Cristina R. Antonescu

Classification and diagnostic prediction of cancers using gene expression profiling and artificial neural networks.

The purpose of this study was to develop a method of classifying cancers to specific diagnostic categories based on their gene expression signatures using artificial neural networks (ANNs). We trained the ANNs using the small, round blue-cell tumors (SRBCTs) as a model. These cancers belong to four distinct diagnostic categories and often present diagnostic dilemmas in clinical practice. The ANNs correctly classified all samples and identified the genes most relevant to the classification. Expression of several of these genes has been reported in SRBCTs, but most have not been associated with these cancers. To test the ability of the trained ANN models to recognize SRBCTs, we analyzed additional blinded samples that were not previously used for the training procedure, and correctly classified them in all cases. This study demonstrates the potential applications of these methods for tumor diagnosis and the identification of candidate targets for therapy.

The landscape of somatic copy-number alteration across human cancers

A powerful way to discover key genes with causal roles in oncogenesis is to identify genomic regions that undergo frequent alteration in human cancers. Here we present high-resolution analyses of somatic copy-number alterations (SCNAs) from 3,131 cancer specimens, belonging largely to 26 histological types. We identify 158 regions of focal SCNA that are altered at significant frequency across several cancer types, of which 122 cannot be explained by the presence of a known cancer target gene located within these regions. Several gene families are enriched among these regions of focal SCNA, including the BCL2 family of apoptosis regulators and the NF-κΒ pathway. We show that cancer cells containing amplifications surrounding the MCL1 and BCL2L1 anti-apoptotic genes depend on the expression of these genes for survival. Finally, we demonstrate that a large majority of SCNAs identified in individual cancer types are present in several cancer types.

Adjuvant imatinib mesylate after resection of localised, primary gastrointestinal stromal tumour: a randomised, double-blind, placebo-controlled trial

BACKGROUND Gastrointestinal stromal tumour is the most common sarcoma of the intestinal tract. Imatinib mesylate is a small molecule that inhibits activation of the KIT and platelet-derived growth factor receptor alpha proteins, and is effective in first-line treatment of metastatic gastrointestinal stromal tumour. We postulated that adjuvant treatment with imatinib would improve recurrence-free survival compared with placebo after resection of localised, primary gastrointestinal stromal tumour. METHODS We undertook a randomised phase III, double-blind, placebo-controlled, multicentre trial. Eligible patients had complete gross resection of a primary gastrointestinal stromal tumour at least 3 cm in size and positive for the KIT protein by immunohistochemistry. Patients were randomly assigned, by a stratified biased coin design, to imatinib 400 mg (n=359) or to placebo (n=354) daily for 1 year after surgical resection. Patients and investigators were blinded to the treatment group. Patients assigned to placebo were eligible to crossover to imatinib treatment in the event of tumour recurrence. The primary endpoint was recurrence-free survival, and analysis was by intention to treat. Accrual was stopped early because the trial results crossed the interim analysis efficacy boundary for recurrence-free survival. This study is registered with ClinicalTrials.gov, number NCT00041197. FINDINGS All randomised patients were included in the analysis. At median follow-up of 19.7 months (minimum-maximum 0-56.4), 30 (8%) patients in the imatinib group and 70 (20%) in the placebo group had had tumour recurrence or had died. Imatinib significantly improved recurrence-free survival compared with placebo (98% [95% CI 96-100] vs 83% [78-88] at 1 year; hazard ratio [HR] 0.35 [0.22-0.53]; one-sided p<0.0001). Adjuvant imatinib was well tolerated, with the most common serious events being dermatitis (11 [3%] vs 0), abdominal pain (12 [3%] vs six [1%]), and diarrhoea (ten [2%] vs five [1%]) in the imatinib group and hyperglycaemia (two [<1%] vs seven [2%]) in the placebo group. INTERPRETATION Adjuvant imatinib therapy is safe and seems to improve recurrence-free survival compared with placebo after the resection of primary gastrointestinal stromal tumour. FUNDING US National Institutes of Health and Novartis Pharmaceuticals.

Acquired resistance to imatinib in gastrointestinal stromal tumor occurs through secondary gene mutation

Most gastrointestinal stromal tumors (GIST) have an activating mutation in either KIT or PDGFRA. Imatinib is a selective tyrosine kinase inhibitor and achieves a partial response or stable disease in about 80% of patients with metastatic GIST. It is now clear that some patients with GIST develop resistance to imatinib during chronic therapy. To identify the mechanism of resistance, we studied 31 patients with GIST who were treated with imatinib and then underwent surgical resection. There were 13 patients who were nonresistant to imatinib, 3 with primary resistance, and 15 with acquired resistance after initial benefit from the drug. There were no secondary mutations in KIT or PDGFRA in the nonresistant or primary resistance groups. In contrast, secondary mutations were found in 7 of 15 (46%) patients with acquired resistance, each of whom had a primary mutation in KIT exon 11. Most secondary mutations were located in KIT exon 17. KIT phosphorylation was heterogeneous and did not correlate with clinical response to imatinib or mutation status. That acquired resistance to imatinib in GIST commonly occurs via secondary gene mutation in the KIT kinase domain has implications for strategies to delay or prevent imatinib resistance and to employ newer targeted therapies.

SYT-SSX gene fusion as a determinant of morphology and prognosis in synovial sarcoma

BACKGROUND Synovial sarcomas account for up to 10 percent of soft-tissue sarcomas and include two major histologic subtypes, biphasic and monophasic, defined respectively by the presence and absence of glandular epithelial differentiation in a background of spindle tumor cells. A characteristic SYT-SSX fusion gene resulting from the chromosomal translocation t(X;18)(p11;q11) is detectable in almost all synovial sarcomas. The translocation fuses the SYT gene from chromosome 18 to either of two highly homologous genes at Xp11, SSX1 or SSX2. SYT-SSX1 and SYT-SSX2 are thought to tunction as aberrant transcriptional regulators. We attempted to determine the influence of the two alternative forms of the SYT-SSX fusion gene on tumor morphology and clinical outcome in patients with this sarcoma. METHODS We analyzed SYT-SSX fusion transcripts in 45 synovial sarcomas (33 monophasic and 12 biphasic) by the reverse-transcriptase polymerase chain reaction and compared the results with relevant clinical and pathological data. RESULTS The SYT-SSX1 and SYT-SSX2 fusion transcripts were detected in 29 (64 percent) and 16 (36 percent) of the tumors, respectively. There was a significant relation (P=0.003) between histologic subtype (monophasic vs. biphasic) and SSX1 or SSX2 involvement in the fusion transcript: all 12 biphasic synovial sarcomas had a SYT-SSX1 fusion transcript, and all 16 tumors that were positive for SYT-SSX2 were monophasic. Kaplan-Meier analysis of 39 patients with localized tumors showed that the 15 patients with SYT-SSX2 had significantly better metastasis-free survival than the 24 patients with SYT-SSX1 (P=0.03 by multivariate analysis; relative risk, 3.0). There were no significant correlations between the type of SYT-SSX transcript and age, sex, tumor location and size, whether there were metastases at diagnosis, or whether patients underwent chemotherapy. Histologic subtype alone was not prognostically important. CONCLUSIONS The type of SYT-SSX fusion transcript correlates with both the histologic subtype and the clinical behavior of synovial sarcoma. SYT-SSX fusion transcripts are a defining diagnostic marker of synovial sarcomas and may also yield important independent prognostic information.

KIT as a Therapeutic Target in Metastatic Melanoma

CONTEXT Some melanomas arising from acral, mucosal, and chronically sun-damaged sites harbor activating mutations and amplification of the type III transmembrane receptor tyrosine kinase KIT. We explored the effects of KIT inhibition using imatinib mesylate in this molecular subset of disease. OBJECTIVE To assess clinical effects of imatinib mesylate in patients with melanoma harboring KIT alterations. DESIGN, SETTING, AND PATIENTS A single-group, open-label, phase 2 trial at 1 community and 5 academic oncology centers in the United States of 295 patients with melanoma screened for the presence of KIT mutations and amplification between April 23, 2007, and April 16, 2010. A total of 51 cases with such alterations were identified and 28 of these patients were treated who had advanced unresectable melanoma arising from acral, mucosal, and chronically sun-damaged sites. INTERVENTION Imatinib mesylate, 400 mg orally twice daily. MAIN OUTCOME MEASURES Radiographic response, with secondary end points including time to progression, overall survival, and correlation of molecular alterations and clinical response. RESULTS Two complete responses lasting 94 (ongoing) and 95 weeks, 2 durable partial responses lasting 53 and 89 (ongoing) weeks, and 2 transient partial responses lasting 12 and 18 weeks among the 25 evaluable patients were observed. The overall durable response rate was 16% (95% confidence interval [CI], 2%-30%), with a median time to progression of 12 weeks (interquartile range [IQR], 6-18 weeks; 95% CI, 11-18 weeks), and a median overall survival of 46.3 weeks (IQR, 28 weeks-not achieved; 95% CI, 28 weeks-not achieved). Response rate was better in cases with mutations affecting recurrent hotspots or with a mutant to wild-type allelic ratio of more than 1 (40% vs 0%, P = .05), indicating positive selection for the mutated allele. CONCLUSIONS Among patients with advanced melanoma harboring KIT alterations, treatment with imatinib mesylate results in significant clinical responses in a subset of patients. Responses may be limited to tumors harboring KIT alterations of proven functional relevance. Trial Registration clinicaltrials.gov Identifier: NCT00470470.

Primary and Secondary Kinase Genotypes Correlate With the Biological and Clinical Activity of Sunitinib in Imatinib-Resistant Gastrointestinal Stromal Tumor

PURPOSE Most gastrointestinal stromal tumors (GISTs) harbor mutant KIT or platelet-derived growth factor receptor alpha (PDGFRA) kinases, which are imatinib targets. Sunitinib, which targets KIT, PDGFRs, and several other kinases, has demonstrated efficacy in patients with GIST after they experience imatinib failure. We evaluated the impact of primary and secondary kinase genotype on sunitinib activity. PATIENTS AND METHODS Tumor responses were assessed radiologically in a phase I/II trial of sunitinib in 97 patients with metastatic, imatinib-resistant/intolerant GIST. KIT/PDGFRA mutational status was determined for 78 patients by using tumor specimens obtained before and after prior imatinib therapy. Kinase mutants were biochemically profiled for sunitinib and imatinib sensitivity. RESULTS Clinical benefit (partial response or stable disease for > or = 6 months) with sunitinib was observed for the three most common primary GIST genotypes: KIT exon 9 (58%), KIT exon 11 (34%), and wild-type KIT/PDGFRA (56%). Progression-free survival (PFS) was significantly longer for patients with primary KIT exon 9 mutations (P = .0005) or with a wild-type genotype (P = .0356) than for those with KIT exon 11 mutations. The same pattern was observed for overall survival (OS). PFS and OS were longer for patients with secondary KIT exon 13 or 14 mutations (which involve the KIT-adenosine triphosphate binding pocket) than for those with exon 17 or 18 mutations (which involve the KIT activation loop). Biochemical profiling studies confirmed the clinical results. CONCLUSION The clinical activity of sunitinib after imatinib failure is significantly influenced by both primary and secondary mutations in the predominant pathogenic kinases, which has implications for optimization of the treatment of patients with GIST.

Primary Renal Neoplasms with the ASPL-TFE3 Gene Fusion of Alveolar Soft Part Sarcoma A Distinctive Tumor Entity Previously Included among Renal Cell Carcinomas of Children and Adolescents

The unbalanced translocation, der(17)t(X;17)(p11.2;q25), is characteristic of alveolar soft part sarcoma (ASPS). We have recently shown that this translocation fuses the TFE3 transcription factor gene at Xp11.2 to ASPL, a novel gene at 17q25. We describe herein eight morphologically distinctive renal tumors occurring in young people that bear the identical ASPL-TFE3 fusion transcript as ASPS, with the distinction that the t(X;17) translocation is cytogenetically balanced in these renal tumors. A relationship between these renal tumors and ASPS was initially suggested by the cytogenetic finding of a balanced t(X;17)(p11.2;q25) in two of the cases, and the ASPL-TFE3 fusion transcripts were then confirmed by reverse transcriptase-polymerase chain reaction. The morphology of these eight ASPL-TFE3 fusion-positive renal tumors, although overlapping in some aspects that of classic ASPS, more closely resembles renal cell carcinoma (RCC), which was the a priori diagnosis in all cases. These tumors demonstrate nested and pseudopapillary patterns of growth, psammomatous calcifications, and epithelioid cells with abundant clear cytoplasm and well-defined cell borders. By immunohistochemistry, four tumors were negative for all epithelial markers tested, whereas four were focally positive for cytokeratin and two were reactive for epithelial membrane antigen (EMA) (one diffusely, one focally). Electron microscopy of six tumors demonstrated a combination of ASPS-like features (dense granules in four cases, rhomboid crystals in two cases) and epithelial features (cell junctions in six cases, microvilli and true glandular lumens in three cases). Overall, although seven of eight tumors demonstrated at least focal epithelial features by electron microscopy or immunohistochemistry, the degree and extent of epithelial differentiation was notably less than expected for typical RCC. We confirmed the balanced nature of the t(X;17) translocation by fluorescence in situ hybridization in all seven renal tumors thus analyzed, which contrasts sharply with the unbalanced nature of the translocation in ASPS. In summary, a subset of tumors previously considered to be RCC in young people are in fact genetically related to ASPS, although their distinctive morphological and genetic features justify their classification as a distinctive neoplastic entity. Finally, the finding of distinctive tumors being associated with balanced and unbalanced forms of the same translocation is to our knowledge, unprecedented.

EWS-FLI1 fusion transcript structure is an independent determinant of prognosis in Ewing's sarcoma.

PURPOSE More than 90% of Ewings sarcomas (ES) contain a fusion of the EWS and FLI1 genes, due to the t(11;22)(q24;q12) translocation. At the molecular level, the EWS-FLI1 rearrangements show great diversity. Specifically, many different combinations of exons from EWS and FLI1 encode in-frame fusion transcripts and result in differences in the length and composition of the chimeric protein, which functions as an oncogenic aberrant transcription factor. In the most common fusion type (type 1), EWS exon 7 is linked in frame with exon 6 of FLI1. As the fundamental pathogenetic lesion in ES, the molecular heterogeneity of these fusion transcripts may have functional and clinical significance. PATIENTS AND METHODS We performed a clinical and pathologic analysis of 112 patients with ES in which EWS-FLI1 fusion transcripts were identified by reverse-transcriptase polymerase chain reaction (RT-PCR). Adequate treatment and follow-up data were available in 99 patients treated with curative intent. Median follow-up in these 99 patients was 26 months (range, 1 to 140 months). Univariate and multivariate survival analyses were performed that included other prognostic factors, such as age, tumor location, size, and stage. RESULTS Among the 99 patients suitable for survival analysis, the tumors in 64 patients contained the type 1 fusion and in 35 patients contained less common fusion types. Stage at presentation was localized in 74 patients and metastatic in 25. Metastases (relative risk [RR] = 2.6; P = .008), and type 1 EWS-FLI1 fusion (RR = 0.37; P = .014) were, respectively, independent negative and positive prognostic factors for overall survival by multivariate analysis. Among 74 patients with localized tumors, the type 1 EWS-FLI1 fusion was also a significant positive predictor of overall survival (RR = 0.32; P = .034) by multivariate analysis. CONCLUSION EWS-FLI1 fusion type appears to be prognostically relevant in ES, independent of tumor site, stage, and size. Further studies are needed to clarify the biologic basis of this phenomenon.

The der(17)t(X;17)(p11;q25) of human alveolar soft part sarcoma fuses the TFE3 transcription factor gene to ASPL, a novel gene at 17q25

Alveolar soft part sarcoma (ASPS) is an unusual tumor with highly characteristic histopathology and ultrastructure, controversial histogenesis, and enigmatic clinical behavior. Recent cytogenetic studies have identified a recurrent der(17) due to a non-reciprocal t(X;17)(p11.2;q25) in this sarcoma. To define the interval containing the Xp11.2 break, we first performed FISH on ASPS cases using YAC probes for OATL1 (Xp11.23) and OATL2 (Xp11.21), and cosmid probes from the intervening genomic region. This localized the breakpoint to a 160 kb interval. The prime candidate within this previously fully sequenced region was TFE3, a transcription factor gene known to be fused to translocation partners on 1 and X in some papillary renal cell carcinomas. Southern blotting using a TFE3 genomic probe identified non-germline bands in several ASPS cases, consistent with rearrangement and possible fusion of TFE3 with a gene on 17q25. Amplification of the 5′ portion of cDNAs containing the 3′ portion of TFE3 in two different ASPS cases identified a novel sequence, designated ASPL, fused in-frame to TFE3 exon 4 (type 1 fusion) or exon 3 (type 2 fusion). Reverse transcriptase PCR using a forward primer from ASPL and a TFE3 exon 4 reverse primer detected an ASPL-TFE3 fusion transcript in all ASPS cases (12/12: 9 type 1, 3 type 2), establishing the utility of this assay in the diagnosis of ASPS. Using appropriate primers, the reciprocal fusion transcript, TFE3-ASPL, was detected in only one of 12 cases, consistent with the non-reciprocal nature of the translocation in most cases, and supporting ASPL-TFE3 as its oncogenically significant fusion product. ASPL maps to chromosome 17, is ubiquitously expressed, and matches numerous ESTs (Unigene cluster Hs.84128) but no named genes. The ASPL cDNA open reading frame encodes a predicted protein of 476 amino acids that contains within its carboxy-terminal portion of a UBX-like domain that shows significant similarity to predicted proteins of unknown function in several model organisms. The ASPL-TFE3 fusion replaces the N-terminal portion of TFE3 by the fused ASPL sequences, while retaining the TFE3 DNA-binding domain, implicating transcriptional deregulation in the pathogenesis of this tumor, consistent with the biology of several other translocation-associated sarcomas.