Kim H. Orchard

Somatic CALR Mutations in Myeloproliferative Neoplasms with Nonmutated JAK2

BACKGROUND Somatic mutations in the Janus kinase 2 gene (JAK2) occur in many myeloproliferative neoplasms, but the molecular pathogenesis of myeloproliferative neoplasms with nonmutated JAK2 is obscure, and the diagnosis of these neoplasms remains a challenge. METHODS We performed exome sequencing of samples obtained from 151 patients with myeloproliferative neoplasms. The mutation status of the gene encoding calreticulin (CALR) was assessed in an additional 1345 hematologic cancers, 1517 other cancers, and 550 controls. We established phylogenetic trees using hematopoietic colonies. We assessed calreticulin subcellular localization using immunofluorescence and flow cytometry. RESULTS Exome sequencing identified 1498 mutations in 151 patients, with medians of 6.5, 6.5, and 13.0 mutations per patient in samples of polycythemia vera, essential thrombocythemia, and myelofibrosis, respectively. Somatic CALR mutations were found in 70 to 84% of samples of myeloproliferative neoplasms with nonmutated JAK2, in 8% of myelodysplasia samples, in occasional samples of other myeloid cancers, and in none of the other cancers. A total of 148 CALR mutations were identified with 19 distinct variants. Mutations were located in exon 9 and generated a +1 base-pair frameshift, which would result in a mutant protein with a novel C-terminal. Mutant calreticulin was observed in the endoplasmic reticulum without increased cell-surface or Golgi accumulation. Patients with myeloproliferative neoplasms carrying CALR mutations presented with higher platelet counts and lower hemoglobin levels than patients with mutated JAK2. Mutation of CALR was detected in hematopoietic stem and progenitor cells. Clonal analyses showed CALR mutations in the earliest phylogenetic node, a finding consistent with its role as an initiating mutation in some patients. CONCLUSIONS Somatic mutations in the endoplasmic reticulum chaperone CALR were found in a majority of patients with myeloproliferative neoplasms with nonmutated JAK2. (Funded by the Kay Kendall Leukaemia Fund and others.).

Deletion of chromosome 13 detected by conventional cytogenetics is a critical prognostic factor in myeloma

In myeloma, the prognostic impact of different strategies used to detect chromosome 13 deletion (Δ13) remains controversial. To address this, we compared conventional cytogenetics and interphase fluorescence in situ hybridization (iFISH) in a large multicenter study (n=794). The ability to obtain abnormal metaphases was associated with a poor prognosis, which was worse if Δ13, p53 deletion or t(4;14) was present, but only Δ13 remained significant on multivariate analysis. Patients with Δ13, by either cytogenetics or iFISH, had a poor prognosis. However, when cases with Δ13 detectable by both cytogenetics and iFISH were separated from those detected by iFISH only, the poor prognosis of iFISH-detectable Δ13 disappeared; their outcome matched that of patients with no detectable Δ13 (P=0.115). Addition of ploidy status to iFISH-Δ13 did not affect the prognostic value of the test. Indeed both cytogenetics and iFISH Δ13 divided both hyperdiploidy and nonhyperdiploidy into two groups with similar prognoses, indicating that the poor prognosis of ploidy is entirely due to its association with Δ13. We conclude that Δ13 detected by metaphase analysis is a critical prognostic factor in myeloma. Absence of Δ13, even in those patients yielding only normal or no metaphases, is associated with a relatively good prognosis.

Allogeneic hematopoietic SCT as treatment option for patients with mitochondrial neurogastrointestinal encephalomyopathy (MNGIE): a consensus conference proposal for a standardized approach.

Allogeneic hematopoietic SCT (HSCT) has been proposed as a treatment for patients with mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). HSCT has been performed in nine patients using different protocols with varying success. Based on this preliminary experience, participants of the first consensus conference propose a common approach to allogeneic HSCT in MNGIE. Standardization of the transplant protocol and the clinical and biochemical assessments will allow evaluation of the safety and efficacy of HSCT as well as optimization of therapy for patients with MNGIE.

Outcome following Reduced-Intensity Allogeneic Stem Cell Transplantation (RIC AlloSCT) for relapsed and refractory mantle cell lymphoma (MCL): a study of the British Society for Blood and Marrow Transplantation.

Reduced-intensity allogeneic stem cell transplantation (RIC-AlloSCT) is being increasingly considered for patients with aggressive lymphoma, but limited evidence exists in mantle cell lymphoma (MCL). We report a retrospective study of transplant outcomes of RIC-AlloSCT for MCL in 70 patients (median age, 48 years, range: 30-67 years), with 57 patients receiving an Alemtuzumab-containing regimen. Thirty-four percent of patients had received a prior autologous stem cell transplant. The 1- and 5-year nonrelapse mortality (NRM) was 18% (95% confidence interval [CI] 10-27) and 21% (95% CI 12-31), respectively. The incidence of severe (grade III and IV) acute graft-versus-host disease (aGVHD) was 10%, and the 5-year incidence of chronic GVHD (cGVHD) was 61%. The cumulative relapse risk was 65% (95% CI 48-77) at 5 years, significantly affected by disease status at transplant (P = .0495), specifically the presence of chemosensitive disease (P = .0364). Fifteen of 18 relapsed patients received donor lymphocyte infustion (DLI) (n = 14) or a second RIC-AlloSCT (n = 1), with 11 of 15 currently in CR. The 5-year overall survival (OS) and progression-free survival (PFS) were 37% (95% CI 25%-56%) and 14% (95% CI 6%-34%), respectively. Age at transplantation and having <2 prior lines of therapy influenced the OS, whereas having <2 prior lines of therapy was the only factor to influence PFS. The use of Alemtuzumab in the conditioning was associated with an improved OS at 3 years (P = .0271). RIC-AlloSCT is a potential treatment modality for aggressive MCL. For patients relapsing post-AlloSCT, the disease is salvageable with DLI. The timing of RIC-AlloSCT should be explored in prospective studies to establish the optimal role in the management of this aggressive lymphoma.

Timing of acquisition of deletion 13 in plasma cell dyscrasias is dependent on genetic context

Monoclonal gammopathy of undetermined significance (MGUS) and smoldering myeloma (SMM) are characterized by an expansion of monoclonal plasma cells and can progress to symptomatic multiple myeloma. This study assessed the incidence and the association of monosomy 13 with IgH translocations, ploidy status and deletions of 16q23 and TP53 in a large series of MGUS and SMM patients. Background Multiple myeloma, monoclonal gammopathy of undetermined significance and smoldering multiple myeloma harbor common chromosomal abnormalities but the prevalence and relative association of aberrations in these diagnostic groups remains controversial. We investigated these aspects in a large series of patients. Design and Methods Chromosome 13 deletion (Δ13), deletion of TP53, ploidy status and immunoglobulin heavy chain (IgH) translocations were evaluated by fluorescence in situ hybridization in patients with monoclonal gammopathy of undetermined significance (n=189), smoldering multiple myeloma (n=127) and multiple myeloma (n=400). Results Overall, Δ13 (25%, 34% and 47%), 16q23 deletions (6%, 8% and 21%) and 17p13 deletions (3%, 1% and 10%) were less frequent in patients with monoclonal gammopathy of undetermined significance and smoldering multiple myeloma than in those with multiple myeloma. When distinct genetic groups were considered, no differences in the prevalence of Δ13 were found with t(4;14)(p16;q32) and t(14;16)(q32;q23) among the three diagnostic groups; in contrast Δ13 was rarer in t(11;14)(q13;q32) in patients with monoclonal gammopathy (1/28) and smoldering myeloma (2/13) than in those with multiple myeloma (40%). Similar results were seen for the few t(6;14)(p21;q32) cases: 0/3 patients with monoclonal gammopathy or smoldering myeloma had the Δ13, whereas 4/6 (67%) patients with multiple myeloma and this translocation also had the deletion. In multiple myeloma patients with both an IgH translocation and Δ13, the proportions of cells affected by the two abnormalities were similar, as was the case for t(4;14) and t(14;16) monoclonal gammopathy patients positive for Δ13. In contrast, in monoclonal gammopathy patients with t(14;20)(q32;q11), the translocation was present in almost all cells, while the Δ13 was present in only a sub-population. Conclusions These results indicate that the presence and time of occurrence of Δ13 depends on the presence of specific concurrent abnormalities. The observation that Δ13 was extremely rare in monoclonal gammopathy of undetermined significance and smoldering multiple myeloma with translocations directly involving cyclin D genes (CCND1 and CCND3) suggest a possible role of Δ13 in the progression of the disease specifically in these genetic sub-groups. (clinicaltrials.gov identifier: ISRCTN 68454111; UKCRN ID 1176).

Selective Apoptotic Killing of Malignant Hemopoietic Cells by Antibody-Targeted Delivery of an Amphipathic Peptide

The alpha-helical amphipathic peptide D-(KLAKLAK)2 is toxic to eukaryotic cells if internalized by a suitable targeting mechanism. We have targeted this peptide to malignant hemopoietic cells via conjugation to monoclonal antibodies, which recognize lineage-specific cell surface molecules. An anti-CD19/peptide conjugate efficiently killed 3/3 B lymphoid lines. However, an anti-CD33/peptide conjugate was cytotoxic to only one of three CD33-positive myeloid leukemia lines. The IC50 towards susceptible lines were in the low nanomolar range. Conjugates were highly selective and did not kill cells that did not express the appropriate cell surface cognate of the antibody moiety. Anti-CD19/peptide conjugates efficiently killed cells from patients with chronic lymphocytic leukemia but anti-CD33/peptide reagents were less effective against fresh acute myeloid leukemia cells. We therefore suggest that amphipathic peptides may be of value as targeted therapeutic agents for the treatment of a subset of hematologic malignancies.

Translocations of 14q32 and deletions of 13q14 are common chromosomal abnormalities in systemic amyloidosis.

Summary.  Systemic monoclonal immunoglobulin light chain amyloidosis (AL) is associated with clonal plasma cell dyscrasias that are often subtle and non‐proliferating. AL shares numerical chromosomal changes with multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS). Illegitimate translocations involving the immunoglobulin heavy chain gene (IGH) at 14q32 and deletions of the long arm of chromosome 13, [del(13q)], commonly occur in MM, MGUS and plasma cell leukaemia. In AL IGH rearrangements have been identified but, to date, there are no reports of del(13q). In this study of 32 patients with AL, 24 with systemic and eight with localized disease, translocations involving IGH and del(13q) were found using dual‐colour interphase fluorescence in situ hybridization (FISH). IGH translocations were observed in 11 patients (37% overall and in 46% with systemic disease), of which nine had the IGH/CCND1 fusion from t(11;14)(q13;q32). Two showed IGH translocations other than the t(11;14) or t(4;14)(p16;q32). In one of these patients a breakpoint within the constant region of IGH between Cα1 and Cα2 was indicated. In the second a deletion covering Cα1 and Cα2 accompanied the translocation. Ten patients (27% overall and 33% of those with systemic disease) showed del(13q). The gain or loss of IGH and CCND1 signals provided evidence of numerical chromosomal changes in three patients.

Fluoro-deoxyglucose positron emission tomography imaging for the detection of occult disease in multiple myeloma

Summary. Positron emission tomography with 2‐deoxy‐2‐[18]fluoro‐d‐glucose (FDG‐PET) imaging has been extensively used to detect occult metastatic malignant lesions in patients with carcinoma. We describe its use in three patients with multiple myeloma, each representing a particular clinical situation in which this imaging modality offered advantages over plain radiography, computerized tomography or magnetic resonance imaging. FDG‐PET provides a whole body image showing sites of occult disease. This is of particular value in patients with non‐secretory myeloma, solitary plasmacytoma or for those that relapse with focal disease following autologous or allogeneic stem cell transplantation.

Frequent upregulation of MYC in plasma cell leukemia.

Plasma cell leukemia (PCL) is a rare form of monoclonal gammopathy, which can originate de novo or evolve from multiple myeloma (MM) as a terminal leukemic phase. Previous cytogenetic studies of PCL have reported the presence of complex karyotypes with involvement of multiple unidentified chromosomal regions. We report here the analysis of 12 PCL (10 primary and two secondary) by metaphase and FISH analysis combined with oligonucleotide array data (244 k, Agilent). Interphase‐FISH results were compared with those from a series of 861 newly diagnosed patients with MM. Cytogenetic analysis was successful on 11 patients, all of whom showed clonal chromosomal abnormalities. Compared with MM, t(11;14)(q13;q32) (42% versus 15%; P = 0.027) and t(14;16)(q32;q23) (25% versus 4%; P = 0.010) were more frequent in PCL, although neither the specific partner chromosome involved in the IgH translocation nor the ploidy status predicted for survival. Chromosomes 1, 8, 13, and 16 showed the highest number of copy number alterations with 8q24 being the chromosomal region most frequently involved. In eight of 12 patients we found abnormalities (translocations, one amplification, small deletions, and duplications) that directly targeted or were very close to MYC. Only four of these changes were detected by routine FISH analysis using commercial probes with the others exclusively detected by arrays. Quantitative reverse transcription polymerase chain reaction demonstrated that these different abnormalities were associated with increased levels of MYC mRNA. We conclude that MYC dysregulation by complex mechanisms is one of the major molecular events in the oncogenesis of PCL.

Loss of 1p and rearrangement of MYC are associated with progression of smouldering myeloma to myeloma: sequential analysis of a single case

This case report suggests that loss of 1p and rearrangement of MYC are associated with progression of smoldering myeloma to to multiple myeloma. We report serial genetic studies on a young female patient initially diagnosed with asymptomatic smouldering myeloma who progressed to symptomatic myeloma 4.5 years after presentation. An unbalanced translocation, der(14)t(4;14)(p16;q32), was initially found in all plasma cells plus deletions of other chromosomal regions as detected by array-based comparative genomic hybridization. Deletion of chromosome 13 was observed in a minor population of plasma cells (<20%) for the first two years, increasing to 100% of plasma cells by the time of multiple myeloma diagnosis. Loss of 1p and a rearrangement of MYC were first observed in a small population of plasma cells one year prior to the clinical diagnosis of multiple myeloma, but these subclones increased rapidly in size to become the major population suggesting that they were directly involved in the transformation process. This case report provides a unique insight into the mechanisms of disease progression from smouldering multiple myeloma to multiple myeloma.