Correlation of mutation profile and response in patients with myelofibrosis treated with ruxolitinib.

Although most patients with myelofibrosis (MF) derive benefit from ruxolitinib, some are refractory, have a suboptimal response, or quickly lose their response. To identify genes that may predict response to ruxolitinib, we performed targeted next-generation sequencing (NGS) of a panel of 28 genes recurrently mutated in hematologic malignancies in a cohort of patients with MF who were treated with ruxolitinib in a phase 1/2 study. We also tested for CALR deletions by standard polymerase chain reaction methods. Ninety-eight percent of patients had a mutation in ≥1 gene. Seventy-nine (82.1%) patients had the JAK2(V617F) mutation, 9 (9.5%) had CALR mutations (7 type 1, 2 type 2), 3 (3.1%) had MPL mutations, and 4 (4.2%) were negative for all 3. ASXL1/JAK2 and TET2/JAK2 were the most frequently comutated genes. Mutations in NRAS, KRAS, PTPN11, GATA2, TP53, and RUNX1 were found in <5% of patients. Spleen response (≥50% reduction in palpable spleen size) was inversely correlated with the number of mutations; patients with ≤2 mutations had ninefold higher odds of a spleen response than those with ≥3 mutations (odds ratio = 9.37; 95% confidence interval, 1.86-47.2). Patients with ≥3 mutations also had a shorter time to treatment discontinuation and shorter overall survival than those with fewer mutations. In multivariable analysis, only number of mutations and spleen response remained associated with time to treatment discontinuation. Patients with ≥3 mutations had the worst outcomes, suggesting that multigene profiling may be useful for therapeutic planning for MF.

MicroRNA-196a is a potential marker of progression during Barrett's metaplasia-dysplasia-invasive adenocarcinoma sequence in esophagus.

Barrett's esophagus (BE)/Barrett's metaplasia (BM) is a recognized precursor of esophageal adenocarcinoma (EA) with an intermediary stage of dysplasia. The low yield and high cost of endoscopic screening of patients with BE underscores the need for novel biomarkers, such as microRNA (miRNA), which have emerged as important players in neoplastic progression for risk assessment of developing dysplasia/adenocarcinoma. Recently, we reported highly elevated levels of miRNA-196a (miR-196a) in EA and demonstrated its growth-promoting and anti-apoptotic functions. Here, we evaluated miR-196a as a marker of BE progression to low-grade dysplasia, high-grade dysplasia, and EA using microdissected paraffin-embedded tissues from 11 patients. Higher levels of miR-196a were observed in EA, BE, and dysplastic lesions compared with normal squamous mucosa, and in high-grade dysplasia compared with BE and low-grade dysplasia. Using frozen tumor tissues from 10 additional patients who had advanced EA, we evaluated the correlation of miR-196a with its in silico-predicted targets, keratin 5 (KRT5), small proline-rich protein 2C (SPRR2C), and S100 calcium-binding protein A9 (S100A9), which are down-regulated during BE progression. MiR-196a levels inversely correlated with the predicted target mRNA levels in EA. We confirmed that miR-196a specifically targets KRT5, SPRR2C, and S100A9 3' UTRs using miR-196a-mimic and luciferase reporter-based assays. In conclusion, this study identified miR-196a as a potential marker of progression of BE and KRT5, SPRR2C, and S100A9 as its targets.

Splenic marginal zone lymphomas are characterized by loss of interstitial regions of chromosome 7q, 7q31.32 and 7q36.2 that include the protection of telomere 1 (POT1) and sonic hedgehog (SHH) genes.

To further characterize the genotypic features of splenic (S) and nodal (N) marginal zone lymphomas (MZL) we compared eight SMZL and five NMZL by array-based comparative genomic hybridization (aCGH). Arbitrarily, aberrations were divided into major imbalances, defined as gains or losses involving five or more contiguous genetic loci, and minor imbalances, defined as those involving four or fewer loci. SMZL, but not NMZL, demonstrated major imbalances. These included deletions involving various lengths of 7q (three cases), and 14q23q24 (one case) and gains of 9p13p21 (one case), 13q21q33 (one case) and 16p13.1 (one case). Common minor imbalances in SMZL were: loss of sonic hedgehog gene (SHH) at 7q36.2 (four cases), loss of protection of telomere 1 gene (POT1) at 7q31.32 (three cases), and gain of glioma associated oncogene 1 (GLI1) at 12q13.2 (three cases). Common minor alterations in NMZL were: loss of the fas-associated via death domain gene (FADD) at 11q13.2 (three cases) and gain of GLI1 (five cases). In conclusion, SMZL, but not NMZL, demonstrates large genomic imbalances and frequent loss of the 7q31.32 and 7q36.2 regions involving POT1 and SHH, respectively. In NMZL, loss of FADD and gain of GLI1 are frequent events.

Identification and validation of biomarkers of IgV(H) mutation status in chronic lymphocytic leukemia using microfluidics quantitative real-time polymerase chain reaction technology.

To develop a model incorporating relevant prognostic biomarkers for untreated chronic lymphocytic leukemia patients, we re-analyzed the raw data from four published gene expression profiling studies. We selected 88 candidate biomarkers linked to immunoglobulin heavy-chain variable region gene (IgV(H)) mutation status and produced a reliable and reproducible microfluidics quantitative real-time polymerase chain reaction array. We applied this array to a training set of 29 purified samples from previously untreated patients. In an unsupervised analysis, the samples clustered into two groups. Using a cutoff point of 2% homology to the germline IgV(H) sequence, one group contained all 14 IgV(H)-unmutated samples; the other contained all 15 mutated samples. We confirmed the differential expression of 37 of the candidate biomarkers using two-sample t-tests. Next, we constructed 16 different models to predict IgV(H) mutation status and evaluated their performance on an independent test set of 20 new samples. Nine models correctly classified 11 of 11 IgV(H)-mutated cases and eight of nine IgV(H)-unmutated cases, with some models using three to seven genes. Thus, we can classify cases with 95% accuracy based on the expression of as few as three genes.

Molecular mechanisms in Barrett's metaplasia and its progression.

The dramatic increase in the incidence and poor overall survival rates of esophageal/gastroesophageal junction adenocarcinoma underscore the necessity to discover molecular markers that can be used for risk assessment, early diagnosis, and targeted therapeutic intervention. Barrett's esophagus (BE) is proposed to represent a precursor of esophageal/gastroesophageal junction adenocarcinoma. BE progression to invasive cancer is defined by a metaplasia-dysplasia-carcinoma progression characterized by an increasing accumulation of genetic changes associated with alterations in molecular gatekeepers of cell circuitries and tissue homeostasis. Using a combination of in situ tissue-based and high-throughput analyses, we investigated alterations of cell-cycle regulators and inflammation-associated molecular effectors. Our data suggest a potential synergistic effect of these alterations for the BE progression to cancer, and underscore the potential use of these markers: (1) in molecular panels assessing cancer risk in BE patients; and (2) as potential therapeutic targets for chemopreventive interventions and to enhance response to anti-neoplastic therapies.

Decreased expression of gene cluster at chromosome 1q21 defines molecular subgroups of chemoradiotherapy response in esophageal cancers.

PURPOSE: Resistance to preoperative chemoradiotherapy (CTXRT) in 75% of patients with esophageal adenocarcinoma (EAC) underscores the need for identification of biomarkers of CTXRT response. We previously noted an association between decreased expression of epidermal differentiation complex (EDC) genes S100A2 and SPRR3 at chromosome 1q21 and CTXRT resistance. In the current study, we did an in-depth investigation of the expression of 1q21-1q25 region genes to uncover the role of the EDC and its flanking genes in CTXRT response. EXPERIMENTAL DESIGN: We compared 19 pretreatment EAC specimens with normal squamous mucosa for the expression of 517 genes at chromosome 1q21-1q25 and selected target genes based on their differential expression. Using the pathologic complete-response (pathCR) status of the resected specimens as a representation of CTXRT sensitivity, we assessed the association between the expression of target genes and CTXRT response and clinical outcomes. RESULTS: On the basis of the expression levels of IVL, CRNN, NICE-1, S100A2, and SPPR3, genes within and in close proximity to the EDC, cancers were segregated into high (subgroup I) or low (subgroup II) expressers. Four of the five pathCRs were high expressers. Thus, low expressers, with one exception, were all nonresponders. Patients in subgroup I also had longer survival than those in subgroup II, although this result was not statistically significant owing to the small study number. CONCLUSIONS: The expression levels of genes mapping within and close to the EDC define CTXRT response subgroups in EACs.

Emerging molecular targets in esophageal cancers.

The phenotypic progression to esophageal cancer is driven by an ongoing process of genomic instability constituting a number of clonal variants and leading to outgrowth of the "fittest" abnormal cell clones. Factors contributing to this process include exposure to chronic tissue damage, host susceptibilities, and alterations of molecular circuitries implicated in tissue homeostasis. Characterization of the host modifiers and molecular alterations will likely lead to the discovery of biomarkers useful for constructing stratified models defining cancer risk, allowing early detection, prediction of response to primary or secondary intervention, and prognostic evaluation of the disease. In addition, identification of key biologic pathways driving esophageal tumorigenesis will lead to development of new targeted interventions. The advent of increasingly sophisticated "omics" (ie, genomics, transcriptomics, proteomics, kinomics, pharmacogenomics), integration of systems biology, and expansion of biologic platforms bridging developmental physio-biology to cancer pathology constitute the backbone of novel tumor classifications and tailored therapies based on molecular signatures and profiles. Promising molecular targets, particularly those implicated in tissue homeostasis and stem cell maintenance, and their potential use in predictive models will be discussed.

Biomarkers of response to preoperative chemoradiation in esophageal cancers.

To identify a panel of biomarkers that predicts response of esophageal cancers to preoperative chemoradiation, our group profiled the gene expression of pretreatment cancer biopsies from patients with esophageal cancer. Six (32%) of these patients had pathologic complete response. All cancers except one that achieved pathologic complete response (83%) clustered in one molecular type (type I), while cancers that achieved less than pathologic complete response with one exception clustered in another molecular type (type II). Activated NF-kappaB was significantly associated with aggressive pathology. These data indicate that expression analysis of a limited set of biomarkers selected from the list of genes that were differentially regulated between the two subtypes can increase predictive power, and suggest that esophageal cancer with activated NF-kappaB may result in poor treatment outcome.

Gene expression profiling of localized esophageal carcinomas: association with pathologic response to preoperative chemoradiation.

PURPOSE: Patients with localized esophageal carcinoma have a 5-year survival rate of less than 20%. Patients are often treated similarly (ie, with preoperative chemoradiotherapy) but the outcomes vary greatly. Chemoradiotherapy and surgery can result in significant undesirable consequences. Currently, however, there are no tools to help select optimum therapy. We hypothesized that gene expression profiling could provide clues and biomarkers for selection of therapy. METHODS: Pretreatment endoscopic cancer biopsies from 19 patients (16 with adenocarcinoma, two with squamous cell carcinoma, and one with adenosquamous carcinoma) enrolled onto a preoperative chemoradiotherapy protocol were profiled using oligonucleotide microarrays. Surgical specimens following therapy were assessed for the degree of pathologic response. On the basis of array data, selected genes were analyzed by polymerase chain reaction. RESULTS: Unsupervised hierarchical cluster analysis segregated the cancers into two molecular subtypes, each consisting 10 and nine specimens, respectively. Most cancers (five of six) that had pathologic complete response (pathCR) clustered in molecular subtype I. Subtype II, with one exception, consisted cancers that had less than pathCR (< pathCR). Using a combination marker approach, levels of PERP, S100A2, and SPRR3 allowed discrimination of pathCR from < pathCR with high sensitivity and specificity (85%). Pathway analysis identified apoptotic pathway as one of the key functions downregulated in molecular type II in comparison with type I. CONCLUSION: These encouraging, albeit preliminary, data suggest that expression profiling may distinguish cancers with different pathologic outcome. This is the first report to show subtypes of esophageal cancers with distinct molecular signatures. The potential of PERP, S100A2, and SPRR3 as biomarkers of pathCR warrants further validation.