Monday, November 28, 2011

The spliceosome: a new factor in bad risk CLL

I mentioned some weeks ago that the spliceosome is going to be important in CLL. The spliceosome is an epigenetic mechanism which is involved in splicing together the separated introns of a gene identified on DNA. It had already been identified as a factor in determining the various subtypes of MDS, but it has now been recognized as an important mechanism underlying fludarabine refractoriness in CLL and also in some cases of Richter syndrome.

I have quoted from this paper from Italy in a recent BLOOD

Mutations of the SF3B1 splicing factor in chronic lymphocytic leukemia:association with progression and fludarabine-refractoriness

The clinical course of chronic lymphocytic leukemia (CLL) ranges from a very indolent
disorder with a normal lifespan, to a rapidly progressive disease leading to death. Occasionally, CLL undergoes transformation to Richter syndrome (RS). The variable clinical course of CLL is driven, at least in part, by the disease immunogenetic and molecular heterogeneity.

Despite recent advances, the genetic lesions identified to date do not entirely explain the development of severe complications, such as chemorefractoriness, which still represent unmet clinical needs. Fludarabine-refractoriness is due to TP53 disruption in ~40% of refractory cases, but in a sizeable fraction of patients the
molecular basis of this aggressive phenotype remains unclear. Recently, two independent studies of the CLL coding genome investigated at disease presentation have revealed a restricted number of mutated genes, including NOTCH1. These
studies have provided a proof of concept that, similar to other malignancies, genome-wide mutational analysis might identify novel lesions of biological and clinical relevance in CLL.

On these grounds, the authors have embarked on the investigation of the coding genome of fludarabine refractory CLL in order to identify genetic lesions associated with chemorefractoriness. The initial phases of this analysis have revealed recurrent mutations of SF3B1, a critical component of the cell spliceosome, pointing to the potential involvement of splicing regulation in CLL pathogenesis and chemo-refractoriness.

The study population comprised 3 cohorts representative of different disease phases: i) fludarabine-refractory CLL (n=59), including cases (n=11) subjected to whole exome sequencing; ii) a consecutive series of newly diagnosed and previously untreated CLL (n=301); and iii) clonally related RS (n=33; all diffuse large B cell lymphomas).

Diagnosis of CLL and of fludarabine-refractoriness were based on IWCLL-NCI criteria; RS was based on histological criteria. Peripheral blood tumor samples were obtained as follows: i) for fludarabine-refractory CLL, immediately before starting treatment to which the patient failed to respond because of stable/progressive disease; ii) for newly diagnosed and previously untreated CLL, at disease presentation. All RS studies were performed on RS diagnostic biopsies. Normal DNAs from the same patients were obtained from saliva or from purified granulocytes and confirmed to be tumor-free by PCR of tumor-specific IGHV-D-J rearrangements. Patients provided informed consent in accordance with local IRB requirements and Declaration of Helsinki. The study was approved by the Local Ethical Committee (Protocol Code 59/CE; Study Number
CE 8/11).

Mutation analysis of SF3B1 (exons 1-25, including splice sites; RefSeq NM_012433.2) was performed on PCR amplimers obtained from genomic DNA by a combination of Sanger sequencing and targeted next generation sequencing. FISH karyotype, mutation analysis of IGHV, TP53 and NOTCH1, copy number analysis, and gene expression profile analysis FISH analysis was performed using probes LSI13 and LSID13S319, CEP12, LSIp53, and LSIATM. IGHV sequences were aligned to ImMunoGeneTics directory and considered mutated if identity to corresponding germline genes was less than 98%.3, TP53 and NOTCH1 mutations were analysed by Sanger sequencing.3,7 Genome-wide DNA profiles were obtained using Affymetrix Genome-Wide Human SNP Array 6.0. Gene expression profile analysis was performed using Affymetrix HG-U133_plus2 arrays.

Statistical analysis

Overall survival was measured from date of diagnosis to date of death (event) or last followup (censoring). Treatment free survival was measured from date of diagnosis to date of progression to symptomatic disease requiring treatment according to IWCLL-NCI guidelines (event), death, or last follow up (censoring).

Results and Discussion

Following the initial observation of recurrent SF3B1 mutations in 3/11 fludarabine refractory CLL analyzed by whole exome sequencing, we performed targeted re-sequencing of the SF3B1 coding sequence and splice sites in 48 additional cases of progressive and fludarabinerefractory CLL (total number of cases analyzed: 59). SF3B1 was altered in 10/59 (17%) fludarabine-refractory CLL by missense mutations (n=9) or in-frame deletions (n=1) clustering in the HEAT3, HEAT4 and HEAT5 repeats of the SF3B1 protein. Two sites that are highly conserved inter-species (codon 662 and codon 700) were recurrently mutated in 3 and 5 cases, respectively. SF3B1 mutations were monoallelic, and were predicted to be functionally significant according to the PolyPhen-2 algorithm. These data document that mutations of SF3B1, a splicing factor that is a critical component of the spliceosome, recurrently associate with fludarabine-refractory CLL.

The biological characteristics of fludarabine-refractory CLL harboring SF3B1 mutations are in summary that mutations occurred irrespective of the IGHV mutation status, CD38 expression and ZAP70 expression. At the time of fludarabine-refractoriness, SF3B1 mutations were enriched in cases harboring a normal FISH karyotype (p=.008). Also, SF3B1 mutations distributed in a mutually exclusive fashion compared to TP53 disruption tested by deletion and/or mutation (mutual information I =0.0609; p=.046). By combining SF3B1 mutations with other genetic lesions enriched in chemorefractory cases (TP53 disruption, NOTCH1 mutations, ATM deletion), fludarabine-refractory CLL appeared to be characterized by multiple molecular alterations that, to some extent, are mutually exclusive.

To investigate whether SF3B1 mutations are restricted to chemorefractory cases, we then compared the prevalence of mutations observed at the time of fludarabine-refractoriness to the prevalence of mutations observed in other disease phases. In a consecutive series evaluated at CLL diagnosis, SF3B1 mutations were rare (17/301; 5%)and occurred irrespective of other molecular and immunogenetic features. Remarkably, 5/17 (29%) CLL mutated at diagnosis were primary fludarabine-refractory patients. In these 5 cases, TP53 disruption and NOTCH1 mutations occurred in 1 cases each. None of the 12 remaining cases harbored TP53 disruption or NOTCH1 mutations.

By univariate analysis, SF3B1 mutations showed a crude association with short treatment free survival (p<.001) and overall survival (p=.011). By multivariate analysis, the increased risk of death predicted by SF3B1 mutations was independent (HR: 3.02; 95% CI: 1.24-7.35; p=.015) of confounding clinical and biological variables. Confirmation within the frame of prospective clinical trials will be helpful to fully assess the generalization of SF3B1 mutations as a CLL prognostic marker. In CLL investigated at diagnosis, the hotspot distribution and molecular spectrum of SF3B1 mutations, as well as their mutual relationship with other genetic lesions, were similar to those observed in fludarabine-refractory CLL. SF3B1 mutations were only found in 2/33 (6.0%) clonally-related RS. Across the different disease phases investigated, mutations were confirmed to be somatically acquired in all cases (n=18) for which germline DNA was available. Among the three SF3B1 mutated cases for which serial samples were analyzed, SF3B1 mutations were acquired in 2 cases. One fludarabine-refractory CLL acquired the c.2044A>G p.K666E mutation at the time of refractoriness, and one RS acquired the c.2146A>G p.K700E mutation at the time of transformation. In the remaining case, the SF3B1 mutation was present in all disease phases. Although the relative expression of SF3B1 in CLL was higher compared to normal B-cell subsets, extensive investigation by SNP array analysis ruled out focal copy number abnormalities of SF3B1 in this leukemia (n=0/323). SF3B1 mutations were consistently absent among mature B-cell neoplasms (n=136) other than CLL. These data document that SF3B1 mutations: i) are specific for CLL among mature B-cell neoplasms; ii) occur at a low rate at CLL presentation, whereas they are enriched in fludarabine-refractory cases; iii) play a minor role in RS transformation, corroborating the notion that CLL histologic shift is molecularly distinct from chemorefractory progression without RS transformation.

The identification of SF3B1 mutations in CLL, and the recent discovery of SF3B1 mutations in myelodysplasia, points to the involvement of splicing regulation as a novel pathogenetic mechanism in hematologic malignancies. SF3B1 is a critical component of both major (U2-like) and minor (U12-like) spliceosomes, which enact the precise excision of introns from premRNA. The precise biological role of SF3B1 mutations in CLL is currently elusive, and will require dedicated studies. The pathogenicity of SF3B1 mutations in CLL is strongly supported by the clustering of these mutations in evolutionarily conserved hotspots localized within HEAT domains, which are tandemly arranged curlicue-like structures serving as flexible scaffolding on which other components can assemble. Also, the observation that SF3B1 regulates the alternative splicing program of genes controlling cell cycle progression and apoptosis points to a potential contribution of SF3B1 mutations in modulating tumor cell proliferation and survival. In addition to pathogenetic implications, SF3B1 mutations might also provide a therapeutic target for SF3B1 inhibitors, which are currently under pre-clinical development as anti-cancer

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