients receiving treatment for acute leukaemia in low-income and middle-income countries (LMICs) to reduce infections despite evidence to the contrary from high-income countries. We, therefore, conducted a randomised controlled trial to ascertain the efficacy of the neutropenic diet in an LMIC setting. METHODS: Patients aged 1-60 years receiving induction chemotherapy for acute leukaemia were randomised to a regular or neutropenic diet. The study's primary objective was to compare the incidence of major infections among patients receiving the two diets during induction chemotherapy. The secondary objectives were to compare stool microbial flora and induction mortality rates. RESULTS: We randomised 200 patients, 98 patients to the regular diet arm and 102 to the neutropenic diet arm. Major infections occurred in 32 (32%) patients in the regular diet arm and 26 (25%) patients in the neutropenic diet arm (p=0.26). There were no statistically significant differences between patients receiving a regular diet versus neutropenic diet for blood culture positivity (n=6 vs 9), inotropic support (17 vs 12), mechanical ventilation (8 vs 5), third-line antibiotic use (28 vs 20), minor infections (12 vs 9), induction mortality (9 vs 4) and remission status (94% vs 94%). The stool culture on day 15 of induction grew multidrug-resistant bacteria in 38% of patients in the regular diet arm and 35% in the neutropenic diet arm (p=0.67). CONCLUSIONS: A neutropenic diet did not prevent infections, reduce mortality or change stool microbial flora in patients with acute leukaemia.

Two-drug versus three-drug induction chemotherapy in pediatric acute myeloid leukemia: a randomized controlled trial.

The benefit of three-drug induction chemotherapy over a two-drug induction has not been evaluated in pediatric acute myeloid leukemia (AML). We, therefore, conducted a randomized controlled trial to ascertain the benefit of a three-drug induction regimen. Patients aged 1-18 years with newly diagnosed AML were randomized to two cycles of induction chemotherapy with daunorubicin and ara-C (DA) or two cycles of ara-C, daunorubicin, and etoposide (ADE). After induction, patients in both arms received consolidation with two cycles of high-dose ara-C. The study's primary objective was to compare the event-free survival (EFS) between the two arms. The secondary objectives included comparing the composite complete remission (cCR) rates, overall survival (OS), and toxicities. The study randomized 149 patients, 77 in the DA and 72 in the ADE arm. The median age was 8.7 years, and 92 (62%) patients were males. The median follow-up was 50.9 months. The cCR rate in the DA and ADE arm were 82% and 79% (p = 0.68) after the second induction. There were 13 (17%) induction deaths in the DA arm and 12 (17%) in the ADE arm (p = 0.97). The 5-year EFS in the DA and ADE arm was 34.4% and 34.5%, respectively (p = 0.66). The 5-year OS in the DA and ADE arms was 41.4% and 42.09%, respectively (p = 0.74). There were no significant differences in toxicities between the regimens. There was no statistically significant difference in EFS, OS, CR, or toxicity between ADE and DA regimens in pediatric AML. The trial was registered with the Clinical Trial Registry of India (Reference number: CTRI/2014/11/005202).

Therapeutic Outcomes in High-Grade B-Cell Lymphoma, NOS: Retrospective Analysis.

The nomenclature high-grade non-Hodgkin's lymphoma was repurposed in the World Health Organization (WHO) 2016 update as high-grade B cell lymphoma (HGBL). However, among the HGBL entities HGBL, not otherwise specified (NOS) remains a poorly described entity with a lack of literature regarding its treatment and prognosis. The baseline characteristics, treatment, and outcome of HGBL, NOS cases were analyzed. Thirty HGBL, NOS patients were diagnosed between January 2017 and December 2019. Their median age was 49.3 years, and 30% had advanced IPI. The majority received R-CHOP chemotherapy, while five patients received dose-adjusted R-EPOCH. At a median follow-up of 15 months, nine patients had disease progression or relapse. EFS and OS were 22 months (12.1-31.9 months) and 37 months (29.4-44.0 months) respectively. Only NCCN-IPI ≤ 2 showed significant influence on the outcome. The results were similar to the outcomes previously reported. This study highlights the importance of NCCN-IPI in ascertaining the prognosis of HGBL, NOS. The literature review suggests that more intensive chemotherapy is ideal for HGBL, NOS. However, prospective trials are needed to prove whether the treatment of HGBL, NOS can be tailored based on NCCN-IPI.

Locally Advanced Breast Cancer (LABC): Real-World Outcome of Patients From Cancer Institute, Chennai.

PURPOSE: There are sparse data on the outcome of patients with locally advanced breast cancer (LABC). This report is on the prognostic factors and long-term outcome from Cancer Institute, Chennai. METHODS: This is an analysis of untreated patients with LABC (stages IIIA-C) who were treated from January 2006 to December 2013. RESULTS: Of the 4,577 patients with breast cancer who were treated, 2,137 patients (47%) with LABC were included for analysis. The median follow-up was 75 months (range, 1-170 months), and 2.3% (n = 49) were lost to follow-up at 5 years. The initial treatment was neoadjuvant concurrent chemoradiation (NACR) (77%), neoadjuvant chemotherapy (15%), or others (8%). Patients with triple-negative breast cancer had a pathologic complete response (PCR) of 41%. The 10-year overall survival was for stage IIIA (65.1%), stage IIIB (41.2%), and stage IIIC (26.7%). Recurrence of cancer was observed in 27% of patients (local 13% and distant 87%). Multivariate analysis showed that patients with a tumor size > 10 cm (hazard ratio [HR], 2.19; 95% CI, 1.62 to 2.98; P = .001), hormone receptor negativity (HR, 1.45; 95% CI, 1.22 to 1.72; P = .001), treatment modality (neoadjuvant chemotherapy, HR, 0.56; 95% CI, 0.43 to 0.73; P = .001), lack of PCR (HR, 2.36; 95% CI, 1.85 to 3.02; P = .001), and the presence of lymphovascular invasion (HR, 1.97; 95% CI, 1.60 to 2.44; P = .001) had decreased overall survival. CONCLUSION: NACR was feasible in inoperable LABC and gave satisfactory long-term survival. PCR was significantly higher in patients with triple-negative breast cancer. The tumor size > 10 cm was significantly associated with inferior survival. However, this report acknowledges the limitations inherent in experience of management of LABC from a single center.

Phase 2 non-randomised trial of secondary cytoreduction and hyperthermic intraperitoneal chemotherapy in recurrent platinum-sensitive ovarian cancer.

BACKGROUND: The role of secondary cytoreduction with hyperthermic intraperitoneal chemotherapy (HIPEC) is not clearly defined in recurrent platinum-sensitive ovarian cancer (PSOC). There is a paucity of studies on secondary cytoreduction with HIPEC in PSOC from developing countries like India. This study was done to assess the feasibility and safety of secondary cytoreduction and HIPEC in recurrent PSOC. METHODS: This was a prospective, non-randomised, open-label, phase 2 trial of secondary cytoreduction and HIPEC (Cisplatin 75 mg/m2 43°C over 60 minutes) in patients with recurrent platinum-sensitive epithelial carcinoma of ovary/fallopian tube/peritoneum done in a tertiary cancer centre from February 2016 to August 2019. The primary outcome was to assess the overall survival (OS) and the secondary outcomes were to assess the progression-free survival (PFS) and toxicity. RESULTS: Twenty-seven patients were screened and among them, 15 patients were included in this analysis with a median follow-up of 25 months. The mean cancer antigen (CA) 125 at the time of recurrence was 149 U/mL (range: 10-2,030 U/mL) and the median platinum-free interval was 21 months. The perioperative chemotherapy used was paclitaxel + carboplatin 53.3% (8/15), liposomal doxorubicin + carboplatin 40% (6/15) and none 6.5% (1/15). The median Peritoneal Carcinomatosis Index score was 8 (range: 3-25). The Clavien Dindo score was I, II and III in 6.7%, 26.7% and 13.3% patients, respectively. Recurrence was radiological and biochemical in 60% (9/15) and 7% (1/15), respectively. The most common site of recurrence was intra-abdominal (peritoneal). The median PFS and OS were 15 months (range: 0-34) and 26 months (range: 23-29), respectively. The grade 3 or 4 toxicity was 40%. CONCLUSION: Secondary cytoreduction with HIPEC is feasible and safe in recurrent PSOC. Conclusive evidence that secondary cytoreduction with HIPEC is essential awaits the results from ongoing randomised controlled trials.

A simple and efficient method for processing of cell lysates for two-dimensional gel electrophoresis.

Sample preparation is one of the major issues in 2-DE for the separation of proteins. Although a 100% representation of cellular proteins onto a 2-DE is virtually impossible, maximum representation of cellular proteins compared with the original cell lysate is important in the subsequent analysis. We demonstrate that lysis of cells in urea/thiourea solution with subsequent sonication to disrupt the nucleic acids and concentration of the lysate using centri-con led to enrichment of proteins. The procedure resulted in minimal nucleic acid contamination with better resolution of spots. 2-DE spot patterns of proteins prepared using urea-thiourea solubilization/centri-con method to other protein enrichment methods such as phenol/chloroform/isoamyl alcohol extraction, methanol/ammonium acetate precipitation, acetone precipitation and ethanol precipitation were compared. Urea-thiourea solubilization combined with centri-con method of protein enrichment represented higher number/unique spots particularly in the 50-250 kDa M(r) compared with others. Lysis of cells in urea/thiourea from the beginning of lysate preparation preserves the proteins from protease activity due to denaturation of proteases. Thus, we demonstrate that the centri-con methodology is simple and effective for the preparation of high-quality sample that can be used for a qualitative representation of cellular proteins on a 2-DE for proteomic analysis.

Oncogenes in high grade serous adenocarcinoma of the ovary.

High grade serous ovarian cancer is characterized by relatively few mutations occurring at low frequency, except in TP53. However other genetic aberrations such as copy number variation alter numerous oncogenes and tumor suppressor genes. Oncogenes are positive regulators of tumorigenesis and play a critical role in cancer cell growth, proliferation, and survival. Accumulating evidence suggests that they are crucial for the development and the progression of high grade serous ovarian carcinoma (HGSOC). Though many oncogenes have been identified, no successful inhibitors targeting these molecules and their associated pathways are available. This review discusses oncogenes that have been identified recently in HGSOC using different screening strategies. All the genes discussed in this review have been functionally characterized both in vitro and in vivo and some of them are able to transform immortalized ovarian surface epithelial and fallopian tube cells upon overexpression. However, it is necessary to delineate the molecular pathways affected by these oncogenes for the development of therapeutic strategies.

A modified olanzapine-based anti-emetic regimen for the control of nausea and vomiting in patients receiving weekly cisplatin.

Background There is limited data on specific antiemetic protocols for control of chemotherapy-induced nausea/vomiting (CINV) caused by weekly cisplatin regimens. Olanzapine is an active agent against CINV and may offer better control of nausea compared to aprepitant/fosaprepitant-based regimens. The usual antiemetic dose of olanzapine (10 mg for four days) causes problems with drowsiness. A lower dose may be as effective with lesser side effects in patients receiving weekly cisplatin. Objective To assess the control of nausea, vomiting, and occurrence of side effects with a modified olanzapine-based antiemetic regimen among patients with carcinoma of the cervix receiving concurrent cisplatin with pelvic radiotherapy. Setting Tertiary cancer hospital in Southern India. Methods We used a modified regimen "mini-OPD", oral olanzapine (5 mg) days 1 and 2, intravenous palonosetron (0.25 mg) and dexamethasone (12 mg) on day 1 of cisplatin administration in patients with carcinoma of the cervix receiving concurrent chemoradiotherapy with weekly cisplatin (40 mg/m2/week). At our centre, these patients remained inpatients throughout chemoradiotherapy. CINV-related outcomes were captured in the patients' records by the treating physician in the subsequent week (up to 6 times per patient depending on the number of cycles). We audited these records to calculate the complete response (CR defined as no emetic episodes and no use of rescue medication) rate. Main outcome measure Grades of nausea, vomiting, and drowsiness as per CTCAE v4.0. Results Data of 65 patients (median age: 48 years) who received mini-OPD regimen (median doses of cisplatin/patient: 4) was available. The CR rate was 55%. Considering all cycles together (217 weekly assessment points), "no nausea" target was attained in 125 (58%) assessments and "no vomiting" in 168 (77%). There were no significant side effects. Conclusions The mini-OPD regimen is an inexpensive, non-toxic and effective regimen for the prevention of CINV in patients receiving weekly cisplatin concurrent with pelvic radiotherapy.

Recurrent cervical cancer treated with palliative chemotherapy: real-world outcome.

INTRODUCTION: Cervical cancer is the third most common cancer in India. There is limited data on the treatment of relapsed cervical cancer from India; therefore, we report the outcomes of patients with recurrent cervical cancer who were treated with palliative chemotherapy (CT). MATERIALS AND METHODS: This was a retrospective study of patients with recurrent cervical cancer who received palliative CT from January 2012 to December 2016. The demographic details, clinical profile and survival outcomes were collected. Patients were treated with carboplatin or paclitaxel and carboplatin. Local radiation was given for symptomatic patients. Patients were assessed for responses clinically and/or radiologically after three and six cycles of CT. Progression-free survival (PFS) and overall survival (OS) were calculated using the Kaplan-Meier method. RESULTS: Forty-six patients with recurrent cervical cancer were included in this analysis, with a median follow-up of 9.4 months. The median age was 49.5 (25-65) years and the median disease-free interval was 31.3 (2-196) months. Biopsy confirmation of relapse was established in 63%. The median number of CT cycles was six. Twenty-four (52.2%) patients completed six cycles of CT. The overall response rate was 56.5%. Patients with a complete or a partial response were more likely to have PFS > 6 months (p < 0.0001). Median PFS and OS were, respectively, 8.4 (95% CI 6.1-10.7) months and 10.3 (95% CI 6.8-13.8) months. The completion of all cycles of CT and the site of metastasis (nodal vs. visceral or combined) were found to be associated with OS. CONCLUSION: Palliative CT with paclitaxel carboplatin is a safe and effective option in Indian patients with recurrent cervical cancer, with more than half of the patients completing the prescribed CT. Further prospective trials may be required to place this treatment in the right context, in this era of immunotherapy and targeted therapy. However, knowing the outcomes in our population and prognostic factors will help in better prognostication of patients, thereby channelling our limited resources where necessary.

The hedgehog pathway regulates cancer stem cells in serous adenocarcinoma of the ovary.

PURPOSE: Signaling by cancer stem cells (CSCs) is known to occur at least in part through conserved developmental pathways. Here, the role of one of these pathways, i.e., the hedgehog pathway, was evaluated in high-grade serous ovarian carcinoma (HGSOC). METHODS AND RESULTS: We found that in HGSOC, hedgehog inhibitors (HHIs) GANT61, LDE225 and GDC0449 reduced or inhibited the formation of spheroids enriched in CSCs. Primary malignant cells (PMCs) in ascites from HGSOC patients cultured in the presence of HHIs showed significant reduction in CSCs. Sonic hedgehog (SHH) significantly increased the expression of ALDH1A1, which was inhibited by GANT61. In the presence of a SHH neutralizing antibody (5E1), a significant reduction in the number of spheroids was observed in HGSOC-derived cell lines. Further, the motility, migration and clonogenic growth of the cells were significantly reduced by HHIs. In the presence of GANT61, a reduction of cells from PMCs in the G0 phase of the cell cycle was observed. The magnitude of difference in expression of Gli1 in tumors from the same HGSOC patients at presentation and at interval debulking surgery was greater in patients who had a recurrence on follow up. GANT61 also significantly inhibited the growth of CSCs in nude mice. Finally, RNA sequencing of HGSOC cells treated with GANT61 showed a significantly reduced expression of CSC markers. CONCLUSIONS: Our results indicate that the hedgehog pathway plays an important role in maintaining the integrity of CSCs in HGSOC and could be a potential therapeutic target.

Flow Cytometry Based MRD and Its Impact on Survival Outcome in Children and Young Adults with ALL: A Prospective Study from a Tertiary Cancer Centre in Southern India.

Presence of minimal residual disease (MRD) following induction chemotherapy is a well-recognized risk factor to predict relapse in acute lymphoblastic leukemia (ALL). There is paucity of data on MRD and outcome in ALL from India. We share our experience in establishing a flow cytometry-based MRD assay for ALL with emphasis on determination of the number of patients who had MRD on day 35 of induction therapy and its correlation with outcome and other prognostic factors. We prospectively studied MRD in patients with ALL less than 25 years who achieved morphological complete remission with induction therapy. The initial series consisted of 104 patients with ALL. Ninety-two patients had bone marrow samples collected on day 35 of remission induction chemotherapy that was adequate for MRD. Strategy of monitoring MRD was based on flow cytometry using six color staining according the leukemia associated immunophenotype found at diagnosis. Data analysis was done using Fisher exact test. The median age was 8.5 years (range 0.9-22 years). Thirty-seven out of ninety-two patients (40.2%) had MRD at end of induction. MRD on day 35 was between 0.01 and 0.1% in 18.9% of patients, between 0.1 and 1% in 59.5% and more than 1% in 21.6% patients. Among the patients who had MRD, 16.7% had favourable cytogenetics, 60% had intermediate and 13.3% had high-risk cytogenetics. The presence or absence of residual leukemia by flow cytometry at day 35 was not significantly related to age (p = 1.0), male gender (p = 0.08) hyperleukocytosis (p = 0.25) or day 8 blast clearance (p = 0.21). However, T cell phenotype (p < 0.001) was significantly associated with MRD. The 5-year event free survival (EFS) for patients who had MRD versus those who did not was 69% and 61.1% respectively (p = 0.41). The 5-year overall survival (OS) for patients who had MRD versus those who did not was 72.5% and 61.1% respectively (p = 0.33). Flow cytometric techniques can be applied to monitor MRD in patients of ALL undergoing induction therapy. Our results suggest MRD correlates with certain known prognostic factors. Though the EFS and OS was lower in MRD positive patients, the results were not statistically significant probably because of the small sample size.

Analysis of hematopoietic stem cells using a composite approach.

Stem cells or Cancer stem cells (CSCs) have now been identified in different type of tissues by using surface markers. Functional assays such as ALDEFLUOR and side population which are marker independent have been additional approaches. However, whether all these approaches identify the same population of cells remain uncertain. To address this issue we have used hematopoietic stem cells as a model. Peripheral blood stem cells enumerated by CD34 are used routinely in bone marrow transplantation which supports the recovery of bone marrow after ablative chemotherapy or radiation. Hematopoietic stem cells (HSCs) were obtained from normal donor bone marrow (n = 5) and G-CSF stimulated peripheral blood stem cells (PBSCs) (n = 5) from patients undergoing leukapheresis prior to bone marrow transplantation. The stem cells were identified by combining CD34 expression with functional assays (ALDEFLUOR and side population). The cell cycle profile was further determined by simultaneous labeling of these cells with Hoechst and Pyronin Y. The simultaneous analysis showed that both CD34+ and CD34- cells co-exist with ALDH1A1+ cells but side population did not segregate with CD34+ cells. Though stem cell populations identified by functional assays were different, the cell cycle analysis showed that both ALDH1A1+ and CD34+ cells were in the G1 phase of cell cycle rather than in the quiescent (G0) phase.

Microvessel Density (MVD) in Locally Advanced Breast Cancer

Background: The aim of this study was to evaluate microvessel density (MVD) by expression of CD31 and CLEC14A in core biopsies from previously untreated patients with locally advanced breast cancer (LABC) and assess its prognostic significance. Methods: MVD was evaluated in core needle biopsies (n = 92), collected prior to any treatment, from patients who were diagnosed with locally advanced breast cancer (LABC). Immunohistochemistry for expression of CD31 and CLEC14A were performed on these tumours. The median duration of follow-up was 9.3 years. The effect of prognostic factors on disease free survival (DFS) and overall survival (OS) was assessed using a Log rank test and Cox regression model. Results: The clinical factors such as age, clinical nodal stage, stage and pathological nodal status were found to be significant in predicting overall survival by multivariate analysis (P<0.05). Out of 92, 52 tumours had blood vessels expressing CD31, whereas in the remainder, there was no expression. The mean and median MVD of CD31 in 92 tumours was 38 and 5.5 respectively, and it was not a significant factor for predicting disease free survival or overall survival. When we considered the tumours (n=52) which expressed CD31, patients who had very high MVD (>100), had inferior progression free survival and overall survival (P=0.5). There was no expression of CLEC14A in any of the core needle biopsies whereas it was expressed in specimens from mastectomy from the same patient. Conclusion: This is the first report of MVD in LABC prior to any treatment. The results suggest angiogenesis could be a prognostic factor in LABC.

Factors that impact the outcomes in testicular germ cell tumors in low-middle-income countries.

Germ cell tumors (GCTs) are one of the most common tumors in adolescents and young adults. There is paucity of data on GCT from low-middle-income countries (LMIC). The present study was conducted to assess the demographic features, clinical manifestations, pathology, and outcomes of GCT patients treated at our center. Patients with testicular GCT above the age of 18 years, treated at our center from 2001 to 2015 were included in the study. Data were extracted retrospectively from the case records. Event-free survival (EFS) and overall survival (OS) were calculated using the Kaplan-Meier method and the variables were compared using the log-rank test. The study included 421 patients among whom 128 (30%) had a histological diagnosis of seminoma and 293 (70%) had non-seminomatous germ cell tumor (NSGCT). Metastatic disease at presentation was observed in 83/128 (65%) with seminoma and 254/293 (87%) with NSGCT. According to the International Germ Cell Cancer Collaborative Group (IGCCCG) risk stratification for metastatic disease, good- and intermediate-risk seminoma were observed in 55/83 (66%) and 28/83 (34%) patients, respectively, and good-, intermediate-, and poor-risk NSGCT were observed in 82/254 (32%), 76/254 (30%), and 96/254 (38%) patients, respectively. The median follow-up was 32.3 months (range 0.03-200 months). The 3-year OS for the entire cohort was 80.3%. The 3-year OS for seminoma was 91.4%, and for NSGCT was 75.3%. Factors significantly associated with inferior EFS and OS on multivariate analysis included poor performance status, scrotal orchidectomy, carboplatin-based regimen, NSGCT histology, and treatment default. Patients with testicular GCT in India present in an advanced stage and higher IGCCCG risk compared to Western data. Factors unique to LMIC like treatment default, bulky disease, dose compromise, and scrotal orchidectomy have a negative impact on the outcome.

Prognostic Utility of the IPS 3 Score for Predicting Outcomes in Advanced Hodgkin Lymphoma.

BACKGROUND: The International Prognostic Scoring System (IPSS) consisting of 7 parameters (IPS7) has been the standard prognostic model used in advanced Hodgkin lymphoma (aHL). However, recent studies have questioned its discriminatory power. For retrospective analyses, its utility might be limited by missing parameters. A recent study has shown that the IPSS consisting of only 3 high-risk features (IPS3; stage IV, age 45 years or older, and hemoglobin <105 g/L) is a simple predictor of survival in aHL. However, there are limited data validating the IPS3. PATIENTS AND METHODS: Outcomes of adults with aHL treated between 2001 and 2015 at a single center were retrospectively analyzed with data from medical records. The prognostic validity of various baseline parameters was assessed individually as well as in combination (IPS7 and IPS3 scores). The Kaplan-Meier method was used to describe the event-free survival (EFS) and overall survival (OS) and univariate (log rank) and multivariate (Cox regression) tests were performed to identify prognostic factors. RESULTS: We identified 314 patients (median age, 32 [range, 18-60] years; male sex [n = 215; 68%]) treated during this period. IPS7 was available in 231 of 314 (73%) and IPS3 in all (100%) patients. Most (71%) were treated with 6 to 8 cycles of ABVD (doxorubicin, bleomycin, vinblastine, dacarbazine) and others received hybrid or cyclophosphamide, vincristine, procarbazine, prednisolone regimens, and 72 (23%) underwent interim positron emission tomography imaging with escalation to bleomycin, etoposide, adriamycin, cyclophosphamide, vincristine, procarbazine, prednisolone in 8 patients. After a median follow-up of 57 months (range, 1.3-167), the 5-year EFS and OS were 72% and 82%, respectively. IPS3 produced a wider separation of survival curves than IPS7 in univariate analysis. In multivariate analysis for EFS, IPS3 (scores of 2 or 3 vs. scores of 0 and 1; hazard ratio, 2.1; P = .004) was the only significant predictor. For OS, no factor emerged as significant. CONCLUSION: The IPS3 is a simple 3-point system that is very useful for prediction of outcomes in aHL and might be particularly suited for retrospective data analysis where all components of the IPS7 might not be available.