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.

In the present study, we compared mesenchymal stem cells (MSCs) derived from 4 different sources, human bone marrow (BM), adipose tissue (AT), umbilical cord Wharton’s Jelly (WJ) and the placenta (PL), in order to determine which population of MSCs displayed the most prominent immunosuppressive effects on phytohemagglutinin-induced T cell proliferation, and which one had the highest proliferative and differentiation potential. MSC and T lymphocyte co-culture (mixed culture) was used to determine whether the MSCs inhibit T cell proliferation, as well as which population of MSCs has the strongest inhibitory ability. The expression of immune-related genes was analyzed by RT-PCR and RT-qPCR. The proliferation and differentiation potential of the MSCs were determined using standard methods. Following MSC and T cell co-culture, mitogen-induced T cell proliferation was effectively suppressed by all 4 populations of MSCs. This occurred through soluble factors rather than direct contact inhibition. Among the 4 populations of MSCs, the WJ-MSC has the strongest suppression effects. On immune related genes, WJ-MSC has the weakest expression of MHC II genes, TLR4, TLR3, JAG1, NOTCH2 and NOTCH3. To compare the proliferation potential, WJ-MSCs showed the most rapid growth rate followed by the AT-, PL- and BM-MSCs. As regards differentiation potential, the WJ-MSCs had the strongest osteogenetic ability followed by PL, AT and BM-MSC. AT-MSC has the strongest adipogenetic ability followed by the WJ-, BM- and PL-MSCs. These data indicated that the WJ-MSCs had the strongest immunomodulatory and immunosuppressive potential. In light of these observations, we suggest that WJ-MSCs are the most attractive cell population for use in immune cellular therapy when immunosuppressive action is required.

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. © 2001 Wiley-Liss, Inc. Key words: microsatellite instability; hMLH1; hMSH2; squamouscell carcinoma; head-and-neck cancerHead-and-neck squamous-cell carcinoma (HNSCC) is a relatively common neoplasm, accounting for 2% to 7% of all tumors in the Western Hemisphere. 1,2 The most prevalent risk factors in this cancer are excessive tobacco and alcohol consumption. 3 However, in young patients with HNSCC, these risk factors are not always present, implicating other factors in the etiology of their disease. Microsatellite sequences are 2 to 5 nucleotide repeats that can be found scattered throughout the eukaryotic genome. They have been widely used as genetic markers due to their high degree of polymorphism. Microsatellite instability (MSI) is characterized by expansion or contraction in the length of short tandem repeats and has been associated with various cancer types and inheritable diseases. MSI was initially described in hereditary non-polyposis colorectal cancer (HNPCC) as well as in sporadic colorectal tumors. 4 In HNPCC, a replication error phenotype (RER ϩ ) has been associated with mutations in the hMSH2, hMLH1, hPMS1 and hPMS2 genes, which are involved in mismatch repair (MMR) mechanisms. 4 -7 The RER ϩ phenotype has also been described in other tumor types, such as gastric, 8 -13 lung, 14,15 ovary, 16,17 pancreatic, 18 endometrial 19,20 and HNSCC. [21][22][23][24][25][26][27] MSI in head-and-neck carcinoma has been found in tumors at a frequency of 7% to 30%. 21,25,28 Head-and-neck tumors generally occur in older men (Ͼ50 years) with a history of smoking and/or alcohol abuse. As in...