ALK-positive large B-cell lymphomas with cryptic SEC31A-ALK and NPM1-ALK fusions.

We report 2 ALK-positive large B-cell lymphoma cases showing granular cytoplasmic and cytoplasmic/nuclear ALK immunostaining in which cryptic ALK rearrangements were identified by fluorescent in situ hybridization and molecular analysis. In the first case, the ALK-involving t(2;3)(p23;q27) masked the cryptic SEC31A-ALK fusion generated by an insertion of the 5' end of SEC31A (4q21) upstream of the 3' end of ALK. This rearrangement was associated with loss of the 5' end of ALK and duplication of SEC31A-ALK on der(20). In the second case with complex rearrangements of both chromosomes 2, a submicroscopic NPM1-ALK fusion created by insertion of the 3' end of ALK into the NPM1 locus was evidenced. Further studies of SEC31A-ALK showed that this variant fusion transforms IL3-dependent Ba/F3 cells to growth factor independence, and that the ALK inhibitor TAE-684 reduces cell proliferation and kinase activity of SEC31A-ALK and its downstream effectors ERK1/2, AKT, STAT3 and STAT5.

Large cleaved and immunoblastic lymphoma may represent two distinct clinicopathologic entities within the group of diffuse large B-cell lymphomas.

PURPOSE: The reliability of immunohistochemistry for subdividing diffuse large B-cell lymphomas (DLBCL) into germinal center B-cell-like (GCB) and non-GCB prognostic subgroups is debated. In this study we evaluated the prognostic significance of such subgrouping on a series of 153 DLBCL patients. Furthermore, we investigated whether both subgroups could comprise clinicopathologic entities recognized by their morphology and characterized by a distinct phenotype, specific genetic abnormalities, and clinical characteristics. PATIENTS AND METHODS: All samples from patients were reviewed and morphologically subdivided into large cleaved, immunoblastic, and not otherwise specified DLBCL. GCB and non-GCB immunohistochemical profiles were established. The presence of chromosomal translocations involving BCL2, BCL6, and MYC and/or rearrangements of these genes was investigated. RESULTS: Subdividing DLBCL with either a GCB or non-GCB immunophenotypic profile was not of prognostic significance. Nevertheless, CD10 expression was a predictor of favorable outcome, whereas high bcl-2 expression and BCL6 rearrangement were adverse predictors of disease-free survival. Interestingly, large cleaved DLBCL was clearly associated with a GCB immunophenotypic profile, CD10 expression, BCL2 rearrangement, age younger than 60 years, and low to low/intermediate International Prognostic Index risk, but was not of prognostic significance. In contrast, immunoblastic morphology was associated with a non-GCB profile and was a significant predictor of unfavorable DFS. CONCLUSION: Subdividing DLBCL into subgroups based on their immunohistochemical profile was not of prognostic significance. Nevertheless, it allowed the additional characterization of two lymphoma subgroups previously recognized in the Working Formulation. Both correspond to two distinct clinicopathologic entities within the DLBCL.

KIT mutations and dose selection for imatinib in patients with advanced gastrointestinal stromal tumours.

A recent randomized EORTC phase III trial, comparing two doses of imatinib in patients with advanced gastrointestinal stromal tumours (GISTs), reported dose dependency for progression-free survival. The current analysis of that study aimed to assess if tumour mutational status correlates with clinical response to imatinib. Pre-treatment samples of GISTs from 377 patients enrolled in phase III study were analyzed for mutations of KIT or PDGFRA by combination of D-HPLC and direct sequencing of tumour genomic DNA. Mutation types were correlated with patients' survival data. The presence of exon 9-activating mutations in KIT was the strongest adverse prognostic factor for response to imatinib, increasing the relative risk of progression by 171% (P<0.0001) and the relative risk of death by 190% (P<0.0001) when compared with KIT exon 11 mutants. Similarly, the relative risk of progression was increased by 108% (P<0.0001) and the relative risk of death by 76% (P=0.028) in patients without detectable KIT or PDGFRA mutations. In patients whose tumours expressed an exon 9 KIT oncoprotein, treatment with the high-dose regimen resulted in a significantly superior progression-free survival (P=0.0013), with a reduction of the relative risk of 61%. We conclude that tumour genotype is of major prognostic significance for progression-free survival and overall survival in patients treated with imatinib for advanced GISTs. Our findings suggest the need for differential treatment of patients with GISTs, with KIT exon 9 mutant patients benefiting the most from the 800 mg daily dose of the drug.

Trisomy 11 in myeloid malignancies is associated with internal tandem duplication of both MLL and FLT3 genes.

In 20 patients with myeloid malignancies and isolated trisomy 11 an internal tandem duplication of the MLL and FLT3 genes was observed in 41% and 31% of the cases, respectively; 80% of the FLT3+ cases showed MLL self-fusion. Concomitant presence of MLL and FLT3 anomalies could be relevant in determining the poor outcome of patients with acute myeloid leukemia with trisomy 11.

Heterogeneity of BCL6 rearrangements in nodular lymphocyte predominant Hodgkin's lymphoma.

BACKGROUND AND OBJECTIVES: Nodular lymphocyte-predominant Hodgkin's lymphoma (NLPHL) showed recurrent rearrangement of the BCL6 which is gene detected in 48% of cases analyzed by interphase-fluorescent in situ hybridization (FISH). These findings point to a critical role for BCL6 in the development of this distinct Hodgkin's lymphoma. We present our results of metaphase-FISH analyses aimed at identifying and characterizing BCL6-related chromosomal translocations in NLPHL. DESIGN AND METHODS: Four NLPHL cases with available metaphase spreads obtained either at the time of diagnosis or during progression to diffuse large B-cell lymphoma (DLBCL) were collected. Extensive metaphase-FISH analysis was performed to identify the affected partner chromosomes and reciprocal breakpoints. RESULTS: Each of the analyzed NLPHL cases showed a different type of BCL6 rearrangement that included the t(3;22)(q27;q11) targeting immunoglobulin (IG) alpha chain locus, complex t(3;7;3;1) involving the 7p12/Ikaros gene region, t(3;9)(q27;p13) affecting an unknown gene in vicinity of PAX5, and t(3;4)(q27;q32) showing the alternative 3q27 breakpoint outside BCL6 and possibly, an internal deletion of BCL6. Retrospective interphase-FISH analysis of 2 cases with subsequent DLBCL showed the same type of BCL6 translocation as in NLPHL samples. INTERPRETATION AND CONCLUSIONS: The spectrum of BCL6 aberrations targeting IG as well as non-IG loci in NLPHL is similar to that found in DLBCL. These findings further support the hypothesis of a germinal center B-cell-derived origin of NLPHL and of a relationship between these two lymphoma entities. This latter issue is additionally illustrated in two NLPHL patients who subsequently developed DLBCL and showed the same type of BCL6 rearrangements in both tumors.

Array CGH analysis in primary gastrointestinal stromal tumors: cytogenetic profile correlates with anatomic site and tumor aggressiveness, irrespective of mutational status.

Gastrointestinal stromal tumors (GISTs) comprise a biologically diverse group of neoplasms with respect to activating mutations in either KIT or PDGFRA, histology, anatomical site of origin, and clinical aggressiveness. In this study, we applied the high resolution array-based comparative genomic hybridization (array-CGH) technology to 66 primary GISTs (40 gastric and 26 nongastric, 48 with KIT and 18 with PDGFRA mutations) for identification of novel high-level alterations and for characterization of genotype-related genomic changes. All cases had genomic imbalances with the highest occurrence of 14q (73%), 1p (62%), 22q (59%), 15q (38%), and 13q (29%) losses. Our data indicate that loss of chromosome 14 and/or 22 is an early change in GIST tumorigenesis irrespective of tumor genotype. Furthermore, DNA copy number changes showed a site dependent pattern. These included lower incidence of losses at 14q (87% vs. 35%), and higher frequency of losses at 1p (45% vs. 85%) and 15q (17% vs. 69%) in nongastric versus gastric site (P<0.001 for all). However, in the multivariate analysis with adjustment to tumor risk stratification, only the 14q loss site-dependent pattern of distribution retained its significance. These findings suggest that loss of 14q is a relatively less frequent genetic event in the development of nongastric GISTs, the lack of which is most likely substituted by the accumulation of 1p/15q and other changes. The novel minimal overlapping regions of deletion at 1p (1p36.32-1p35.2, 1p34.1, and 1p22.1-1p21.3), 13q (13q14.11-q14.2 and 13q32.3-q33.1), and 15q23 were delineated, which point to chromosomal regions that may harbor genes relevant to the development of these neoplasms.

Mechanisms of resistance to imatinib mesylate in gastrointestinal stromal tumors and activity of the PKC412 inhibitor against imatinib-resistant mutants.

BACKGROUND AND AIMS: Resistance is a major challenge in the treatment of patients with gastrointestinal stromal tumors (GISTs). We investigated the mechanisms of resistance in patients with progressive GISTs with primary KIT mutations and the efficacy of the kinase inhibitor PKC412 for the inhibition of imatinib-resistant mutants. METHODS: We performed a cytogenetic analysis and screened for mutations of the KIT and PDGFRA kinase domains in 26 resistant GISTs. KIT autophosphorylation status was assessed by Western immunoblotting. Imatinib-resistant GIST cells and Ba/F3 cells expressing these mutant proteins were tested for sensitivity to imatinib and PKC412. RESULTS: Six distinct secondary mutations in KIT were detected in 12 progressive tumors, with V654A and T670I found to be recurrent. One progressive tumor showed acquired PDGFRA -D842V mutation. Amplification of KIT or KIT / PDGFRA was found in 2 patients. Eight of 10 progressive tumors available for analysis showed phosphorylated KIT. Two remaining progressive tumors lost KIT protein expression. GIST cells carrying KIT -del557-558/T670I or KIT -InsAY502-503/V654A mutations were resistant to imatinib, while PKC412 significantly inhibited autophosporylation of these mutants. Resistance to imatinib and sensitivity to PKC412 of KIT -T670I and PDGFRA -D842V mutants was confirmed using Ba/F3 cells. CONCLUSIONS: This study shows the high frequency of KIT/PDGFRA kinase domain mutations in patients with secondary resistance and defines genomic amplification of KIT / PDGFRA as an alternative cause of resistance to the drug. In a subset of patients, cancer cells lost their dependence on the targeted tyrosine kinase. Our findings show the sensitivity of the imatinib-resistant KIT -T670I and KIT -V654A and of PDGFRA -D842V mutants to PKC412.

Activity and safety of combined rituximab with chlorambucil in patients with mantle cell lymphoma.

We evaluated the combination of rituximab with chlorambucil in patients with mantle cell lymphoma (MCL) not eligible for aggressive therapy. Fourteen patients (male/female: 9/5) were included (two newly diagnosed, 12 relapsed/refractory). The toxicities were neutropenia, thrombopenia and infection. Nine (64%) patients responded; five (36%) achieved complete remission and four (29%) achieved partial remission. The median progression-free survival for responders was 26 months (95% CI, 4-48). Marrow polymerase chain reaction negativity was attained in seven responders. These results suggest that this schedule may have notable antitumour activity in patients with MCL, including patients in relapse after autologous stem cell transplantation.

Gastrointestinal stromal tumours (GISTs) negative for KIT (CD117 antigen) immunoreactivity.

Gastrointestinal stromal tumours (GISTs) are currently defined as mesenchymal tumours of the gastrointestinal tract that express KIT receptor tyrosine kinase. However, a small subgroup of tumours that fulfil the clinical and morphological criteria for GISTs lack KIT expression. So far, the biological features of these tumours have rarely been addressed. The present study describes seven gastrointestinal stromal neoplasms that presented clinicopathological features typical of GISTs but showed absence of CD117 expression as detected by immunohistochemistry. The tumours originated from the stomach (n = 5), duodenum (n = 1), and colon (n = 1), showing histologically either predominantly epithelioid (n = 3), mixed spindled and epithelioid (n = 2), or anaplastic/spindle cell (n = 2) type features. CD34 and alpha-smooth muscle actin (alpha-SMA) positivity was present in four and three tumours, respectively. Chromosomal analysis was performed in two cases, both showing losses of chromosomes 14, 22, and 1p, which is the characteristic feature of GISTs. Dual-colour interphase fluorescence in situ hybridization (FISH) analysis, utilizing chromosome 1p-, 14-, and 22-specific probes, revealed a similar cytogenetic profile in the remaining five tumour specimens. Mutational analysis of exons 9, 11, 13, and 17 of KIT, and exons 12 and 18 of PDGFRA was performed in all cases by denaturing high-pressure liquid chromatography (DHPLC) pre-screening, followed by direct sequencing. None of the tumours showed KIT mutant isoforms. Three tumours harboured PDGFRA exon 18 activating mutations; two were Asp --> Val(842) missense substitutions and one was a DIM842-844 amino acid deletion. KIT and PKC theta (protein activated in interstitial cells of Cajal and GISTs) expression was determined by western immunoblotting of the total cell lysates from three tumour biopsies. None of these three tumours expressed KIT, while all specimens showed expression of PKC theta protein. These findings indicate that there is a subgroup of KIT-negative GISTs that exhibit the same morphological, cytogenetic, and molecular features as KIT-positive tumours. While intragenic PDGFRA activating mutations are present in some of these tumours, the oncogenic events underlying the pathogenesis of the others remain unknown.

Differential expression of KIT/PDGFRA mutant isoforms in epithelioid and mixed variants of gastrointestinal stromal tumors depends predominantly on the tumor site.

Gastrointestinal stromal tumors (GISTs) form a distinctive group of mesenchymal neoplasms, showing differentiation towards the interstitial cells of Cajal. Morphologically, GISTs vary from cellular spindle cell tumors to epithelioid or mixed, epithelioid and spindle cell variants. The genotypic features underlying the morphologic differences of GISTs with vs without epithelioid components are not well defined. Acquisition of activating mutations in KIT and PDGFRA has been reported as alternative oncogenic events in the pathogenesis of GISTs. In this study, a comprehensive KIT and PDGFRA mutational analysis was performed in a group of 28 epithelioid/mixed type tumors, in order to explore whether a specific KIT/PDGFRA mutational status segregates these neoplasms from spindle cell variant GISTs. All GISTs were primary neoplasms, 16 (57.1%) originated from the stomach and 12 (42.8%) from other locations. Histomorphologically, 14 GISTs showed an epithelioid and 14 a mixed cell type pattern. Mutational analysis included KIT exons 9, 11, 13, and 17, and PDGFRA exons 12 and 18 prescreening by denaturing high-performance liquid chromatography, followed by direct sequencing. Activating mutations of KIT were found in 14 (50%) GISTs, the majority being within exon 11 (n=11; 39.2%), and the other comprised exon 9 AY 502-503 duplications (n=2; 7.2%) and exon 17 Lys --> Aln822 missense mutations (n=1; 3.6%). Most of the KIT mutant tumors (n=11; 78.6%) originated from nongastric sites. Seven (25.0%) GISTs with no detectable KIT mutations demonstrated PDGFRA mutant isoforms, carrying either D842 V mutations (n=5) or exon 18 deletions (n=2). All GISTs harboring PDGFRA mutant isoforms originated from the stomach. In seven tumors, no detectable mutations were found; all but one of nonmutant tumors initiated from the stomach and exhibited an epithelioid morphology. These findings indicate that the mutational status of epithelioid/mixed GISTs associates with the anatomical site of the tumor.

Two new translocations involving the 11q23 region map outside the MLL locus in myeloid leukemias.

BACKGROUND AND OBJECTIVES: In acute leukemias, chromosomal translocations involving the 11q23 band are frequently, but not invariably, associated with MLL gene rearrangement and their finding is associated with a poor prognosis. We observed two new translocations with a breakpoint in the 11q23 region at standard cytogenetic analysis: a previously undescribed t(3;11)(q21;q23) in a 70-year old woman with a fulminating form of AML-M1 and a new translocation t(6;11)(q15;q23) in a 61-year old man with an atypical chronic myelogenous leukemia. In these two patients, involvement of the MLL gene was analyzed by molecular cytogenetic techniques which also allowed a more precise mapping of the breakpoints. DESIGN AND METHODS: The MLL gene was analyzed by Southern blot and by fluorescent in situ hybridization (FISH) with a double-color MLL probe. A panel of 11q, 3q and 6q cosmid/YAC probes mapping around the breakpoints was used for breakpoint mapping. RESULTS: In both patients, FISH analysis and Southern blot showed that the MLL gene was not rearranged; in patient 1, MLL was retained on the 11q+ derivative, whereas in patient 2 it moved to the 6q- chromosome. In the t(3;11) we localized the chromosome 11 breakpoint at 11q23.3, in a region flanked by CP-939H3 and cos1p3, distal to the MLL locus; in the t(6;11) the break occurred at 11q21, in a region flanked by CP-819A5 and CP-829A6, proximal to the MLL locus. INTERPRETATION AND CONCLUSIONS: Our cases add two new translocations to the list of chromosomal anomalies involving the long arm of chromosome 11, and show that apparent translocation t(11q23) may involve loci and genes other than MLL. Characterizing the molecular heterogeneity of 11q23 translocations may identify some prognostic significance.

ALK activation by the CLTC-ALK fusion is a recurrent event in large B-cell lymphoma.

We present 3 cases of large B-cell lymphoma (LBCL) with a granular cytoplasmic staining for anaplastic lymphoma kinase (ALK). All of the cases showed striking similarities in morphology and immunohistochemical profile characterized by a massive monomorphic proliferation of CD20-/CD138+ plasmablast-like cells. In one of the cases, initially diagnosed as a null-type anaplastic large cell lymphoma (ALCL), the B-cell phenotype became evident only at recurrence. Fluorescent in situ hybridization (FISH) and molecular studies led to the detection of a CLTC-ALK rearrangement in all 3 cases, without any evidence of full-length ALK receptor expression. The associated t(2;17)(p23;q23) was demonstrated in the karyotype of 2 cases. Although a similar CLTC-ALK aberration was previously identified in ALK-positive T-/null cell ALCL and inflammatory myofibroblastic tumor, its association with ALK-positive LBCL seems to be specific and intriguing.