Reducing cytogenetic testing in the era of next generation sequencing: Are we choosing wisely?

INTRODUCTION: In most laboratories, next generation sequencing (NGS) has been added without consideration for redundancy compared to conventional cytogenetics (CG). We tested a streamlined approach to genomic testing in patients with suspected myeloid and plasma cell neoplasms using next generation sequencing ("NGS first") as the primary testing modality and limiting cytogenetics (CG) to samples with morphologic abnormalities in the marrow aspirate. METHODS: Based on morphologic interpretation of bone marrow aspirate and flow cytometry, samples were triaged into four groups: (a) Samples with dysplasia or excess blasts had both NGS and karyotyping; (b) Samples without excess blasts or dysplasia had NGS only; (c) Repeat samples with previous NGS and/or CG studies were not retested; (d) Samples for suspected myeloma with less than 5% plasma cell had CG testing cancelled. RESULTS: Seven hundred eleven adult bone marrow (BM) samples met the study criteria. The NGS first algorithm eliminated CG testing in 229/303 (75.6%) of patients, primarily by reducing repeat testing. Potential cost avoided was approximately $124 000 per annum. Hematologists overruled the triage comment in only 11/303 (3.6%) cases requesting CG testing for a specific indication. CONCLUSIONS: Utilizing NGS as the primary genomic testing modality NGS was feasible and well accepted, reducing over three quarters of all CG requests and improving the financial case for adoption of NGS. Key factors for the success of this study were collaboration of clinical and genomic diagnostic teams in developing the algorithm, rapid turnaround time for BM interpretation for triage, and communication between laboratories.

Clinical value of next-generation sequencing compared to cytogenetics in patients with suspected myelodysplastic syndrome.

Next-generation sequencing (NGS) increasingly influences diagnosis, prognosis and management of myelodysplastic syndrome (MDS). In addition to marrow morphology and flow cytometry, our institution performs cytogenetics (CG) and NGS-based testing routinely in patients with suspected MDS. We evaluated the relative value of NGS in the assessment of patients with suspected MDS. We initially compared the diagnostic and prognostic information derived from CG and NGS in 134 patients. NGS enhanced the diagnostic yield compared to CG for clonal myeloid disorders (sensitivity 77% vs. 42·2%; specificity 90·2% vs. 78%; positive predictive value 92·8% vs. 76%; and negative predictive value 70·8% vs. 45·5%). The identification of poor prognosis mutations by NGS altered risk category in 27/39 (69·2%) patients with MDS with good/intermediate risk CG. Subsequently, we prospectively evaluated 70 patients with suspected MDS using an 'NGS-first approach' with CG restricted to samples with morphological abnormalities. We rarely identified mutations or CG abnormalities in patients without dysplastic features. NGS has a superior diagnostic performance compared to CG in patients with suspected MDS. We estimate that by using an 'NGS-first approach' we could reduce karyotyping by approximately 30%.

Effect of the addition of rituximab to salvage chemotherapy prior to autologous stem cell transplant in aggressive CD20+ lymphoma: a cohort comparison from the NCIC Clinical Trials Group Study LY.12.

The impact of the addition of rituximab to salvage chemotherapy prior to autologous stem cell transplant (ASCT) was evaluated in a retrospective subgroup analysis of NCIC CTG LY.12. Among 414 patients who relapsed following R-CHOP, 96 received salvage chemotherapy alone [R - cohort]; and 318 received rituximab with chemotherapy [R + cohort] following a protocol amendment. The R-cohort had a higher proportion of patients with PS ≥2 and relapse <1 year after R-CHOP. The response rate (45.6% vs. 25.0%, p = 0.0003), CR/CRu (15.7% vs. 4.2%, p = 0.003) and transplantation rate (51.9% vs. 31.3%, p = 0.0004) was higher in the R + cohort. Event-free (27% vs. 22%, p = 0.0954) and overall survival at four years (43% vs. 31%; p = 0.045) were greater in the R + cohort when the patients with best response SD/PD to R-CHOP were excluded. Addition of rituximab to salvage therapy before ASCT appears to improve the response rate, transplantation rate, and overall survival in patients with CD20+ lymphoma who responded to R-CHOP.

Filgrastim-Stimulated Bone Marrow Compared with Filgrastim-Mobilized Peripheral Blood in Myeloablative Sibling Allografting for Patients with Hematologic Malignancies: A Randomized Canadian Blood and Marrow Transplant Group Study.

In adult hematopoietic cell transplantation (HCT), filgrastim-mobilized peripheral blood (G-PB) has largely replaced unstimulated marrow for allografting. Although the use of G-PB results in faster hematopoietic recovery, it is also associated with more chronic graft-versus-host disease (cGVHD). A potential alternative allograft is filgrastim-stimulated marrow (G-BM), which we hypothesized may be associated with prompt hematopoietic recovery but with less cGVHD. We conducted a phase 3, open-label, multicenter randomized trial of 230 adults with hematologic malignancies receiving allografts from siblings after myeloablative conditioning to compare G-PB with G-BM. The primary endpoint was time to treatment failure, defined as a composite of extensive cGVHD, relapse/disease progression, and death. With a median follow-up of 36 months (range, 9.6 to 48), comparing G-BM with G-PB, there was no difference between the 2 arms with respect to the primary outcome of this study (hazard ratio [HR], .91; 95% confidence interval [CI], .68 to 1.22; P = .52). However, the cumulative incidence of overall cGVHD was lower with G-BM (HR, .66; 95% CI, .46 to .95; P = .007) and there was no difference in the risk of relapse or progression (P = .35). The median times to neutrophil recovery (P = .0004) and platelet recovery (P = .012) were 3 days shorter for recipients allocated to G-PB compared with those allocated to G-BM, but there were no differences in secondary engraftment-related outcomes, such as time to first hospital discharge (P = .17). In addition, there were no graft failures in either arm. This trial demonstrates that, compared with G-PB, the use of G-BM allografts leads to a significantly lower rate of overall cGVHD without a loss of the graft-versus-tumor effect and comparable overall survival. Our findings suggest that further study of this type of allograft is warranted.

Randomized comparison of gemcitabine, dexamethasone, and cisplatin versus dexamethasone, cytarabine, and cisplatin chemotherapy before autologous stem-cell transplantation for relapsed and refractory aggressive lymphomas: NCIC-CTG LY.12.

PURPOSE: For patients with relapsed or refractory aggressive lymphoma, we hypothesized that gemcitabine-based therapy before autologous stem-cell transplantation (ASCT) is as effective as and less toxic than standard treatment. PATIENTS AND METHODS: We randomly assigned 619 patients with relapsed/refractory aggressive lymphoma to treatment with gemcitabine, dexamethasone, and cisplatin (GDP) or to dexamethasone, cytarabine, and cisplatin (DHAP). Patients with B-cell lymphoma also received rituximab. Responding patients proceeded to stem-cell collection and ASCT. Coprimary end points were response rate after two treatment cycles and transplantation rate. The noninferiority margin for the response rate to GDP relative to DHAP was set at 10%. Secondary end points included event-free and overall survival, treatment toxicity, and quality of life. RESULTS: For the intention-to-treat population, the response rate with GDP was 45.2%; with DHAP the response rate was 44.0% (95% CI for difference, -9.0% to 6.7%), meeting protocol-defined criteria for noninferiority of GDP (P = .005). Similar results were obtained in a per-protocol analysis. The transplantation rates were 52.1% with GDP and 49.3% with DHAP (P = .44). At a median follow-up of 53 months, no differences were detected in event-free survival (HR, 0.99; stratified log-rank P = .95) or overall survival (HR, 1.03; P = .78) between GDP and DHAP. Treatment with GDP was associated with less toxicity (P < .001) and need for hospitalization (P < .001), and preserved quality of life (P = .04). CONCLUSION: For patients with relapsed or refractory aggressive lymphoma, in comparison with DHAP, treatment with GDP is associated with a noninferior response rate, similar transplantation rate, event-free survival, and overall survival, less toxicity and hospitalization, and superior quality of life.

Phase II study of targeted therapy with temozolomide in acute myeloid leukaemia and high-risk myelodysplastic syndrome patients pre-screened for low O(6) -methylguanine DNA methyltransferase expression.

Resistance to temozolomide is largely mediated by the DNA repair enzyme O(6) -methylguanine DNA methyltransferase (MGMT). We conducted a prospective multicentre study of patients with previously untreated acute myeloid leukaemia (AML) or high-risk myelodysplastic syndrome (MDS) who were not candidates for intensive therapy. Patient selection was based on MGMT expression by Western blot. Patients with MGMT:ACTB (ß-actin) ratio <0·2 were eligible to receive temozolomide 200 mg/m(2) /d ×7 d. Patients achieving a complete response (CR) could receive up to 12 monthly cycles of temozolomide ×5/28 d. Of 166 patients screened, 81 (49%) demonstrated low MGMT expression; 45 of these were treated with temozolomide. The overall response rate was 53%; 36% achieved complete clearance of blasts, with 27% achieving a CR/CR with incomplete platelet recovery (CRp). Factors associated with a trend toward a higher response rate included MDS, methylated MGMT promoter and standard cytogenetic risk group. Induction and post-remission cycles were well-tolerated and most patients were treated on an outpatient basis. Patient who achieved CR/CRp had a superior overall survival compared to partial or non-responders. In conclusion, targeted therapy based on pre-selection for low MGMT expression was associated with a higher response rate to temozolomide compared to previous reports of unselected patients.

Predictors of unsuccessful mobilization with granulocyte colony-stimulating factor alone in patients undergoing autologous hematopoietic stem cell transplantation.

Mobilization of hematopoietic stem cells is achieved with hematopoietic growth factors with or without chemotherapy or other agents. Although studies comparing granulocyte colony-stimulating factor (G-CSF) alone to combined regimens demonstrate an increase in stem cell yield in the latter, mobilization with G-CSF alone is still effective and has been widely practiced. We conducted a retrospective cohort study of consecutive patients at our institution who underwent at least one mobilization attempt with G-CSF between January 2000 and December 2008 to identify the proportion of patients failing one or more mobilization attempts and the potential predictors of mobilization failure with this regime. Out of 293 patients, 251 (86.6%) were successfully mobilized and 244 (83.6%) underwent hematopoietic stem cell transplantation. Median yield was 3.55 × 106 CD34⁺ cells/kg. On univariate analysis, mobilization success was influenced by degree of previous treatment and underlying diagnosis (P < 0.001 each) but not by age (P = 0.114), sex (P = 0.860), or radiotherapy (P = 0.454). A diagnosis of non-Hodgkin's lymphoma (NHL) and number of previous chemotherapy regimens were predictors of failure on multivariate analysis. CD34⁺ yield was influenced by diagnosis and previous chemotherapy (P < 0.001 each). Mobilization with G-CSF alone yields adequate collections for most patients; however, heavily pretreated NHL patients with one failed attempt had high rates of remobilization failure and should be considered for alternative regimens.

Hodgkin lymphoma with cutaneous involvement.

We report a case of a 54-year-old previously healthy man with Hodgkin lymphoma who presented initially with a solitary cutaneous ulcer. Unlike non-Hodgkin lymphoma subtypes, skin involvement of Hodgkin lymphoma is extremely rare. Furthermore, the prognosis of Hodgkin lymphoma with skin infiltration is felt to be extremely poor. Contrary to other reports, this case demonstrates that a good response with standard therapy is possible.

Results of a multicenter randomized phase II trial of thalidomide and prednisone maintenance therapy for multiple myeloma after autologous stem cell transplant.

We report a multicenter, randomized phase II trial conducted to assess the tolerability of combined thalidomide and prednisone maintenance in multiple myeloma. Eligibility required administration of melphalan (200 mg/m2) with blood stem cell support within 1 year of treatment onset and initiation of maintenance within 60 to 100 days after stem cell infusion. All patients received 50 mg of prednisone by mouth on alternate days and thalidomide at a starting dose of either 200 or 400 mg daily by mouth. The primary end point was the incidence of dropout or dose reduction due to treatment toxicity within 6 months. Sixty-seven patients were enrolled. Median follow-up is 36.8 months. The primary end point was reached by 31% of patients on the 200 mg of thalidomide arm and 64% of patients on the 400 mg of thalidomide arm. Allowing for dose reduction, 76% of patients assigned to the 200 mg of thalidomide arm and 41% of patients assigned to the 400 mg of thalidomide arm remained on any maintenance therapy 18 months after registration. Eighty-eight percent of all patients dose-reduced thalidomide and 72% of all patients dose-reduced prednisone within 2 years of beginning maintenance. The median progression-free survival post-transplant is 32.3 months, or 42.2 months from diagnosis. Only the 200 mg of thalidomide arm of this trial met our definition of a tolerable maintenance therapy, defined as no dose reductions or discontinuation due to toxicity in at least 65% of patients for a minimum of 6 months, thus establishing a dosing schedule for phase III trials.

Gemcitabine, dexamethasone, and cisplatin in patients with recurrent or refractory aggressive histology B-cell non-Hodgkin lymphoma: a Phase II study by the National Cancer Institute of Canada Clinical Trials Group (NCIC-CTG).

BACKGROUND: Gemcitabine has been shown to have activity as a single agent in lymphoma and, when combined with cisplatin, is effective therapy for a number of solid tumors. The authors wished to determine the response rate and toxicity of gemcitabine, dexamethasone, and cisplatin for recurrent or refractory non-Hodgkin lymphoma (NHL). METHODS: Patients with recurrent or refractory diffuse large B-cell NHL or variants (REAL classification), measurable disease, and one previous chemotherapy regimen were eligible. Treatment consisted of gemcitabine 1000 mg/m(2) intravenously (i.v.) on Days 1 and 8, dexamethasone 40 mg orally on Days 1-4, and cisplatin 75 mg/m(2) i.v. on Day 1 (GDP), every 21 days as an outpatient. The primary end point was a response after two cycles. Patients could then proceed to stem cell transplantation (SCT) or receive up to six treatment cycles. RESULTS: Fifty-one eligible patients were evaluable for toxicity and response. The median age of the patients was 57 years (range, 18-84 years) and most had diffuse large-cell lymphoma. After 2 cycles, there were 8 complete responses (CR; 16%) and 17 partial responses (PR; 33%). There was an overall response rate (RR) of 49% (95% confidence interval = 37-63%). The RR afer completion of all protocol chemotherapy (including those who received > 2 cycles of GDP) was 53% (11 CR, 16 PR). Grade 3 and 4 neutropenia occurred in 33% and 39% of patients, respectively. Grade 3 and 4 thrombocytopenia occurred in 24% and 4% of patients, respectively. Seven patients (14%) experienced febrile neutropenia. Of the 35 patients < 66 years, 22 (63%) proceeded to SCT. CONCLUSIONS: GDP is an active regimen in B-cell NHL and can be administered with acceptable toxicity to outpatients. A Phase III trial comparing GDP with standard cisplatin-based chemotherapy is now ongoing through the National Cancer Institute of Canada Clinical Trials Group.

An evaluation of the donor experience in the canadian multicenter randomized trial of bone marrow versus peripheral blood allografting.

We compared the donation of bone marrow (BM) versus recombinant human granulocyte colony-stimulating factor-mobilized peripheral blood progenitor cells (PBPC) in HLA-matched sibling donors. Donors randomized to donate BM or PBPC completed questionnaires (Profile of Mood States [POMS] and Short-Form 36 Health Survey) assessing peridonation health-related quality of life (QoL), donation experience, and acceptability of donation before and 1 week and 4 weeks after donation. Between January 1996 and March 1999, 184 patients and their donors were randomized. Predonation and postdonation data were available on 52 (56%) and 35 (38%) of the BM and PBPC donors, respectively. The median donor age was 45 years, and 44% were female. The median time (range) to return to full activity for the BM and PBPC donors was 4 days (1-21 days) and 2 days (0-21 days), respectively (P = .01). One week after donation, BM donors reported more fatigue and less energy than the PBPC donors. BM donors' POMS total mood disturbance scores were worse 1 week after versus before donation, whereas the PBPC donors' scores did not change. POMS subscores indicated more fatigue and less energy in the BM versus PBPC donors. Anxiety improved in both groups, but more in PBPC donors. Four weeks after donation, the Short-Form 36 Health Survey indicated persistent moderate negative effects on QoL with BM donation versus small effects with PBPC donation. BM donation was associated with more physical morbidity and negative effects on QoL up to 1 month after donation than was PBPC donation. Despite this, most donors would donate again. Further work is needed to decrease donor anxiety and symptoms. If both BM and PBPC donation are feasible, then the graft source should be dictated by the predicted patient outcome as determined from the results of randomized trials.

High-Dose Chemotherapy Followed by Peripheral and/or Bone Marrow Stem Cell Transplant in Patients With Advanced Sarcoma: Experience of a Canadian Centre.

PURPOSE: Few reports have been published on the evaluation of stem cell auto transplantation for chemosensitive sarcomas. Some suggest benefit, others do not. We present results of 24 patients with sarcoma undergoing autotransplantation at a Canadian institution. PATIENTS AND METHODS: Twenty-four patients were treated between 1988 and 1998: 23 were >/=18 years (median 27; range 12-56); genders were equal; 12 patients had Ewing's sarcoma. At diagnosis, 12 (50%) had metastatic disease. Prior to autotransplant, all had >/=1 chemotherapy regimen. Fourteen (58%) were in complete remission (CR) and seven (29%) had minimal residual disease. All received etoposide 60 mg/kg (Day -4), melphalan 140 mg/malpha(2) on (Day -3) and a stem cell reinfusion (Day 0). RESULTS: Three patients (12.5%) were alive and disease-free with median follow-up of 92 months (80-142); one was alive with disease 32 months post-autotransplant. Twenty had died (disease, 17; transplant-related, 2; unknown, 1). Of the four alive, three had Ewing's sarcoma, one alveolar rhabdomyosarcoma, and all were in CR at transplant. Median time to relapse was 6 months (2-59). Sixteen of 18 (89%) relapsed within 1 year. Median overall survival was 10 months (0-137). A trend towards improved survival (P=0.07) was evident for patients in CR prior to autotransplant. CONCLUSIONS: Stem cell autotransplantation does not benefit most patients with sarcoma. A subgroup of high-risk patients in CR may fare better and warrant further study.

A randomized multicenter comparison of bone marrow and peripheral blood in recipients of matched sibling allogeneic transplants for myeloid malignancies.

Cytokine-mobilized peripheral blood is increasingly used instead of bone marrow as the source of cells for allogeneic transplantation. Although cells lead to faster hematologic recovery, their effects on graft-versus-host disease, relapse, and survival are less certain. Between January 1996 and February 2000, 228 patients with chronic myeloid leukemia, acute myeloid leukemia, or myelodysplasia were randomized to receive either bone marrow or peripheral blood allografts from HLA-matched siblings. All patients received busulfan and cyclophosphamide as conditioning chemotherapy and cyclosporine and methotrexate as graft-versus-host disease prophylaxis. We compared the times to neutrophil and platelet recovery, acute and chronic graft-versus-host disease, relapse, and overall survival between the groups. The median times to neutrophil recovery were 19 days and 23 days and the times to platelet recovery were 16 days and 22 days in the peripheral blood and bone marrow groups, respectively (P <.0001 for both comparisons). The cumulative incidence of grades II to IV acute graft-versus-host disease 100 days after transplantation was 44% in both groups (hazard ratio, 0.99; 95% confidence interval, 0.66-1.49; P >.9), and the incidence of extensive chronic graft-versus-host disease at 30 months after transplantation was 40% with peripheral blood and 30% with bone marrow (hazard ratio, 1.23; 95% confidence interval, 0.78-1.96; P =.37). There was no statistically significant difference in the probability of relapse of the underlying disease between the groups. The probabilities of survival at 30 months after transplantation were 68% and 60% in the peripheral blood and bone marrow groups, respectively (hazard ratio, 0.62; 95% confidence interval, 0.39-0.97; P =.04). In patients with chronic myeloid leukemia, acute myeloid leukemia, and myelodysplasia undergoing allogeneic transplantation from matched siblings, the use of peripheral blood instead of bone marrow leads to faster hematologic recovery, similar risk of graft-versus-host disease, and improved survival.