Amrita Krishnan

Initial genome sequencing and analysis of multiple myeloma

Multiple myeloma is an incurable malignancy of plasma cells, and its pathogenesis is poorly understood. Here we report the massively parallel sequencing of 38 tumour genomes and their comparison to matched normal DNAs. Several new and unexpected oncogenic mechanisms were suggested by the pattern of somatic mutation across the data set. These include the mutation of genes involved in protein translation (seen in nearly half of the patients), genes involved in histone methylation, and genes involved in blood coagulation. In addition, a broader than anticipated role of NF-κB signalling was indicated by mutations in 11 members of the NF-κB pathway. Of potential immediate clinical relevance, activating mutations of the kinase BRAF were observed in 4% of patients, suggesting the evaluation of BRAF inhibitors in multiple myeloma clinical trials. These results indicate that cancer genome sequencing of large collections of samples will yield new insights into cancer not anticipated by existing knowledge.

Multicenter Phase II Study of Bortezomib in Patients With Relapsed or Refractory Mantle Cell Lymphoma

PURPOSE Evaluate response rate, duration of response (DOR), time-to-progression (TTP), overall survival (OS), and safety of bortezomib treatment in patients with relapsed or refractory mantle cell lymphoma (MCL). PATIENTS AND METHODS Bortezomib 1.3 mg/m(2) was administered on days 1, 4, 8, and 11 of a 21-day cycle, for up to 17 cycles. Response and progression were determined using International Workshop Response Criteria, both using data from independent radiology review and by the investigators. Primary efficacy analyses were based on data from independent radiology review. RESULTS In total, 155 patients were treated. Median number of prior therapies was one (range, one to three). Response rate in 141 assessable patients was 33% including 8% complete response (CR)/unconfirmed CR. Median DOR was 9.2 months. Median TTP was 6.2 months. Results by investigator assessments were similar. Median OS has not been reached after a median follow-up of 13.4 months. The safety profile of bortezomib was similar to previous experience in relapsed multiple myeloma. The most common adverse events grade 3 or higher were peripheral neuropathy (13%), fatigue (12%), and thrombocytopenia (11%). Death from causes that were considered to be treatment related was reported for 3% of patients. CONCLUSION These results confirm the activity of bortezomib in relapsed or refractory MCL, with predictable and manageable toxicities. Bortezomib provides significant clinical activity in terms of durable and complete responses, and may therefore represent a new treatment option for this population with usually very poor outcome. Studies of bortezomib-based combinations in MCL are ongoing.

Widespread Genetic Heterogeneity in Multiple Myeloma: Implications for Targeted Therapy

We performed massively parallel sequencing of paired tumor/normal samples from 203 multiple myeloma (MM) patients and identified significantly mutated genes and copy number alterations and discovered putative tumor suppressor genes by determining homozygous deletions and loss of heterozygosity. We observed frequent mutations in KRAS (particularly in previously treated patients), NRAS, BRAF, FAM46C, TP53, and DIS3 (particularly in nonhyperdiploid MM). Mutations were often present in subclonal populations, and multiple mutations within the same pathway (e.g., KRAS, NRAS, and BRAF) were observed in the same patient. In vitro modeling predicts only partial treatment efficacy of targeting subclonal mutations, and even growth promotion of nonmutated subclones in some cases. These results emphasize the importance of heterogeneity analysis for treatment decisions.

RNA-Based Gene Therapy for HIV with Lentiviral Vector–Modified CD34+ Cells in Patients Undergoing Transplantation for AIDS-Related Lymphoma

Transfected stem cells transplanted into patients with HIV infection resulted in sustained RNA expression of introduced genes in blood cells for up to 2 years. Steps Toward a Stable Source of Therapeutic RNA Gene therapy in humans has not been easy to implement. Genes inserted into complex human cells have triggered serious unintended consequences and have often proven to be short-lived. Yet perseverance may be paying off. DiGiusto et al. report a step toward workable gene therapy in the form of stable expression of a lentiviral vector encoding anti-HIV RNAs in blood stem cells transplanted into AIDS patients. None of these patients is cured, but the vector seems to stably express the potentially therapeutic RNAs. Putting exogenous gene sequences into humans is risky, and review boards are appropriately conservative. But DiGiusto et al. took advantage of a clinical situation to design a trial that minimized extra risk to the subjects. Blood cancer (lymphoma) is common in AIDS patients, and they are often treated by ablation of their diseased bone marrow with chemotherapy followed by a transplant with their own previously saved blood stem cells. Because these patients were being transplanted with their own blood cells anyway, the authors were able to get permission to transfect a few of the blood cells of four patients with a vector carrying anti-HIV entities and reinfuse them along with the normally transplanted cells. The vector made RNAs that could counteract viral replication in several ways: inhibition of viral entry (with a CCR5 ribozyme), inhibition of RNA transport [by a small interfering RNA (siRNA) to tat/rev], and inhibition of viral transcription initiation with a decoy RNA. The good news was that the patients showed no signs of toxicity besides problems usually associated with transplantation and that blood cells from all four patients contained signs of the transplanted genes, with the amounts increasing in two of the patients after 18 months. Although the fraction of cells containing the genes was <0.2%, this was not too different from the fraction of transfected cells that was infused into the patients. The three anti-HIV RNAs could also be detected as long as 1 year after the initial infusion, and examination of T cells, monocytes, and B cells from one patient confirmed the presence of vector in these three cell types. These cells that survived for long periods of time in patients, although too scarce to cure or even improve their HIV infections, nevertheless offer lessons for future applications of gene therapy. We know that this procedure is seemingly safe and that cells given new genetic material via a lentiviral vector outside the patient can survive once reimplanted. Continued perseverance can only bring us closer to realizing the potential of this promising therapy. AIDS patients who develop lymphoma are often treated with transplanted hematopoietic progenitor cells. As a first step in developing a hematopoietic cell–based gene therapy treatment, four patients undergoing treatment with these transplanted cells were also given gene-modified peripheral blood–derived (CD34+) hematopoietic progenitor cells expressing three RNA-based anti-HIV moieties (tat/rev short hairpin RNA, TAR decoy, and CCR5 ribozyme). In vitro analysis of these gene-modified cells showed no differences in their hematopoietic potential compared with nontransduced cells. In vitro estimates of successful expression of the anti-HIV moieties were initially as high as 22% but declined to ~1% over 4 weeks of culture. Ethical study design required that patients be transplanted with both gene-modified and unmanipulated hematopoietic progenitor cells obtained from the patient by apheresis. Transfected cells were successfully engrafted in all four infused patients by day 11, and there were no unexpected infusion-related toxicities. Persistent vector expression in multiple cell lineages was observed at low levels for up to 24 months, as was expression of the introduced small interfering RNA and ribozyme. Therefore, we have demonstrated stable vector expression in human blood cells after transplantation of autologous gene-modified hematopoietic progenitor cells. These results support the development of an RNA-based cell therapy platform for HIV.

Solid Cancers After Bone Marrow Transplantation

PURPOSE To evaluate the incidence and associated risk factors of solid cancers after bone marrow transplantation (BMT). PATIENTS AND METHODS We analyzed 2,129 patients who had undergone BMT for hematologic malignancies at the City of Hope National Medical Center between 1976 and 1998. A retrospective cohort and nested case-control study design were used to evaluate the role of pretransplantation therapeutic exposures and transplant conditioning regimens. RESULTS Twenty-nine patients developed solid cancers after BMT, which represents a two-fold increase in risk compared with a comparable normal population. The estimated cumulative probability (+/- SE) for development of a solid cancer was 6.1% +/- 1.6% at 10 years. The risk was significantly elevated for liver cancer (standardized incidence ratio [SIR], 27.7; 95% confidence interval [CI], 1.9 to 57.3), cancer of the oral cavity (SIR, 17.4; 95% CI, 6.3 to 34.1), and cervical cancer (SIR, 13.3; 95% CI, 3.5 to 29.6). Each of the two patients with liver cancer had a history of chronic hepatitis C infection. All six patients with squamous cell carcinoma of the skin had chronic graft-versus-host disease. The risk was significantly higher for survivors who were younger than 34 years of age at time of BMT (SIR, 5.3; 95% CI, 2.7 to 8.6). Cancers of the thyroid gland, liver, and oral cavity occurred primarily among patients who received total-body irradiation. CONCLUSION The risk of radiation-associated solid tumor development after BMT is likely to increase with longer follow-up. This underscores the importance of close monitoring of patients who undergo BMT.

Bortezomib in patients with relapsed or refractory mantle cell lymphoma: updated time-to-event analyses of the multicenter phase 2 PINNACLE study

BACKGROUND We previously reported results of the phase 2, multicenter PINNACLE study, which confirmed the substantial single-agent activity of bortezomib in patients with relapsed or refractory mantle cell lymphoma (MCL). MATERIALS AND METHODS We report updated time-to-event data, in all patients and by response to treatment, after extended follow-up (median 26.4 months). RESULTS Median time to progression (TTP) was 6.7 months. Median time to next therapy (TTNT) was 7.4 months. Median overall survival (OS) was 23.5 months. In responding patients, median TTP was 12.4 months, median duration of response (DOR) was 9.2 months, median TTNT was 14.3 months, and median OS was 35.4 months. Patients achieving complete response had heterogeneous disease characteristics; among these patients, median TTP and DOR were not reached, and median OS was 36.0 months. One-year survival rate was 69% overall and 91% in responding patients. Median OS from diagnosis was 61.1 months, after median follow-up of 63.7 months. Activity was seen in patients with refractory disease and patients relapsing following high-intensity treatment. Toxicity was generally manageable. CONCLUSIONS Single-agent bortezomib is associated with lengthy responses and notable survival in patients with relapsed or refractory MCL, with considerable TTP and TTNT in responding patients, suggesting substantial clinical benefit.

Daratumumab monotherapy in patients with treatment-refractory multiple myeloma (SIRIUS): an open-label, randomised, phase 2 trial.

BACKGROUND New treatment options are needed for patients with multiple myeloma that is refractory to proteasome inhibitors and immunomodulatory drugs. We assessed daratumumab, a novel CD38-targeted monoclonal antibody, in patients with refractory multiple myeloma. METHODS In this open-label, multicentre, phase 2 trial done in Canada, Spain, and the USA, patients (age ≥18 years) with multiple myeloma who were previously treated with at least three lines of therapy (including proteasome inhibitors and immunomodulatory drugs), or were refractory to both proteasome inhibitors and immunomodulatory drugs, were randomly allocated in a 1:1 ratio to receive intravenous daratumumab 8 mg/kg or 16 mg/kg in part 1 stage 1 of the study, to decide the dose for further assessment in part 2. Patients received 8 mg/kg every 4 weeks, or 16 mg/kg per week for 8 weeks (cycles 1 and 2), then every 2 weeks for 16 weeks (cycles 3-6), and then every 4 weeks thereafter (cycle 7 and higher). The allocation schedule was computer-generated and randomisation, with permuted blocks, was done centrally with an interactive web response system. In part 1 stage 2 and part 2, patients received 16 mg/kg dosed as in part 1 stage 1. The primary endpoint was overall response rate (partial response [PR] + very good PR + complete response [CR] + stringent CR). All patients who received at least one dose of daratumumab were included in the analysis. The trial is registered with ClinicalTrials.gov, number NCT01985126. FINDINGS The study is ongoing. In part 1 stage 1 of the study, 18 patients were randomly allocated to the 8 mg/kg group and 16 to the 16 mg/kg group. Findings are reported for the 106 patients who received daratumumab 16 mg/kg in parts 1 and 2. Patients received a median of five previous lines of therapy (range 2-14). 85 (80%) patients had previously received autologous stem cell transplantation, 101 (95%) were refractory to the most recent proteasome inhibitors and immunomodulatory drugs used, and 103 (97%) were refractory to the last line of therapy. Overall responses were noted in 31 patients (29.2%, 95% CI 20.8-38.9)-three (2.8%, 0.6-8.0) had a stringent CR, ten (9.4%, 4.6-16.7) had a very good PR, and 18 (17.0%, 10.4-25.5) had a PR. The median time to first response was 1.0 month (range 0.9-5.6). Median duration of response was 7.4 months (95% CI 5.5-not estimable) and progression-free survival was 3.7 months (95% CI 2.8-4.6). The 12-month overall survival was 64.8% (95% CI 51.2-75.5) and, at a subsequent cutoff, median overall survival was 17.5 months (95% CI 13.7-not estimable). Daratumumab was well tolerated; fatigue (42 [40%] patients) and anaemia (35 [33%]) of any grade were the most common adverse events. No drug-related adverse events led to treatment discontinuation. INTERPRETATION Daratumumab monotherapy showed encouraging efficacy in heavily pretreated and refractory patients with multiple myeloma, with a favourable safety profile in this population of patients. FUNDING Janssen Research & Development.

Hepatic Veno-Occlusive Disease following Stem Cell Transplantation: Incidence, Clinical Course, and Outcome

The occurrence of hepatic veno-occlusive disease (VOD) has been reported in up to 60% of patients following stem cell transplantation (SCT), with incidence varying widely between studies depending on the type of transplant, conditioning regimen, and criteria used to make the diagnosis. Severe VOD is characterized by high mortality and progression to multiorgan failure (MOF); however, there is no consensus on how to evaluate severity. This review and analysis of published reports attempts to clarify these issues by calculating the overall mean incidence of VOD and mortality from severe VOD, examining the effect of changes in SCT practice on the incidence of VOD over time, and discussing the methods used to evaluate severity. Across 135 studies performed between 1979 and October 2007, the overall mean incidence of VOD was 13.7% (95% confidence interval [CI]=13.3%-14.1%). The mean incidence of VOD was significantly lower between 1979-1994 than between 1994-2007 (11.5% [95% CI, 10.9%-12.1%] vs 14.6% [95% CI, 14.0%-15.2%]; P <.05). The mortality rate from severe VOD was 84.3% (95% CI, 79.6%-88.9%); most of these patients had MOF, which also was the most frequent cause of death. Thus, VOD is less common than early reports suggested, but the current incidence appears to be relatively stable despite recent advances in SCT, including the advent of reduced-intensity conditioning. The evolution of MOF in the setting of VOD after SCT can be considered a reliable indication of severity and a predictor of poor outcome.

Autologous haemopoietic stem-cell transplantation followed by allogeneic or autologous haemopoietic stem-cell transplantation in patients with multiple myeloma (BMT CTN 0102): a phase 3 biological assignment trial

BACKGROUND Autologous haemopoietic stem-cell transplantation (HSCT) improves survival in patients with multiple myeloma, but disease progression remains an issue. Allogeneic HSCT might reduce disease progression, but can be associated with high treatment-related mortality. Thus, we aimed to assess effectiveness of allogeneic HSCT with non-myeloablative conditioning after autologous HSCT compared with tandem autologous HSCT. METHODS In our phase 3 biological assignment trial, we enrolled patients with multiple myeloma attending 37 transplant centres in the USA. Patients (<70 years old) with adequate organ function who had completed at least three cycles of systemic antimyeloma therapy within the past 10 months were eligible for inclusion. We assigned patients to receive an autologous HSCT followed by an allogeneic HSCT (auto-allo group) or tandem autologous HSCTs (auto-auto group) on the basis of the availability of an HLA-matched sibling donor. Patients in the auto-auto group subsequently underwent a random allocation (1:1) to maintenance therapy (thalidomide plus dexamethasone) or observation. To avoid enrolment bias, we classified patients as standard risk or high risk on the basis of cytogenetics and β2-microglobulin concentrations. We used the Kaplan-Meier method to estimate differences in 3-year progression-free survival (PFS; primary endpoint) between patients with standard-risk disease in the auto-allo group and the best results from the auto-auto group (maintenance, observation, or pooled). This study is registered with ClinicalTrials.gov, number NCT00075829. FINDINGS Between Dec 17, 2003, and March 30, 2007, we enrolled 710 patients, of whom 625 had standard-risk disease and received an autologous HSCT. 156 (83%) of 189 patients with standard-risk disease in the auto-allo group and 366 (84%) of 436 in the auto-auto group received a second transplant. 219 patients in the auto-auto group were randomly assigned to observation and 217 to receive maintenance treatment, of whom 168 (77%) completed this treatment. PFS and overall survival did not differ between maintenance and observation groups and pooled data were used. Kaplan-Meier estimates of 3-year PFS were 43% (95% CI 36-51) in the auto-allo group and 46% (42-51) in the auto-auto group (p=0·671); overall survival also did not differ at 3 years (77% [95% CI 72-84] vs 80% [77-84]; p=0·191). Within 3 years, 87 (46%) of 189 patients in the auto-allo group had grade 3-5 adverse events as did 185 (42%) of 436 patients in the auto-auto group. The adverse events that differed most between groups were hyperbilirubinaemia (21 [11%] patients in the auto-allo group vs 14 [3%] in the auto-auto group) and peripheral neuropathy (11 [6%] in the auto-allo group vs 52 [12%] in the auto-auto group). INTERPRETATION Non-myeloablative allogeneic HSCT after autologous HSCT is not more effective than tandem autologous HSCT for patients with standard-risk multiple myeloma. Further enhancement of the graft versus myeloma effect and reduction in transplant-related mortality are needed to improve the allogeneic HSCT approach. FUNDING US National Heart, Lung, and Blood Institute and the National Cancer Institute.

Defibrotide for the treatment of severe hepatic veno-occlusive disease and multiorgan failure after stem cell transplantation: a multicenter, randomized, dose-finding trial.

Therapeutic options for severe hepatic veno-occlusive disease (VOD) are limited and outcomes are dismal, but early phase I/II studies have suggested promising activity and acceptable toxicity using the novel polydisperse oligonucleotide defibrotide. This randomized phase II dose-finding trial determined the efficacy of defibrotide in patients with severe VOD following hematopoietic stem cell transplantation (HSCT) and identified an appropriate dose for future trials. Adult and pediatric patients received either lower-dose (arm A: 25 mg/kg/day; n = 75) or higher-dose (arm B: 40 mg/kg/day; n = 74) i.v. defibrotide administered in divided doses every 6 hours for > or =14 days or until complete response, VOD progression, or any unacceptable toxicity occurred. Overall complete response and day +100 post-HSCT survival rates were 46% and 42%, respectively, with no significant difference between treatment arms. The incidence of treatment-related adverse events was low (8% overall; 7% in arm A, 10% in arm B); there was no significant difference in the overall rate of adverse events between treatment arms. Early stabilization or decreased bilirubin was associated with better response and day +100 survival, and decreased plasminogen activator inhibitor type 1 (PAI-1) during treatment was associated with better outcome; changes were similar in both treatment arms. Defibrotide 25 or 40 mg/kg/day also appears effective in treating severe VOD following HSCT. In the absence of any differences in activity, toxicity or changes in PAI-1 level, defibrotide 25 mg/kg/day was selected for ongoing phase III trials in VOD.