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To classify cancer specimens by their gene expression profiles, we created a statistical method based on Bayes' rule that estimates the probability of membership in one of two cancer subgroups. We used this method to classify diffuse large B cell lymphoma (

We used gene expression profiling to establish a molecular diagnosis of mantle cell lymphoma (MCL), to elucidate its pathogenesis, and to predict the length of survival of these patients. An MCL gene expression signature defined a large subset of MCLs that expressed cyclin D1 and a novel subset that lacked cyclin D1 expression. A precise measurement of tumor cell proliferation, provided by the expression of proliferation signature genes, identified patient subsets that differed by more than 5 years in median survival. Differences in cyclin D1 mRNA abundance synergized with INK4a/ARF locus deletions to dictate tumor proliferation rate and survival. We propose a quantitative model of the aberrant cell cycle regulation in MCL that provides a rationale for the design of cell cycle inhibitor therapy in this malignancy.

Leptin and its receptor, obese receptor (OB-R), comprise an important signaling system for the regulation of body weight. Splice variants of OB-R mRNA encode proteins that differ in the length of their cytoplasmic domains. We cloned a long isoform of the wild-type leptin receptor that is preferentially expressed in the hypothalamus and show that it can activate signal transducers and activators of transcription (STAT)-3, and . A point mutation within the OB-R gene of diabetic (db) mice generates a new splice donor site that dramatically reduces expression of this long isoform in homozygous db/db mice. In contrast, an OB-R protein with a shorter cytoplasmic domain is present in both db/db and wild-type mice. We show that this short isoform is unable to activate the STAT pathway. These data provide further evidence that the mutation in OB-R causes the db/db phenotype and identify three STAT proteins as potential mediators of the anti-obesity effects of leptin.Leptin, the product of the obese (ob) gene, is a 16-kDa secreted protein primarily produced by adipocytes (1). There is a good correlation between the percentage of body fat and serum leptin levels suggesting that leptin production is regulated by the mass of adipocytes (2, 3). Leptin levels were normal or elevated in obese individuals (2, 4) arguing against a simple leptin deficiency as the cause of obesity in the majority of humans (5). Serum leptin concentrations increased under a fatty diet but failed to prevent weight gain (3). Therefore, insensitivity to the action of leptin appears to be a common mechanism in obese individuals and in several rodent models. This suggests that dysregulation at the level of the leptin receptor, the downstream signaling pathway, or an unknown modifying mechanism may constitute the basis for weight disorders. The crucial role of leptin and its receptor in obesity is well illustrated by two phenotypically very similar mutants obese (ob) and diabetes (db) (6). Mice homozygous for a loss of function mutation of ob display obesity, hyperglycemia, and insulin resistance resembling type II diabetes. Administration of recombinant leptin to ob mice corrected these abnormalities (7-9). Based on early parabiosis experiments it was expected that db would be caused by a mutation in the ob receptor (OB-R) (6).OB-R was cloned by virtue of its high affinity to leptin through an expression cloning strategy (10). The OB-R gene was mapped to the same 5-centimorgan interval on mouse chromosome 4 to which db had been localized (10). Surprisingly, no mutation in the coding region of OB-R cDNA of db/db mice was found and leptin binding sites were unaltered in db/db mice (10). However, the cloned mouse OB-R cDNA encoded a protein with a much shorter cytoplasmic domain than the human homologue, suggesting that a longer mouse isoform exists. We cloned this longer form of OB-R from wild-type mice and found that the mRNA for this isoform is dramatically reduced in db/db mice. A G to T mutation in db mice generates a new splice donor and su...

The presence or absence of somatic mutations in the expressed immunoglobulin heavy chain variable regions (IgVH) of chronic lymphocytic leukemia (CLL) cells provides prognostic information. Patients whose leukemic cells express unmutated IgVH regions (Ig-unmutated CLL) often have progressive disease, whereas patients whose leukemic cells express mutated IgVH regions (Ig-mutated CLL) more often have an indolent disease. Given the difficulty in performing IgVH sequencing in a routine diagnostic laboratory, this prognostic distinction is currently unavailable to most patients. Pilot gene expression profiling studies in patients with CLL identified genes that were differentially expressed between the Ig-unmutated and Ig-mutated CLL subtypes. Here, we have profiled an expanded cohort of 107 patients and show that ZAP-70 is the gene that best distinguishes the CLL subtypes. Ig-unmutated CLL expressed ZAP-70 5.54-fold more highly than Ig-mutated CLL (P < 10 -21 ). ZAP-70 expression correctly predicted IgVH mutation status in 93% of patients. ZAP-70 expression and IgVH mutation status were comparable in their ability to predict time to treatment requirement following diagnosis. In 7 patients, ZAP-70 expression and IgVH mutation status were discordant: 4 Ig-mutated CLLs had high ZAP-70 expression and 3 Igunmutated CLLs had low ZAP-70 expression. Among these ZAP-70 "outliers," those with Ig-mutated CLL had clinical features that are uncharacteristic of this CLL subtype: 2 required early treatment and 2 used a mutated VH3-21 gene, an IgVH gene that has been associated with progressive disease. We developed reverse transcriptase-polymerase chain reaction and immunohistochemical assays for ZAP-70 expression that can be applied clinically and would yield important prognostic information for patients with CLL. IntroductionThe clinical course of chronic lymphocytic leukemia (CLL) is heterogeneous. [1][2][3] Although some patients have an indolent disease without any need for therapeutic interventions, other patients may succumb rapidly despite intensive treatment. There has been an intense search for good prognostic markers in early-stage disease that might facilitate risk-adapted treatment strategies. 4,5 Although cytogenetic abnormalities, especially deletions of 11q and 17p, have been shown to correlate with short survival, 6,7 these changes may not be present in early disease and may only be acquired during disease progression. 8 The presence or absence of somatic mutations in the variable region of the B-cell-receptor heavy chain gene (IgVH) has been shown to distinguish between 2 disease subsets conferring important prognostic information. 9,10 Median survival in patients whose CLL cells express unmutated IgVH genes (Ig-unmutated CLL) ranges between 79 and 119 months. 7,[9][10][11] In contrast, patients whose CLL cells express mutated IgVH genes (Ig-mutated CLL) have a distinctly longer median survival, reaching 293 months in one study, and many may never require treatment. 11 Several lines of evidence indicate that th...

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Mantle cell lymphoma (MCL) is an aggressive tumor, but a subset of patients may follow an indolent clinical course. To understand the mechanisms underlying this biological heterogeneity, we performed whole-genome and/or whole-exome sequencing on 29 MCL cases and their respective matched normal DNA, as well as 6 MCL cell lines. Recurrently mutated genes were investigated by targeted sequencing in an independent cohort of 172 MCL patients. We identified 25 significantly mutated genes, including known drivers such as ataxia-telangectasia mutated (ATM), cyclin D1 (CCND1), and the tumor suppressor TP53; mutated genes encoding the anti-apoptotic protein BIRC3 and Toll-like receptor 2 (TLR2); and the chromatin modifiers WHSC1, MLL2, and MEF2B. We also found NOTCH2 mutations as an alternative phenomenon to NOTCH1 mutations in aggressive tumors with a dismal prognosis. Analysis of two simultaneous or subsequent MCL samples by whole-genome/whole-exome (n = 8) or targeted (n = 19) sequencing revealed subclonal heterogeneity at diagnosis in samples from different topographic sites and modulation of the initial mutational profile at the progression of the disease. Some mutations were predominantly clonal or subclonal, indicating an early or late event in tumor evolution, respectively. Our study identifies molecular mechanisms contributing to MCL pathogenesis and offers potential targets for therapeutic intervention.next-generation sequencing | cancer genetics | cancer heterogeneity M antle cell lymphoma (MCL) is a mature B-cell neoplasm characterized by the t(11;14)(q13;q32) translocation leading to the overexpression of cyclin D1 (1). CCND1 is a weak oncogene that requires the cooperation of other oncogenic events to transform lymphoid cells (2). Molecular studies have identified alterations in components of the cell-cycle regulation, DNA damage response, and cell survival pathways (3, 4), but the profile of mutated genes contributing to the pathogenesis of MCL and cooperating with CCND1 is not well defined (1). Most MCL cases have a rapid evolution and an aggressive behavior with an unfavorable outcome with current therapies (5). Paradoxically, a subset of patients follows an indolent clinical evolution with stable disease even in the absence of chemotherapy (6, 7). This favorable behavior has been associated with IGHV-mutated (8, 9) and lack of expression of SOX11 (10, 11), a transcription factor highly specific of MCL that contributes to the aggressive behavior of this tumor (12). However, the molecular mechanisms responsible for this clinical heterogeneity are not well understood.To gain insight into the molecular pathogenesis of MCL we performed whole-genome sequencing (WGS) and whole-exome sequencing (WES) of 29 MCL and further investigated mutated genes in an expanded series of patients. We also analyzed the subclonal heterogeneity of the tumors and their modulation during the evolution of the disease. Results Landscape of Mutations in MCL.We performed WGS and WES of 4 and 29 MCL, respectively. These patients were re...

The oncogene c-maf is translocated in approximately 5%-10% of multiple myelomas. Unexpectedly, we observed c-maf expression in myeloma cell lines lacking c-maf translocations and in 50% of multiple myeloma bone marrow samples. By gene expression profiling, we identified three c-maf target genes: cyclin D2, integrin beta7, and CCR1. c-maf transactivated the cyclin D2 promoter and enhanced myeloma proliferation, whereas dominant inhibition of c-maf blocked tumor formation in immunodeficient mice. c-maf-driven expression of integrin beta7 enhanced myeloma adhesion to bone marrow stroma and increased production of VEGF. We propose that c-maf transforms plasma cells by stimulating cell cycle progression and by altering bone marrow stromal interactions. The frequent overexpression of c-maf in myeloma makes it an attractive target for therapeutic intervention.