Yali Xuan

mRNA transcript quantification in archival samples using multiplexed, color-coded probes

BackgroundA recently developed probe-based technology, the NanoString nCounter™ gene expression system, has been shown to allow accurate mRNA transcript quantification using low amounts of total RNA. We assessed the ability of this technology for mRNA expression quantification in archived formalin-fixed, paraffin-embedded (FFPE) oral carcinoma samples.ResultsWe measured the mRNA transcript abundance of 20 genes (COL3A1, COL4A1, COL5A1, COL5A2, CTHRC1, CXCL1, CXCL13, MMP1, P4HA2, PDPN, PLOD2, POSTN, SDHA, SERPINE1, SERPINE2, SERPINH1, THBS2, TNC, GAPDH, RPS18) in 38 samples (19 paired fresh-frozen and FFPE oral carcinoma tissues, archived from 1997-2008) by both NanoString and SYBR Green I fluorescent dye-based quantitative real-time PCR (RQ-PCR). We compared gene expression data obtained by NanoString vs. RQ-PCR in both fresh-frozen and FFPE samples. Fresh-frozen samples showed a good overall Pearson correlation of 0.78, and FFPE samples showed a lower overall correlation coefficient of 0.59, which is likely due to sample quality. We found a higher correlation coefficient between fresh-frozen and FFPE samples analyzed by NanoString (r = 0.90) compared to fresh-frozen and FFPE samples analyzed by RQ-PCR (r = 0.50). In addition, NanoString data showed a higher mean correlation (r = 0.94) between individual fresh-frozen and FFPE sample pairs compared to RQ-PCR (r = 0.53).ConclusionsBased on our results, we conclude that both technologies are useful for gene expression quantification in fresh-frozen or FFPE tissues; however, the probe-based NanoString method achieved superior gene expression quantification results when compared to RQ-PCR in archived FFPE samples. We believe that this newly developed technique is optimal for large-scale validation studies using total RNA isolated from archived, FFPE samples.

High frequency of microsatellite instability in young patients with head-and-neck squamous-cell carcinoma: Lack of involvement of the mismatch repair genes hMLH1 AND hMSH2

The most prevalent risk factors in the development of head‐and‐neck squamous‐cell carcinoma (HNSCC) are excessive tobacco and alcohol consumption. In young patients with HNSCC, these risk factors are often absent. Our purpose was to investigate the risk factors, microsatellite instability (MSI) changes and status of the mismatch repair genes hMLH1 and hMSH2 in a cohort of young patients with HNSCC. Fifty‐seven HNSCC tumors were examined for the presence of MSI at 16 microsatellite sites using PCR. In the young patient group (24 cases, ≤44 years old), 100% of tumors had MSI at 1 site at least and 88% had MSI at 2 or more loci. In older patients (33 cases, ≥45 years), MSI at 1 or more sites was found in 61% of tumors (young vs. old, p = 0.0003) and instability at 2 or more sites was found in 36% of tumors (young vs. old, p = 0.0001). The involvement of the mismatch repair genes was investigated by examining promoter methylation, exon mutation and gene expression of hMLH1 and hMSH2. All results were negative, indicating that inactivation of these 2 genes does not play a role in the development of MSI in tumors from this patient group. Furthermore, the young patient group had a significantly lower incidence of smoking (46% young, 88% old; p = 0.001) and alcohol consumption (33% young, 67% old; p = 0.0169), emphasizing the probable importance of other environmental and/or genetic factors in the development of their disease.

A gene signature in histologically normal surgical margins is predictive of oral carcinoma recurrence

BackgroundOral Squamous Cell Carcinoma (OSCC) is a major cause of cancer death worldwide, which is mainly due to recurrence leading to treatment failure and patient death. Histological status of surgical margins is a currently available assessment for recurrence risk in OSCC; however histological status does not predict recurrence, even in patients with histologically negative margins. Therefore, molecular analysis of histologically normal resection margins and the corresponding OSCC may aid in identifying a gene signature predictive of recurrence.MethodsWe used a meta-analysis of 199 samples (OSCCs and normal oral tissues) from five public microarray datasets, in addition to our microarray analysis of 96 OSCCs and histologically normal margins from 24 patients, to train a gene signature for recurrence. Validation was performed by quantitative real-time PCR using 136 samples from an independent cohort of 30 patients.ResultsWe identified 138 significantly over-expressed genes (> 2-fold, false discovery rate of 0.01) in OSCC. By penalized likelihood Cox regression, we identified a 4-gene signature with prognostic value for recurrence in our training set. This signature comprised the invasion-related genes MMP1, COL4A1, P4HA2, and THBS2. Over-expression of this 4-gene signature in histologically normal margins was associated with recurrence in our training cohort (p = 0.0003, logrank test) and in our independent validation cohort (p = 0.04, HR = 6.8, logrank test).ConclusionGene expression alterations occur in histologically normal margins in OSCC. Over-expression of the 4-gene signature in histologically normal surgical margins was validated and highly predictive of recurrence in an independent patient cohort. Our findings may be applied to develop a molecular test, which would be clinically useful to help predict which patients are at a higher risk of local recurrence.

Myeloid leukemia with promyelocytic features in transgenic mice expressing hCG-NuMA-RARα

Acute promyelocytic leukemia (APL) is characterized by the accumulation of abnormal promyelocytes in the bone marrow (BM), and by the presence of a reciprocal chromosomal translocation involving retinoic acid receptor alpha (RARα). To date, five RARα partner genes have been identified in APL. NuMA-RARα was identified in a pediatric case of APL carrying a translocation t(11;17)(q13;q21). Using a construct containing the NuMA-RARα fusion gene driven by the human cathepsin G promoter (hCG-NuMA-RARα), two transgenic mouse lines were generated. Transgenic mice were observed to have a genetic myeloproliferation (increased granulopoiesis in BM) at an early age, and rapidly developed a myeloproliferative disease-like myeloid leukemia. This leukemia was morphologically and immunophenotypically indistinguishable from human APL, with a penetrance of 100%. The phenotype of transgenic mice was consistent with a blockade of neutrophil differentiation. NuMA-RARα is therefore sufficient for disease development in this APL model.

MicroRNA Signature Obtained From the Comparison of Aggressive With Indolent Non-Hodgkin Lymphomas: Potential Prognostic Value in Mantle-Cell Lymphoma

PURPOSE Mantle-cell lymphoma (MCL) has a variable natural history but is incurable with current therapies. MicroRNAs (miRs) are useful in prognostic assessment of cancer. We determined an miR signature defining aggressiveness in B-cell non-Hodgkin lymphomas (NHL) and assessed whether this signature aids in MCL prognosis. METHODS We assessed miR expression in a training set of 43 NHL cases. The miR signature was validated in 44 additional cases and examined on a training set of 119 MCL cases from four institutions in Canada. miRs significantly associated with overall survival were examined in an independent cohort of 114 MCL cases to determine association with patient outcome. miR expression was combined with current clinical prognostic factors to develop an enhanced prognostic model in patients with MCL. RESULTS Fourteen miRs were differentially expressed between aggressive and indolent NHL; 11 of 14 were validated in an independent set of NHL (excluding MCL). miR-127-3p and miR-615-3p were significantly associated with overall survival in the MCL training set. Their expression was validated in an independent MCL patient set. In comparison with Ki-67, expression of these miRs was more significantly associated with overall survival among patients with MCL. miR-127-3p was combined with Ki-67 to create a new prognostic model for MCL. A similar model was created with miR-615-3p and Mantle Cell Lymphoma International Prognostic Index scores. CONCLUSION Eleven miRs are differentially expressed between aggressive and indolent NHL. Two novel miRs were associated with overall survival in MCL and were combined with clinical prognostic models to generate novel prognostic data for patients with MCL.

Evidence of functional interaction between NuMA-RARα and RXRα in an in vivo model of acute promyelocytic leukemia

Acute promyelocytic leukemia (APL) is characterized by reciprocal balanced chromosomal translocations involving retinoic acid receptor-α (RARα). RARα heterodimerizes with the retinoid X receptor-α (RXRα) and transcriptionally regulates myeloid differentiation in response to ATRA (all-trans retinoic acid). Several lines of evidence suggest that APL fusion proteins interact with RXRα. To elucidate the role of RXRα in APL, we conditionally knocked out RXRα in the hCG-NuMA-RARα APL mouse model. Phenotype analysis of NuMA-RARα+ mice demonstrated that these mice developed a myeloproliferative disease-like myeloid leukemia within 4 months of birth. While hemizygous and homozygous RXRα conditional knockout mice were phenotypically normal as late as 12 months of age, we observed that the leukemic phenotype in NuMA-RARα+ mice was dependent on the presence of functional RXRα. Bone marrow promyelocyte counts were significantly reduced in NuMA-RARα+ mice with RXRα knocked down. Significant differences in the accumulations of Gr-1+ and Mac-1+ cells were also seen. We further observed that genes previously identified to be cooperating events in APL were also regulated in an RXRα-dependent manner. We therefore propose that the APL fusion protein NuMA-RARα cooperates with RXRα in the development of leukemia in hCG-NuMA-RARα transgenic mice and suggest a novel role for RXRα in the pathogenesis of APL.

Correlation among nuclear localization of NuMA-RARα, deregulation of gene expression and leukemic phenotype of hCG-NuMA-RARα transgenic mice.

Acute promyelocytic leukemia (APL) is a model system of aberrant transcription in cancer. We sought to elucidate the mechanism of action of the variant fusion NuMA-RARα in APL, using the hCG-NuMA-RARα transgenic model. We report that subcellular localization of NuMA-RARα in transgenic mice is dependent upon its protein expression and transgene dosage. Subcellular localization of the fusion is inversely correlated with extent of gene deregulation at the mRNA level for Cebpα, Cebpɛ and Pu.1. Finally, we report that phenotype onset is correlated with NuMA-RARα copy number; mice with higher copy number developing disease later than those with lower copy number.

Abstract 1240: Functional deregulation of NF-kB and abnormal TNFa response in acute promyelocytic leukemia

Acute promyelocytic leukemia (APL) accounts for approximately 10% of acute myelogenous leukemia (AML) cases, and is characterized by accumulation of abnormal promyelocytes in patient bone marrow and peripheral blood. APL is associated with balanced chromosomal translocations involving retinoic acid receptor alpha (RARA), giving rise to fusion oncoproteins referred to as X-RARA. As deregulation of retinoid signaling is insufficient for leukemia development, our studies aim to determine other signaling pathways involved in APL by assessing the gene expression profiles and cell biology of X-RARA. We previously determined, using gene expression microarray analysis, common downstream targets of the variant APL fusion proteins NPM- and NuMA-RARA. We observed an over-representation of NF-κB target genes within this dataset. In these cells, a number of NF-κB target genes were commonly over-expressed. A subset of commonly deregulated genes were validated in our APL cell lines and 23 primary APL patient samples by real-time quantitative RT-PCR (RQ-PCR). 13/16 genes that were tested were significantly altered in APL compared to normal BM (n=11) p + cells to survive and proliferate in the presence of TNFα. Our data provides the first evidence of the functional deregulation of the NF-κB-mediated signaling pathway and the TNFα response in cells expressing the variant APL fusion proteins. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1240.