Detection of serum M-protein in acetonitrile precipitates by MALDI-TOF mass spectrometry: A novel, low-cost methodology.

BACKGROUND: Several studies have demonstrated the analytical sensitivity of MALDI-TOF mass spectrometry (MALDI-TOF MS) by immunoenrichment for M-protein analysis. We report the results of a novel, low-cost, reagent-based extraction process using acetonitrile (ACN) precipitation to enrich for κ and λ light chains which can be analysed by MALDI-TOF MS. METHODS: Institutional Ethics committee approval was obtained. Serum samples from patients with monoclonal gammopathy of undetermined significance (MGUS), multiple myeloma (MM), plasmacytoma, AL amyloidosis and Waldenström macroglobulinemia (WM) underwent ACN precipitation. The images obtained were overlaid on apparently healthy donor serum samples to confirm the presence of M-protein. A sample was considered positive for M-protein if there was a sharp or broad peak within the κ or λ mass/charge (m/z) range: m/z- [M + 2H]2+: 11,550-12,300 Da and λ m/z- [M + 2H]2+: 11,100-11,500 Da. Images were acquired at a m/z range of 10,000-29,000 Da. Corresponding serum protein electrophoresis (SPEP), serum immunofixation electrophoresis (IFE) and serum free light chain (sFLC) assay by nephelometry were performed for all the samples. RESULTS: Two-hundred-and-two serum samples were included in the study: MM- 184 (91%); AL amyloidosis- 2 (1%); plasmacytoma- 8 (4%); MGUS- 6 (3%) and WM- 2 (1%). All the SPEP positive samples were identified by MALDI-TOF MS. Out of 179 samples positive for M-protein by IFE, MALDI-TOF MS was positive in 176 samples (98%). Compared to IFE, the sensitivity and specificity of M-protein identification by MALDI-TOF MS were 98.3% and 52.2%, respectively. CONCLUSIONS: This study demonstrates the feasibility of qualitatively identifying M-protein without the need for antibody-based immunoenrichment, making the technique cost-effective.

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.

Autologous Stem Cell Transplantation in Testicular Germ Cell Tumor-Preliminary Experience from a Single Center.

Background Germ cell tumor (GCT) of the testis is one of the highly curable solid organ malignancies. Those who experience relapse after platinum-based chemotherapy can be salvaged with systemic therapy followed by high-dose chemotherapy (HDCT) and autologous stem cell transplantation (ASCT). Complete remission can be obtained in approximately 50 to 60% of patients treated with HDCT. Our experience reports the efficacy and safety of HDCT followed by ASCT in relapsed GCT. Methods Analysis of patient records (2012-2019) showed that three patients had received HDCT and ASCT. Results All the three patients were treated with BEP (bleomycin, etoposide, and cisplatin) as first-line therapy. HDCT was done in Case 1 after third-line salvage and in other two patients after second-line salvage chemotherapies. High-dose carboplatin and etoposide were used as conditioning regimen. Granulocyte colony-stimulating factor was used for the mobilization of stem cells. After ASCT, complete remission was documented in all the patients. All were alive and disease-free till the last follow-up. Grade ¾ toxicities including myelosuppression, diarrhea, and mucositis were observed in all three patients. Conclusion This is the first report from India on HDCT with ASCT in GCT. HDCT/ASCT seems to be feasible, safe, and effective in relapsed testicular GCTs.

Haploidentical Hematopoietic Stem Cell Transplantation in Leukemia's: Experience from a Cancer Center in India.

There has been a surge in haploidentical hematopoietic stem cell transplantation (HSCT) in India recently. However, there is a paucity of data on haploidentical HSCT from India. The report is an analysis of data of haploidentical HSCT performed at our center. Analysis of patients with acute leukemia or chronic myeloid leukemia who underwent haploidentical HSCT during 2014-2019 was performed. The graft versus host disease (GVHD) prophylaxis was post-transplant Cyclophosphamide with Mycophenolate-mofetil and Cyclosporine. All patients were transfused peripheral blood stem cells from donors. Overall survival (OS) was calculated using the Kaplan-Meier method. Twenty-one patients underwent haploidentical HSCT. Fourteen-patients were males. The median age of patients was 15 years. Fludarabine with total body irradiation was the most common conditioning regimen (n = 15, 71.4%). The median duration for neutrophil and platelet engraftment was 14 days. Cumulative incidence of acute and chronic GVHD was 19%, and 38% respectively. The median follow-up was 26 months and the two-year OS was 38%. Twelve (57%) patients died during the study period, 8 patients (38%) died from transplant-related mortality (TRM), and 4 from disease relapse. Sepsis was the cause of death in six of the eight TRM. Nine out of 21 patients (42.8%) are leukemia-free on follow-up. Haploidentical HSCT is a promising modality of treatment in patients who have no suitable matched donors. Though the TRM remains high, good disease control was achieved in 42.8% of patients. Multi-drug resistant bacterial infection remains a challenge in performing haploidentical HSCT in developing countries.

"Hairy Cell Leukemia (HCL): 'Real World' Outcome".

HCL is an uncommon B cell lympho-proliferative disorder with high remission rates. There is paucity of data on the long-term outcome of HCL from India. We retrospectively collected data from individual case records of patients with HCL who were treated in Cancer Institute, Chennai from January 2001 until January 2018. Sixteen patients were diagnosed with HCL and were treated with cladribine (81%), interferon (13%) and one patient received only best supportive care (6%). All the treated patients achieved complete response. More than half of the patients developed febrile neutropenia but there were no treatment related mortality. The 5-year DFS was 77% and 5-year OS was 80%. Relapse of disease was seen in 27%. HCL is a curable malignancy with high remission rates and survival comparable to patient treated in west.

Evaluation of Cytogenetic Abnormalities in Patients with Acute Lymphoblastic Leukemia.

Acute lymphoblastic leukemia (ALL) accounts for 20% of all adult leukemias and is the most common leukemia during childhood (80%). We present data on cytogenetics of ALL from a tertiary centre in India correlating it with clinical factors. Karyotyping of bone marrow samples of 204 patients with newly diagnosed ALL was performed with standard G-banding technique. Clinical data of patients was obtained from case records. Survival was estimated using Kaplan-Meir curves and compared by the log-rank test. Univariate and multivariate analysis was done for survival with age, sex, immunophenotype, hyperleukocytosis, risk type, remission status and cytogenetics. The most common karyotypes observed were normal in 39.7% (N = 81), hyperdiploidy in 12.7% (N = 26), t(9;22) in 4.4% (N = 9), t(1;19) in 3.9% (N = 8). Adults with ALL had worse survival compared with pediatric patients (HR 3.62; 2.03-6.45 95% CI, p < 0.001). Patients not in morphologic remission after induction chemotherapy fared poorly (HR 4.86; 2.67-8.84 95% CI, p < 0.001). Patients with favourable cytogenetics had better overall survival (HR 0.36; 0.12-1.05 95% CI, p < 0.05). On multivariate analysis, achievement of morphologic remission emerged as single most significant predictor of survival (p < 0.001). MLL gene rearrangement and t(12;21) were seen less commonly as compared to Western data. However, incidence rates of various cytogenetic abnormalities were similar to that reported from other centres from India. Age, morphologic remission at end of induction chemotherapy and favourable cytogenetics correlated significantly with survival.

Gemcitabine, vinorelbine and dexamethasone: A safe and effective regimen for treatment of relapsed/refractory hodgkin's lymphoma.

BACKGROUND: Salvage regimens in relapsed/refractory Hodgkin's lymphoma (HL) differ in their efficacy and toxicity profiles. Gemcitabine (G), vinorelbine (V) and liposomal doxorubicin (GVDoxil) is one regimen with high response rates but has high toxicity and cost. We devised a regimen of GVDex by substituting the more expensive liposomal doxorubicin with the cheaper high-dose dexamethasone (Dex). PATIENTS AND METHODS: We analyzed the data of 48 adult and paediatric patients of relapsed/refractory HL who received GVDex as salvage therapy. GVDex was delivered as outpatient once in 3 weeks (Q3 weekly) (G 1000 mg/m2 IV over 30 min on D1, 8; V 25 mg/m2 IV fast infusion on D1, 8; Dex40 mg PO D1-4) for 2-3 cycles. We present the overall response rate, toxicity, progression-free (PFS) and overall survival (OS) from the time of start of GVDex. RESULTS: Forty-eight patients [median age: 24 years (5-63)] received GVDex [(median cycles:3(1-6)] in this period. Median time from diagnosis to the first relapse was 18.9 (2-119) months. Overall response rate [ORR = complete (CR)+partial (PR)] was 63%. Eleven (23%) patients developed febrile neutropenia. After a median follow-up of 20 months, the Kaplan-Meier estimates of patients alive and progression-free at 24 months were 60% and 49%, respectively. CONCLUSIONS: The response rates with GVDex were comparable to those reported with GVDoxil when used as a first-line salvage regimen in relapsed/refractory HL. It was an effective regimen even in patients who failed 2 lines of therapy for HL.

Outcomes of Intensive Treatment of Adult Acute Myeloid Leukemia Patients: A Retrospective Study From a Single Centre.

BACKGROUND: Acute Myeloid Leukemia (AML) is a very aggressive cancer with difficult treatment and poor outcomes. The treatment of these patients is quite challenging due to various reasons including the need for extensive supportive care, and high cost of therapy. Reports on outcomes from India are few. METHODS: We analyzed 93 adult patients (≥ 18 years) with AML who were treated with curative intent between 2007 and 2014. Patients received daunorubicin at dose of 60-90 mg/m2 and cytarabine 100 mg/m2 during induction and consolidation with 3 courses of high dose cytarabine (1.5-3 g/m2per dose for 6 doses per cycle). Only 4 patients underwent consolidation allogenic stem cell transplantation in first remission (CR1). RESULTS: The median age was 37 (18-66) years; males: 52%. Conventional cytogenetics (N = 63) showed 23% (N = 15), 56% (N = 35), 27% (N = 13) in good, intermediate risk and poor risk category respectively. FLT3-ITD was positive in 12/33 (36%) and NPM mutation in 7/23 (30%). Daunorubicin dose was 60 mg/m2 in 75% (N = 70) and 90 mg/m2 in 25% (N = 23) patients. Induction mortality was 17% (16/93) [60 mg/m2:19% (13/70), and 90 mg/m2:13% (3/23); p = 0.39)]. Complete remission was achieved by 60% (56/93) [60 mg/m2:53% (37/70), and 90 mg/m2:83% (19/23); p = 0.09)]. The median overall survival was 9.2 months and the actuarial survival at 2 years was 30%. By univariate analysis, FLT3-ITD positivity, white cell counts higher than 100,000/mm3 at presentation, and use of lower dose of daunorubicin in induction were associated with poorer outcomes. CONCLUSIONS: Outcomes in adult AML are generally poor. Many patients with high risk disease don't receive allogenic transplantation in CR1. Increased availability of allogenic stem cell transplantation may help to improve outcomes.

Chronic Myeloid Leukemia: Long-Term Outcome Data in the Imatinib Era.

Recent reports suggest that in the TKI era, the survival of chronic myeloid leukemia approaches that of general population. The real-world situation may be different. We analyzed patients (≥ 18 years) with chronic phase (CP) CML enrolled over a 7-year period (2002-2008) in an imatinib access program. Event was defined as non-achievement/loss of complete hematological response (CHR), loss of cytogenetic response or progression to accelerated (AP)/blast phase (BC). Progression was defined as development of AP/BC. Any delay of ≥ 1 week in reporting for drug refills was categorized as non-adherence. Of the 443 patients with CP-CML who started imatinib [median age: 36 years (18-70); High risk: 32% (Sokal) and 14% (Hasford/EUTOS)], 162 (37%) had received prior therapy [mostly hydroxyurea (N = 153]. CHR was achieved by 430 (97%). After a median follow up of 109.5 months (3.4-184.3), the EFS, PFS and OS at 10 years was 43%, 75% and 76% respectively. Superior EFS was predicted by low-risk Hasford score and adherence to therapy. Adherence to therapy was the only factor which predicted EFS on multivariate analysis (HR 0.64, 95% CI 0.50-0.83, P = 0.001). Long-term follow up of patients with CP-CML reflects poorer survival than those reported from clinical trials and reflects multiple issues that affect "real-world" patients. The continued drop in EFS, noted during long-term follow up, might take time to impact the PFS and OS due to the chronic nature of the disease. Sustained adherence to therapy is important for optimum long-term outcomes.

Outcomes of Oral Metronomic Therapy in Patients with Lymphomas.

Oral Metronomic chemotherapy (OMC) is used in patients with lymphoma who may not tolerate intravenous chemotherapy or have refractory disease. It is cheaper, less toxic and easy to administer. Adult patients with lymphoma who received OMC (combination of cyclophosphamide, etoposide and prednisolone) were included in this retrospective analysis. Response assessment was clinical with limited use of radiology. Progression free and overall survival (PFS and OS) were calculated from the time of start of OMC until documentation of disease progression or death. Between 2007 and 2017, 149 patients were given OMC [median age: 62 years (19-87); 94 patients (63.1%) male]. Majority [112 patients (75.2%)] had stage III/IV disease. The most common subtype of lymphoma was diffuse large B cell lymphoma (40.9%). OMC was used at diagnosis in 41 patients (27.5%) and after relapse in 108 patients (72.5%). Overall response rates were 43.9 and 41.7% with clinical CR in 14 (34.1%) and 21 (19.4%) in patients given first line and later lines of OMC respectively. After a median follow up of 12 months (range 1-123 months), median PFS and OS were 10.5 (95% CI 8.6-12.5) and 18.8 (95% CI 12.1-25.5) months respectively. PFS and OS at 12 months were 47.6 and 64.2% respectively. Though OMC is used in many centers in India, there is scanty published information on its efficacy in lymphoma. In this analysis, we demonstrate its activity in a subset of patients with predominantly high-grade and advanced stage NHL. OMC is a useful option in frail patients and a small proportion can achieve deep and long lasting responses.

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.

Acute Lymphoblastic Leukemia in Young Adults Treated with Intensive "Pediatric" Type Protocol.

Young adults with acute lymphoblastic leukemia do better when treated on "pediatric" protocols. Young adults (18-30 years) with Ph-negative ALL treated between 2000 and 2014 were retrospectively analyzed. Two-hundred and thirty-two patients were included [median age 21 years (18-30); 176 (76%) males; median WBC 16,000/cmm]. Protocols used were: BFM 95 (N = 147, 63%), MCP-841 (N = 51, 22%), GMALL (N = 21, 9%), INCTR (N = 9, 4%) and UKALL (N = 4, 2%). Complete remission was achieved in 194/232 (84%). Twenty patients (9%) died due to toxicity which was higher with BFM versus others (18/147 vs. 2/85; p = 0.031). After a median follow-up of 48 months, median RFS and OS were 35.5 months (25-46), and 25 months (18-31) and actuarial RFS and OS (5-years) were 45% (37-53) and 39% (32-46). BFM protocol improved RFS (51 vs. 35%, p = 0.027) but not OS (43 vs. 33%, p = 0.2). The survival outcomes reported are 15-20% lower than those reported from West. Better supportive care and risk-adapted therapy may improve outcomes.

DA-EPOCH-R in Aggressive CD 20 Positive B Cell Lymphomas: Real-World Experience.

Recent reports have shown that excellent survival outcomes can be achieved in adult Burkitt's lymphoma with the use of DA-EPOCH-R regimen. When compared to earlier intense pediatric-type protocols, this regimen is less toxic. There are limited reports available on the use of this regimen outside the context of clinical trials. We analyzed the outcomes of patients who were treated with the DA-EPOCH-R regimen [Burkitt's lymphoma (BL), primary mediastinal B cell lymphoma (PMBCL) and HIV-positive patients with diffuse large B cell lymphoma (DLBCL)] at our center over a 3 year period. Baseline characters, responses, and toxicity data was captured from records. Event-free survival (EFS-from therapy initiation till occurrence of event (non-achievement of complete response or relapse) and overall survival (OS-from therapy initiation till death due to any cause) were estimated using Kaplan-Meier method. Among 41 patients [median age 40 years (18-76)], the following diagnoses were included-HIV negative patients (N = 29): BL (N = 24), PMBCL (N = 5); HIV positive patients (N = 12): BL (N = 8), and DLBCL (N = 4). Among those with BL, majority had stage III/IV disease (N = 21/32, 65%). At the completion of planned therapy, 33 had achieved CR (81%). One patient died due to toxicity. The actuarial EFS and OS at 2 years were 80 and 77% respectively for all patients. The OS at 2 years was 100% for PMBCL, 80% for BL and 50% for HIV-positive DLBCL. Majority of the failures in BL were in patients with advanced disease. DA-EPOCH-R can be used in real-world setting and allows treatment of older patients with BL.

Voriconazole is a safe and effective anti-fungal prophylactic agent during induction therapy of acute myeloid leukemia.

BACKGROUND: Antifungal prophylaxis (AFP) reduces the incidence of invasive fungal infections (IFIs) during induction therapy of acute myeloid leukemia (AML). Posaconazole is considered the standard of care. Voriconazole, a generic cheaper alternative is a newer generation azole with broad anti-fungal activity. There is limited data on the use of voriconazole as a prophylactic drug. MATERIALS AND METHODS: A single-center, prospective study was performed during which patients with AML undergoing induction chemotherapy received voriconazole as AFP (April 2012 to February 2014). Outcomes were compared with historical patients who received fluconazole as AFP (January 2011-March 2012, n = 66). RESULTS: Seventy-five patients with AML (median age: 17 years [range: 1-75]; male:female 1.6:1) received voriconazole as AFP. The incidence of proven/probable/possible (ppp) IFI was 6.6% (5/75). Voriconazole and fluconazole cohorts were well-matched with respect to baseline characteristics. Voriconazole (when compared to fluconazole) reduced the incidence of pppIFI (5/75, 6.6% vs. 19/66, 29%; P < 0.001), need to start therapeutic (empiric + pppIFI) antifungals (26/75, 34% vs. 51/66, 48%; P < 0.001) and delayed the start of therapeutic antifungals in those who needed it (day 16 vs. day 10; P < 0.001). Mortality due to IFI was also reduced with the use of voriconazole (1/75, 1.3% vs. 6/66, 9%; P = 0.0507), but this was not significant. Three patients discontinued voriconazole due to side-effects. CONCLUSION: Voriconazole is an effective and safe oral agent for IFI prophylaxis during induction therapy of AML. Availability of generic equivalents makes this a more economical alternative to posaconazole.

Nonadherence to imatinib adversely affects event free survival in chronic phase chronic myeloid leukemia.

There is limited data on the impact of treatment interruptions due to nonadherence in patients with chronic phase chronic myeloid leukemia (CP-CML) treated with Imatinib. We looked at factors (including adherence to therapy) affecting the outcome in a large cohort of patients with CP-CML. All the 516 patients received Imatinib free-of-cost through a company sponsored scheme, which mandated regular three monthly visits for drug procurement. Data regarding the disease characteristics, adherence to treatment and outcomes, were obtained from patients records. Unwarranted interruption of treatment for more than 1 week was defined as nonadherence. With a median follow-up of 39 months, the estimated 5-year event free survival (EFS) was 70.8% (95%, CI = 63.3-78.3). Nearly one-third of the patients (29.6%) were found to be nonadherent at some point during their treatment. On univariate analysis, the factors adversely affecting the EFS were prolonged symptom duration before diagnosis, treatment with hydroxyurea for more than 1 month before start of Imatinib, and nonadherence to therapy. Only nonadherence was significant in multivariate analysis (HR1.6; P = 0.048). The 5-year EFS in adherent and nonadherent patients was 76.7% and 59.8% respectively (P = 0.011, log rank test). Nonadherent patients were less likely to achieve complete cytogenetic responses (26% versus 44%; P = 0.004; χ(2) test) at any point. A significant proportion of patients with CP-CML have drug interruptions due to nonadherence during therapy and this compromises the EFS. Adherence to therapy must be included as an important evaluation parameter in all future studies of CML.