Ketterling, Rhett P.

AbstractWe report the results of a prospective, randomized phase 3 trial evaluating the use of gemtuzumab ozogamicin (GO) in an intensive consolidation approach in 657 patients 17-60 years of age. Patients in first complete remission (CR1) after cytarabine and standard- or high-dose daunorubicin induction received 2 cycles of consolidation with high-dose cytarabine followed by peripheral blood progenitor cell collection. The 352 patients who entered consolidation were randomized to receive GO (n = 132) or not (n = 138) and then proceeded to autologous hematopoietic cell transplantation (HCT). GO was given to 67 patients. Median follow-up was 50.9 months. Results of the intention-to-treat analysis demonstrated a 4-year disease-free survival (DFS) of 33.6% versus 35.9% (P = .54) and an overall survival (OS) of 41.3% versus 41.9% (P = .52) for those randomized to receive GO versus no GO, respectively. Patients with favorable- and intermediate-risk acute myeloid leukemia (AML) treated with high-dose daunorubicin and autologous HCT had 4-year DFS rates of 60% and 40% and OS rates of 80% and 49.3%, respectively. For younger AML patients in CR1, autologous HCT should be considered in favorable- and intermediate-cytogenetic risk patients who do not have an allogeneic donor. The addition of a single dose of GO in this setting did not improve outcomes. This trial is registered at http://www.clinicaltrials.gov as NCT00049517.

Purpose Findings from the human epidermal growth factor receptor 2 (HER2) –positive National Surgical Adjuvant Breast and Bowel Project (NSABP) B31 trial suggested that MYC/HER2 coamplification (> 5.0 copies/nucleus) was associated with additional benefit from adjuvant trastuzumab in patients with early-stage breast cancer. To further explore this relationship, we investigated associations between MYC amplification and disease-free survival (DFS) in a similar adjuvant trastuzumab HER2-positive breast cancer trial—North Central Cancer Treatment Group (NCCTG) N9831. Patients and Methods This analysis included 799 patients randomly assigned to receive chemotherapy alone or with concurrent trastuzumab on N9831. Fluorescence in situ hybridization (FISH) was performed by using a dual-probe mixture for MYC and centromere 8 (MYC:CEP8) on tissue microarrays. MYC amplification was prespecified as MYC:CEP8 ratio > 2.2 or average MYC copies/nucleus > 5.0. Exploratory variables included polysomy 8. Results In comparing DFS (median follow-up, 4.0 years) between treatments, patients with MYC:CEP8 ratio ≤ 2.2 (n = 618; 77%) and > 2.2 (n = 181; 23%) had hazard ratios (HRs) of 0.46 (P < .001) and 0.67 (P = .33), respectively (interaction P = .38). Patients with MYC copies/nucleus ≤ 5.0 (n = 534; 67%) and > 5.0 (n = 265; 33%) had HRs of 0.52 (P = .002) and 0.48 (P = .02), respectively (interaction P = .94). Patients with MYC:CEP8 ratio < 1.3 with normal chromosome 8 copy number (n = 141; 18%) and ≥ 1.3 or < 1.3 with polysomy 8 (n = 658; 82%) had HRs of 0.66 (P = .28) and 0.44 (P < .001), respectively (interaction P = .23). Patients with MYC copies/nucleus < 2.5 (n = 130; 16%) and ≥ 2.5 (n = 669; 84%) had HRs of 1.07 (P = .87) and 0.42 (P < .001), respectively (interaction P = .05). Conclusion We did not confirm the B31 association between MYC amplification and additional trastuzumab benefit. Exploratory analyses revealed potential associations between alternative MYC/chromosome 8 copy number alterations and differential benefit of adjuvant trastuzumab.

Abstract Zosuquidar, which modulates P-glycoprotein (P-gp) with minimal delay of anthracycline clearance, may reverse P-gp–mediated resistance in acute myeloid leukemia without increased toxicity. A total of 449 adults older than 60 years with acute myeloid leukemia or high-risk myelodysplastic syndrome enrolled in a randomized placebo-controlled double-blind trial (Eastern Cooperative Oncology Group 3999). Overall survival was compared between patients receiving conventional-dose cytarabine and daunorubicin and either zosuquidar (550 mg; 212 patients) or placebo (221 patients). Median and 2-year overall survival values were 7.2 months and 20% on zosuquidar and 9.4 months and 23% on placebo, respectively (P = .281). Remission rate was 51.9% on zosuquidar and 48.9% on placebo. All cause mortality to day 42 was not different (zosuquidar 22.2% vs placebo 16.3%; P = .158). In vitro modulation of P-gp activity by zosuquidar and expression of P-gp, multidrug resistance-related protein 1, lung resistance protein, and breast cancer resistance protein, were comparable in the 2 arms. Poor-risk cytogenetics were more common in P-gp+ patients. P-gp expression and cytogenetics were correlated, though independent prognostic factors. We conclude that zosuquidar did not improve outcome in older acute myeloid leukemia, in part, because of the presence P-gp independent mechanisms of resistance. This trial is registered at www.clinicaltrials.gov as #NCT00046930.

Purpose We examined associations between tumor characteristics (human epidermal growth factor receptor 2 [HER2] protein expression, HER2 gene and chromosome 17 copy number, hormone receptor status) and disease-free survival (DFS) of patients in the N9831 adjuvant trastuzumab trial. Patients and Methods All patients (N = 1,888) underwent chemotherapy with doxorubicin and cyclophosphamide, followed by weekly paclitaxel with or without concurrent trastuzumab. HER2 status was determined by immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH) at a central laboratory, Mayo Clinic, Rochester, MN. Patients with conflicting local positive HER2 expression results but normal central laboratory testing were included in the analyses (n = 103). Results Patients with HER2-positive tumors (IHC 3+, FISH HER2/centromere 17 ratio ≥ 2.0, or both) benefited from trastuzumab, with hazard ratios (HRs) of 0.46, 0.49, and 0.45, respectively (all P < .0001). Patients with HER2-amplified tumors with polysomic (p17) or normal (n17) chromosome 17 copy number also benefited from trastuzumab, with HRs of 0.52 and 0.37, respectively (P < .006). Patients who received chemotherapy alone and had HER2-amplified and p17 tumors had a longer DFS than those who had n17 (78% v 68%; P = .04), irrespective of hormone receptor status or tumor grade. Patients with HER2-normal tumors by central testing (n = 103) seemed to benefit from trastuzumab, but the difference was not statistically significant (HR, 0.51; P = .14). Patients with hormone receptor–positive or –negative tumors benefited from the addition of trastuzumab, with HRs of 0.42 (P = .005) and 0.60 (P = .0001), respectively. Conclusion These results confirm that IHC or FISH HER2 testing is appropriate for patient selection for adjuvant trastuzumab therapy. Trastuzumab benefit seemed independent of HER2/centromere 17 ratio and chromosome 17 copy number.

Abstract Pathologic examination of products of conception (POC) is used to differentiate hydropic abortus (HA), partial hydatidiform mole (PM), and complete hydatidiform mole (CM). Histologic classification of POC specimens can be difficult, and ancillary testing is often required for a definitive diagnosis. This study evaluated 66 POC specimens by flow cytometry, digital image analysis, p57 immunohistochemical analysis, and fluorescence in situ hybridization (FISH). The final diagnosis, based on the combined analysis of all test results, included 33 HAs, 24 PMs, and 9 CMs. The p57 immunostain identified 9 CMs that were evaluated as nontriploid by all other techniques. FISH seems to have the best accuracy (100%) for determining whether a specimen contains a triploid chromosome complement. These data suggest that the combination of p57 and FISH seems to be the best ancillary testing strategy to aid pathologists in the appropriate identification of CM, PM, and HA in POC specimens.

AbstractThe biology and outcome of adult T-cell acute lymphoblastic leukemia are poorly understood. We present here the clinical and biologic features of 356 patients treated uniformly on the prospective trial (UKALL XII/ECOG 2993) with the aim of describing the outcome and identifying prognostic factors. Complete remission was obtained in 94% of patients, and 48% survived 5 years. Positivity of blasts for CD1a and lack of expression of CD13 were associated with better survival (P = .01 and < .001, respectively). NOTCH1 and CDKN2A mutations were seen in 61% and 42% of those tested. Complex cytogenetic abnormalities were associated with poorer survival (19% vs 51% at 5 years, P = .006). Central nervous system involvement at diagnosis did not affect survival (47% vs 48%, P = not significant). For 99 patients randomized between autograft and chemotherapy, 5-year survival was 51% in each arm. Patients with a matched sibling donor had superior 5-year survival to those without donors (61% vs 46%, χ2, P = .02); this was the result of less relapse (25% vs 51% at 5 years, P < .001). Only 8 of 123 relapsed patients survive. This study provides a baseline for trials of new drugs, such as nelarabine, and may allow risk-adapted therapy in patients with poor-prognosis T-cell ALL.

Abstract IER3 (formerly IEX-1) encodes a 27-kDa glycoprotein that regulates death receptor–induced apoptosis, interacts with NF-κB pathways, and increases expression rapidly in response to cellular stresses such as irradiation. Animal models, gene expression microarray experiments, and functional studies in cell lines have suggested a potential role for IER3 in oncogenesis, but, to date, no abnormalities of IER3 at the DNA level have been reported in patients with neoplasia. Here, we describe breakpoint cloning of a t(6;9)(p21;q34) translocation from a patient with a myelodysplastic syndrome (MDS), facilitated by conversion technology and array-based comparative genomic hybridization, which revealed a rearrangement translocating the IER3 coding region away from critical flanking/regulatory elements and to a transcript-poor chromosomal region, markedly decreasing expression. Using split-signal and locus-specific fluorescence in situ hybridization (FISH) probes, we analyzed 204 patients with diverse hematological malignancies accompanied by clonal chromosome 6p21 abnormalities, and found 8 additional patients with MDS with IER3 rearrangements (translocations or amplification). Although FISH studies on 157 additional samples from patients with MDS and a normal-karyotype were unrevealing, and sequencing the IER3 coding and proximal promoter regions of 74 MDS patients disclosed no point mutations, reverse transcription-PCR results suggested that dysregulated expression of IER3 is common in MDS (61% &gt;4-fold increase or decrease in expression with decreased expression primarily in early MDS and increased expression primarily in later MDS progressing toward leukemia), consistent with findings in previous microarray experiments. These data support involvement of IER3 in the pathobiology of MDS. [Cancer Res 2009;69(19):7518–23]

Abstract The t(2;14)(p16;q32) has been reported previously in only 12 cases of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL). The clinicopathologic features have been incompletely described. We describe 6 new cases of CLL/SLL with t(2;14)(p16;q32). All had marrow involvement, 4 had absolute lymphocytosis, 4 had lymphadenopathy, and 3 had hepatosplenomegaly. All showed atypical lymphocyte morphologic features with plasmacytoid differentiation and irregular nuclei; 3 had increased prolymphocytes. Flow cytometry demonstrated typical immunophenotypes in 5 and an atypical immunophenotype in 1. All expressed ZAP70; 5 assessed showed unmutated IgVH genes. Karyotyping identified t(2;14)(p16;q32) as the sole abnormality in 1, primary abnormality in 2, and part of a complex karyotype in 3. Fluorescence in situ hybridization analysis revealed BCL11A/IgH rearrangement in all. After chemotherapy, 3 patients died of disease and 3 were alive with disease (median follow-up, 80 months). We conclude that CLL/SLL with t(2;14) (p16;q32) and BCL11A/IgH rearrangement is characterized by atypical morphologic features and unmutated IgVH genes.

AbstractPrimary systemic amyloidosis (AL) is a rare monoclonal plasma cell (PC) disorder characterized by the deposition of misfolded immunoglobulin (Ig) light chains (LC) in vital organs throughout the body. To our knowledge, no cell lines have ever been established from AL patients. Here we describe the establishment of the ALMC-1 and ALMC-2 cell lines from an AL patient. Both cell lines exhibit a PC phenotype and display cytokine-dependent growth. Using a comprehensive genetic approach, we established the genetic relationship between the cell lines and the primary patient cells, and we were also able to identify new genetic changes accompanying tumor progression that may explain the natural history of this patient's disease. Importantly, we demonstrate that free lambda LC secreted by both cell lines contained a beta structure and formed amyloid fibrils. Despite absolute Ig LC variable gene sequence identity, the proteins show differences in amyloid formation kinetics that are abolished by the presence of Na2SO4. The formation of amyloid fibrils from these naturally secreting human LC cell lines is unprecedented. Moreover, these cell lines will provide an invaluable tool to better understand AL, from the combined perspectives of amyloidogenic protein structure and amyloid formation, genetics, and cell biology.

Abstract JAK2V617F and MPLW515L/K are myeloproliferative disorder (MPD)-associated mutations. We genotyped 552 individual hematopoietic colonies obtained by CD34+ cell culture from 16 affected patients (13 JAK2V617F and 3 MPLW515L/K) to determine (a) the proportion of colonies harboring a particular mutation in the presence or absence of cytokines, (b) the lineage distribution of endogenous colonies for each mutation, and (c) the differences (if any) in the pattern of mutation among the various MPDs, as established by genotyping of individual colonies. Genotyping analysis revealed cohabitation of mutation-negative and mutation-positive endogenous colonies in polycythemia vera as well as other MPDs. Culture of progenitor cells harboring MPLW515L/K yielded virtually no endogenous erythroid colonies in contrast to JAK2V617F-harboring progenitor cells. The mutation pattern (i.e., relative distribution of homozygous, heterozygous, or wild-type colonies) was not a distinguishing feature among the MPDs, and MPLW515 mutations were detected in B and/or T lymphocytes in all three patients tested. These observations suggest that clonal myelopoiesis antedates acquisition of JAK2V617F or MPLW515L/K mutations and that the latter is acquired in a lympho-myeloid progenitor cell. Disclosure of potential conflicts of interest is found at the end of this article.