Microsatellite Alterations Research Articles

How strong is the association between IPF and lung cancer? An answer from airway's DNA.

Idiopathic pulmonary fibrosis is a chronic progressive disease of lung interstitium of unknown etiology with poor prognosis. In patients with IPF, the incidence of lung cancer is much higher than that in the general population. The identification of noninvasive biomarkers for early diagnosis of IPF is of great relevance in consideration of the management of these patients. Among the noninvasive omic markers, an increasing interest has been directed toward the study of genetic alterations of microsatellites (MAs) in exhaled breath condensate (EBC). The aim of this preliminary study was to investigate the MAs, located in chromosomal regions 8p21.3-q11.1 and 17q11.2-q21, that harbor tumor suppressor genes, in EBC and in the paired whole blood (WB) of IPF patients. Eleven IPF patients were compared with 10 healthy control subjects. All subjects underwent collection of the EBC and WB. The EBC was collected using a condenser. Four microsatellite markers (THRA1, D17S579, D17S250 and D8S137) were used for the analysis of MAs. The EBC-DNA and WB-DNA were amplified by PCR; PCR products were analyzed using the ABI Prism 310 DNA. Microsatellite alterations were found in 58.82% of EBC-DNA and 12.50% of WB-DNA in patients with IPF (p<0.01). None of the healthy subjects exhibited MAs in the studied markers. Our findings suggest that these genetic alterations, studied in EBC, may play an important role in the complex genetic basis of IPF. Since these MAs are frequently detected in cancer, they might explain the higher relative risk of tumorigenesis in this disease.

Microsatellite alteration and immunohistochemical expression profile of chromosome 9p21 in patients with sporadic renal cell carcinoma following surgical resection.

BackgroundLong-term prognostic significance of loss of heterozygosity on chromosome 9p21 for localized renal cell carcinoma following surgery remains unreported. The study assessed the frequency of deletions of different loci of chromosome 9p along with immunohistochemical profile of proteins in surgically resected renal cancer tissue and correlated this with long-term outcomes.MethodsDNA was extracted from renal tumours and corresponding normal kidney tissues in prospectively collected samples of 108 patients who underwent surgical resection for clinically localized disease between January 2001 and December 2005, providing a minimum of 9 years follow-up for each participant. After checking quality of DNA, amplified by PCR, loss of heterozygosity (LOH) on chromosome 9p was assessed using 6 microsatellite markers in 77 clear cell carcinoma. Only 5 of the markers showed LOH (D9S1814, D9S916, D9S974, D9S942, and D9S171). Protein expression of p15(INK4b), p16(INK4a), p14(ARF), CAIX, and adipose related protein (ADFP) were demonstrated by immunostaining in normal and cancer tissues. Loss of heterozygosity for microsatellite analysis was correlated with tumour characteristics, recurrence free, cancer specific, and overall survival, including significance of immunohistochemical profile of protein expressions.ResultsThe main deletion was found at loci telomeric to CDKN2A region at D9S916. There was a significant correlation between frequency of LOH stage (p = 0.005) and metastases (p = 0.006) suggesting a higher LOH for advanced and aggressive renal cell carcinoma. Most commonly observed LOH in the 3 markers: D9S916, D9S974, and D9S942 were associated with poor survival, and were statistically significant on multivariate analysis. Immunohistochemical expression of p14, p15, and p16 proteins were either low or absent in cancer tissue compared to normal.ConclusionsLoss of heterozygosity of p921 chromosome is associated with aggressive tumours, and predicts cancer specific or recurrence free survival on long-term follow-up.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2514-8) contains supplementary material, which is available to authorized users.

Impact of microsatellite alteration in surgical margins on local recurrence in oral cavity cancer patients.

The aim of this study was to investigate the association between microsatellite alteration in the surgical margins and local recurrence of oral cavity squamous cell carcinoma patients. Surgical specimens confirmed by pathological examination and corresponding surgical margins were collected from 120 oral cavity squamous cell carcinoma patients. Ten microsatellite markers were examined in the tumor specimens and paired surgical margins, which proved to be negative on pathological assessment. The specimens and surgical margins were amplified by polymerase chain reaction followed by computerized analysis. Forty-two specimens (35.0%) with microsatellite instability (MSI) in at least one marker were found, and more than half of the specimens (n=73, 60.8%) had loss of heterozygosity (LOH) in at least one marker. Although MSI and LOH were not associated with the prognosis of oral cavity squamous cell carcinoma patients, presence of MSI in the tumor-free surgical margins increased the risk of local recurrence (hazard ratio: 9.549; 95% confidence interval: 4.143-22.01). Genetic analysis of tumor-free surgical margins is a useful tool for identifying oral cavity squamous cell carcinoma patients who are vulnerable to local recurrence.

Assessment of clinically related outcomes and biomarker analysis for translational integration in colorectal cancer (ACROBATICC): study protocol for a population-based, consecutive cohort of surgically treated colorectal cancers and resected colorectal liver metastasis.

BackgroundMore accurate predictive and prognostic biomarkers for patients with colorectal cancer (CRC) primaries or colorectal liver metastasis (CLM) are needed. Outside clinical trials, the translational integration of emerging pathways and novel techniques should facilitate exploration of biomarkers for improved staging and prognosis.MethodsAn observational study exploring predictive and prognostic biomarkers in a population-based, consecutive cohort of surgically treated colorectal cancers and resected colorectal liver metastases. Long-term outcomes will be cancer-specific survival, recurrence-free survival and overall survival at 5 years from diagnosis. Beyond routine clinicopathological and anthropometric characteristics and laboratory and biochemistry results, the project allows for additional blood samples and fresh-frozen tumour and normal tissue for investigation of circulating tumour cells (CTCs) and novel biomarkers (e.g. immune cells, microRNAs etc.). Tumour specimens will be investigated by immunohistochemistry in full slides. Extracted DNA/RNA will be analysed for genomic markers using specific PCR techniques and next-generation sequencing (NGS) panels. Flow cytometry will be used to characterise biomarkers in blood. Collaboration is open and welcomed, with particular interest in mutual opportunities for validation studies.Status and perspectivesThe project is ongoing and recruiting at an expected rate of 120–150 patients per year, since January 2013. A project on circulating tumour cells (CTCs) has commenced, with analysis being prepared. Investigating molecular classes beyond the TNM staging is under way, including characteristics of microsatellite instability (MSI) and elevated microsatellite alterations in selected tetranucleotides (EMAST). Hot spot panels for known mutations in CRC are being investigated using NGS. Immune-cell characteristics are being performed by IHC and flow cytometry in tumour and peripheral blood samples. The project has ethical approval (REK Helse Vest, #2012/742), is financially supported with a Ph.D.-Grant (EMAST project; Folke Hermansen Cancer Fund) and a CTC-project (Norwegian Research Council; O. Nordgård). The ACROBATICC clinical and molecular biobank repository will serve as a long-term source for novel exploratory analysis and invite collaborators for mutual validation of promising biomarker results. The project aims to generate results that can help better discern prognostic groups in stage II/III cancers; explore prognostic and predictive biomarkers, and help detail the biology of colorectal liver metastasis for better patient selection and tailored treatment. The project is registered at http://www.ClinicalTrials.gov NCT01762813.

Elevated microsatellite alterations at selected tetranucleotides in early-stage colorectal cancers with and without high-frequency microsatellite instability: same, same but different?

Microsatellite instability (MSI) is associated with better prognosis in colorectal cancer (CRC). Elevated microsatellite alterations at selected tetranucleotides (EMAST) is a less‐understood form of MSI. Here, we aim to investigate the role of EMAST in CRC±MSI related to clinical and tumor‐specific characteristics. A consecutive, population‐based series of stage I–III colorectal cancers were investigated for MSI and EMAST using PCR primers for 10 microsatellite markers. Of 151 patients included, 33 (21.8%) had MSI and 35 (23.2%) were EMAST+, with an overlap of 77% for positivity, (odds ratio [OR] 61; P < 0.001), and 95% for both markers being negative. EMAST was more prevalent in colon versus rectum (86% vs. 14%, P = 0.004). EMAST+ cancers were significantly more frequent in proximal colon (77 vs. 23%, P = 0.004), had advanced t‐stage (T3–4 vs. T1–2 in 94% vs. 6%, respectively; P = 0.008), were larger (≥5 cm vs. <5 cm in 63% and 37%, respectively; P = 0.022) and had poorly differentiated tumor grade (71 vs. 29%, P < 0.01). Furthermore, EMAST+ tumors had a higher median number of harvested lymph nodes than EMAST− (11 vs. 9 nodes; P = 0.03). No significant association was found between EMAST status and age, gender, presence of distant metastases or metastatic lymph nodes, and overall survival. A nonsignificant difference toward worse survival in node‐negative colon cancers needs confirmation in larger cohorts. EMAST+ cancers overlap and share features with MSI+ in CRC. Overall, survival was not influenced by the presence of EMAST, but may be of importance in subgroups such as node‐negative disease of the colon.

Microsatellite alteration in head and neck squamous cell carcinoma patients from a betel quid-prevalent region.

We investigated the frequency of microsatellite alteration and their impact on survival in head and neck squamous cell carcinoma patients from an endemic betel quid chewing area. We collected 116 head and neck squamous cell carcinoma specimens along with corresponding surgical margins which were confirmed by pathological examination. Ten oligonucleotide markers were chosen for the assessment of microsatellite alteration. The specimens were amplified by polymerase chain reaction followed by automatic fragment analysis. There were 44 specimens (37.9%) with microsatellite instability (MSI) in at least one marker while more than half of the specimens (n = 68, 58.6%) had loss of heterozygosity (LOH) in at least one marker. Though MSI/LOH was not correlated with the survival of head and neck squamous cell carcinoma patients, presence of MSI in the tumor-free surgical margins was associated with local recurrence (odds ratio: 15.14; 95% confidence interval: 6.451 ~ 35.53; P < 0.001). Genomic assessment of surgical margin can help surgeons to identify head and neck squamous cell carcinoma patients who are at risk of developing local recurrence in a betel quid-prevalent region.

Microsatellite Alterations With Allelic Loss at 9p24.2 Signify Less-Aggressive Colorectal Cancer Metastasis

Molecular events that lead to recurrence and/or metastasis after curative treatment of patients with colorectal cancers (CRCs) are poorly understood. Patients with stage II or III primary CRC with elevated microsatellite alterations at selected tetranucleotide repeats and low levels of microsatellite instability (E/L) are more likely to have disease recurrence after treatment. Hypoxia and/or inflammation not only promote metastasis, but also induce elevated microsatellite alterations at selected tetranucleotide repeats by causing deficiency of MSH3 in the cancer cell nucleus. We aimed to identify genetic alterations associated with metastasis of primary colorectal tumors to liver and to determine their effects on survival. We obtained 4 sets of primary colorectal tumors and matched liver metastases from hospitals in Korea and Japan. Intragenic microsatellites with large repeats at 141 loci were examined for frame-shift mutations and/or loss of heterozygosity (LOH) as possible consequences of MSH3 deficiency. Highly altered loci were examined for association with E/L in liver metastases. We analyzed data from 156 of the patients with stage II or III primary colorectal tumors to determine outcomes and whether altered loci were associated with E/L. LOH at several loci at chromosome 9p24.2 (9p24.2-LOH) was associated with E/L in liver metastases (odds ratio= 10.5; 95% confidence interval: 2.69-40.80; P= .0007). We found no significant difference in the frequency of E/L, 9p24.2-LOH, mutations in KRAS or BRAF, or the combination of E/L and 9p24.2-LOH, between primary colorectal tumors and their matched metastases. Patients with stage II or III colorectal tumors with E/L and 9p24.2-LOH had increased survival after CRC recurrence (hazard ratio= 0.25; 95% CI: 0.12-0.50; P= .0001), compared with patients without with E/L and 9p24.2-LOH. E/L with 9p24.2-LOH appeared to be an independent prognostic factor for overall survival of patients with stage III CRC (hazard ratio= 0.06; 95% CI: 0.01-0.57; P= .01). E/L with 9p24-LOH appears to be a biomarker for less aggressive metastasis from stage III primary colorectal tumors.

New panel of microsatellite alterations detectable in the EBC for lung cancer prognosis.

Our research group demonstrated, in a precedent study, the prognostic power of the 3p microsatellites alterations (MAs) detectable in exhaled breath condensate (EBC) in NSCLC patients. The analysis of genetic markers in the EBC might have precious clinical and economic consequences when inserted in diagnostic and follow up programs for lung cancer.The aim of this study was to evaluate the prognostic value of a new panel of MAs in the EBC of patients with NSCLC.We enrolled 45 NSCLC patients during a period of 36 months and the follow-up period was 156 weeks. We analyzed MAs for eight markers in EBC samples: D3S2338, D3S1266, D3S1300, D3S1304, D3S1289, D5S2094, D3S1313, and AFMa305ye1.Our study showed that the presence of more than 2 simultaneous MAs reduces outcome in NSCLC patients. The new panel of eight microsatellites markers proposed in EBC samples could have a potential clinical role in assessing survival in lung cancer patients.

Advances in Diagnosis and Treatment of Multiple Primary Lung Cancer

多原发性肺癌的发病率和检出率逐年升高。目前临床上诊断多原发性肺癌(multiple primary lung cancer, MPLC)主要参照Martini-Melamed标准和美国胸科医师协会(American College of Chest Physicians, ACCP)标准, 综合考虑临床表现、影像学特征、组织学类型和分子遗传学特征。组织学类型不同的MPLC诊断相对容易, 而组织学类型相同的MPLC诊断仍相当困难。DNA倍体分析、基因突变检测、微卫星多态性分析等分子生物学技术为MPLC的正确诊断提供了新手段, 可评估各病灶的克隆性关系, 帮助鉴别MPLC与转移。MPLC的首选治疗方案为根治性手术, 术式应考虑患者肺功能储备等因素, 选择肺叶切除、肺段切除或楔形切除; 对于不能根治性切除的病灶, 可综合化疗、放疗、立体定向放疗(stereotactic ablative radiotherapy, SABR)、射频消融(radiofrequency ablation, RFA)、分子靶向治疗等。

Modal variety of microsatellite instability in human endometrial carcinomas.

PurposeMicrosatellite instability (MSI) in human endometrial cancer (EC) was analysed using a unique fluorescent technique. MSI is associated with various human neoplasms. However, the reported frequency of MSI differs widely in each malignancy. Methodological difficulties have in fact been pointed out in its assay techniques.MethodsWe previously established a sensitive fluorescent technique in which the major methodological problems are overcome. Application of this technique has revealed two distinct modes of microsatellite alterations, i.e. Type A and Type B. In the present study, we have applied this technique to 94 ECs.ResultsSignificant microsatellite alterations were observed in 38 (40.4 %) tumours of the panel. The two modes, Type A and Type B, were indeed observed in this malignancy. More importantly, we found that the modes more closely correlated with the molecular and clinicopathological backgrounds of the tumours than the established and widely used MSI grades, MSI-H and MSI-L. Type B MSI widely correlated with family history of hereditary non-polyposis colorectal cancer-associated cancers, whereas MSI-H only did with that of colorectal cancer. Furthermore, mutation in the KRAS oncogene, which has been regarded as generally infrequent in microsatellite-unstable tumours, was clearly associated with Type A MSI.ConclusionsOur observations may suggest a biological relevance and a potential utility of the modal classification of MSI and, furthermore, added complexities to genomic instability underlying tumourigenesis in human endometrium.Electronic supplementary materialThe online version of this article (doi:10.1007/s00432-015-2030-2) contains supplementary material, which is available to authorized users.

Efficacy of Adjuvant 5-Fluorouracil Therapy for Patients with EMAST-Positive Stage II/III Colorectal Cancer.

Elevated Microsatellite Alterations at Selected Tetranucleotide repeats (EMAST) is a genetic signature found in up to 60% of colorectal cancers (CRCs) that is caused by somatic dysfunction of the DNA mismatch repair (MMR) protein hMSH3. We have previously shown in vitro that recognition of 5-fluorouracil (5-FU) within DNA and subsequent cytotoxicity was most effective when both hMutSα (hMSH2-hMSH6 heterodimer) and hMutSβ (hMSH2-hMSH3 heterodimer) MMR complexes were present, compared to hMutSα > hMutSβ alone. We tested if patients with EMAST CRCs (hMutSβ defective) had diminished response to adjuvant 5-FU chemotherapy, paralleling in vitro findings.We analyzed 230 patients with stage II/III sporadic colorectal cancers for which we had 5-FU treatment and survival data. Archival DNA was analyzed for EMAST (>2 of 5 markers mutated among UT5037, D8S321, D9S242, D20S82, D20S85 tetranucleotide loci). Kaplan-Meier survival curves were generated and multivariate analysis was used to determine contribution to risk.We identified 102 (44%) EMAST cancers. Ninety-four patients (41%) received adjuvant 5-FU chemotherapy, and median follow-up for all patients was 51 months. Patients with EMAST CRCs demonstrated improved survival with adjuvant 5FU to the same extent as patients with non-EMAST CRCs (P<0.05). We observed no difference in survival between patients with stage II/III EMAST and non-EMAST cancers (P = 0.36).There is improved survival for stage II/III CRC patients after adjuvant 5-FU-based chemotherapy regardless of EMAST status. The loss of contribution of hMSH3 for 5-FU cytotoxicity may not adversely affect patient outcome, contrasting patients whose tumors completely lack DNA MMR function (MSI-H).

EMAST is associated with a poor prognosis in microsatellite instable metastatic colorectal cancer.

PurposeTo determine the frequency and prognostic value of elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) in metastatic colorectal cancer (mCRC) patients in relation to microsatellite instability (MSI) status and MSH3 protein expression.Material and MethodsThe frequency of EMAST was evaluated in mCRC patients with MSI tumors and microsatellite stable (MSS) tumors. A literature overview was performed to compare the frequency of EMAST in our study with existing data. Immunohistochemistry for MSH3 was compared with EMAST status. Outcome was studied in terms of overall survival (OS) of mCRC patients with MSI and MSS tumors.ResultsEMAST was evaluated in 89 patients with MSI tumors (including 39 patients with Lynch syndrome) and 94 patients with MSS tumors. EMAST was observed in 45.9% (84 out of 183) of patients, with an increased frequency in MSI tumors (79.8% versus 13.8%, p < 0.001). We found no correlation between EMAST and MSH3 protein expression. There was no effect of EMAST on prognosis in patients with MSS tumors, but patients with MSI / non-EMAST tumors had a significantly better prognosis than patients with MSI / EMAST tumors (OS: HR 3.22, 95% CI 1.25-8.30).ConclusionFrequency of EMAST was increased in mCRC patients with MSI tumors, compared to MSS tumors. Our data suggest that the presence of EMAST correlates with worse OS in these patients. There was no effect of EMAST on the prognosis of patients with MSS tumors. A limitation of our study is the small number of patients in our subgroup analysis.

EMAST is a Form of Microsatellite Instability That is Initiated by Inflammation and Modulates Colorectal Cancer Progression.

DNA mismatch repair (MMR) function is critical for correcting errors coincident with polymerase-driven DNA replication, and its proteins are frequent targets for inactivation (germline or somatic), generating a hypermutable tumor that drives cancer progression. The biomarker for defective DNA MMR is microsatellite instability-high (MSI-H), observed in ~15% of colorectal cancers, and defined by mono- and dinucleotide microsatellite frameshift mutations. MSI-H is highly correlated with loss of MMR protein expression, is commonly diploid, is often located in the right side of the colon, prognosticates good patient outcome, and predicts poor efficacy with 5-fluorouracil treatment. Elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) is another form of MSI at tetranucleotide repeats that has been observed in multiple cancers, but its etiology and clinical relevance to patient care has only been recently illuminated. Specifically, EMAST is an acquired somatic defect observed in up to 60% of colorectal cancers and caused by unique dysfunction of the DNA MMR protein MSH3 (and its DNA MMR complex MutSβ, a heterodimer of MSH2-MSH3), and in particular a loss-of-function phenotype due to a reversible shift from its normal nuclear location into the cytosol in response to oxidative stress and the pro-inflammatory cytokine interleukin-6. Tumor hypoxia may also be a contributor. Patients with EMAST colorectal cancers show diminished prognosis compared to patients without the presence of EMAST in their cancer. In addition to defective DNA MMR recognized by tetranucleotide (and di- and tri-nucleotide) frameshifts, loss of MSH3 also contributes to homologous recombination-mediated repair of DNA double stranded breaks, indicating the MSH3 dysfunction is a complex defect for cancer cells that generates not only EMAST but also may contribute to chromosomal instability and aneuploidy. Areas for future investigation for this most common DNA MMR defect among colorectal cancers include relationships between EMAST and chemotherapy response, patient outcome with aneuploid changes in colorectal cancers, target gene mutation analysis, and mechanisms related to inflammation-induced compartmentalization and inactivation for MSH3.

Microsatellite alterations are also present in the less aggressive types of adult T-cell leukemia-lymphoma.

BackgroundAdult T-cell leukemia/lymphoma (ATL) is a mature T-cell neoplasia etiologically linked to HTLV-1. Manifestations of ATL are diverse and different clinical types with different tissue involvement and aggressiveness have been described. The mechanisms that lead to the development of ATL clinical types have not yet been clarified. Considering that in ATL patients HTLV-1 infection generally occurs in childhood, a multistep carcinogenesis model has been proposed. Microsatellite alterations are important genetic events in cancer development and these alterations have been reported in the aggressive types of ATL. Little is known about oncogenesis of the less aggressive types.Methodology/Principal FindingsIn this study we investigated the role of the microsatellite alterations in the pathogenesis mediated by HTLV-1 in the different types of ATL. We examined the presence of microsatellite instability (MSI) and loss of heterozigosity (LOH) in matched pair samples (tumoral and normal) of 24 patients with less aggressive types (smoldering and chronic) and in aggressive types (acute and lymphoma) of ATL. Four microsatellite markers D10S190, D10S191, D1391 and DCC were analyzed. MSI was found in four patients, three smoldering and one chronic, and LOH in four patients, three smoldering and one acute. None of the smoldering patients with microsatellite alterations progressed to aggressive ATL.Conclusions/SignificanceTo our knowledge, this is the first report describing the presence of MSI and LOH in the less aggressive types of ATL. These results indicate that microsatellite alterations may participate in the development of the less aggressive types of ATL.

Microsatellite instability in D2S123 flanking the hMSH2 gene in oral squamous cell carcinoma in South India

Background: Oral cancer is the third most common cancer in India. It is a multifactorial disease. Cells with defective mismatch repair gene hMSH2 can result in genomic instability in oral squamous cell carcinoma (OSCC) tumors. The objective of this study was to investigate the incidence of microsatellite instability (MSI) flanking the hMSH2 in OSCC tumors and relate the MSI status with the mutation profile of hMSH2 in Malayalam speaking population from Kerala. Materials and Methods: Patients diagnosed with OSCC, without superimposed premalignant and other malignant conditions, were recruited for the study based on strict selection criteria. 37 subjects from Malayalam speaking ethnic background of Kerala in India were selected. Blood and carcinoma tissues from were obtained from each patient diagnosed with OSCC. Big adenine tract 26 (BAT26) and D2S123 microsatellite flanking the hMSH2 gene were assessed for their peak patterns in each patient's blood and tissue DNA to analyze MSI and loss of heterozygosity (LOH). Results: No MSI was observed in any of the patient at BAT26 loci. Though BAT26 is reported to be quasi monomorphic, but interestingly it was found to be polymorphic in one patient. In D2S123 loci microsatellite alterations (MA) were observed in 50% of the OSCC patients, which comprised of both LOH and MSI. MA was observed to be significantly increased in moderately differentiated OSCC tumors. These MSI and LOH were independent of clinicopathological characteristics and mutation profile of hMSH2. Conclusion: Higher incidence of MSI at D2S123 in OSCC tumors could be indicative of diagnostic significance. However, this needs to be validated further in increased sample size and across different ethnic population.

Prevalence of cell-free DNA mutations in patients with renal cell carcinoma: Preliminary findings

I quantity of circulating cell-free DNA (cfDNA) has been shown to correspond to many solid tumors. Circulating cell-free DNA is elevated in patients with renal cell carcinoma (RCC), and limited data suggests potential utilityof cfDNA quantificationin RCC prognosis. While there are select studies analyzing hypermethylation or microsatellite alterations of cfDNA, many studies in RCC analyze total amount and/or length of cfDNA.As there are other non-malignant causes of elevated cfDNA, including endothelial dysfunction, trauma, and autoimmune disease, a more specific method to detect RCCassociated cfDNA would be advantageous.

Interleukin 6 Alters Localization of hMSH3, Leading to DNA Mismatch Repair Defects in Colorectal Cancer Cells

Elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) is the most common DNA mismatch repair defect in colorectal cancers, observed in approximately 60% of specimens. This acquired genotype correlates with metastasis and poor outcomes for patients, and is associated with intra-epithelial inflammation and heterogeneous nuclear levels of the mismatch repair protein hMSH3. Inflammation and accompanying oxidative stress can cause hMSH3 to change its intracellular location, but little is known about the source of oxidative stress in cancer cells. We investigated whether cytokines mediate this process. We analyzed levels of interleukin 6 (IL6) and its receptor (IL6R) in human colon and lung cancer cell lines by flow cytometry and enzyme-linked immunosorbent assay; proteins were localized by immunofluorescence and immunoblot analyses. IL6 signaling was blocked with antibodies against IL6, soluble glycoprotein 130 Fc fragments, and the signal transducers and activators of transcription 3 inhibitor NSC74859; a constitutively active form of STAT3 was expressed in colon and lung cancer cell lines to replicate IL6R signaling. EMAST was detected by DNA fragment analysis. Immunohistochemistry was used to examine levels of IL6 in 20 colorectal tumor and adjacent nontumor tissues. Incubation of colon and lung cancer cell lines with IL6, but not other cytokines, caused hMSH3, but no other mismatch repair proteins, to move from the nucleus to the cytosol after generation of oxidative stress; inhibition of IL6 signaling prevented this shift. Expression of constitutively active STAT3 also caused hMSH3 to translocate from the nucleus to the cytoplasm in cancer cell lines. Incubation of cells with IL6 led to tetranucleotide frameshifts, the signature for EMAST. EMAST-positive colorectal tumors had significantly higher levels of IL6 than EMAST-negative tumors. IL6 signaling disrupts the nuclear localization of hMSH3 and DNA repair, leading to EMAST in cancer cell lines. Inflammatory cytokines might therefore promote genetic alterations in human cancer cells.

Microsatellite alteration in multiple primary lung cancer.

Patients with pulmonary neoplasms have an increased risk for developing a second tumor of the lung, either at the same time or different times. It is important to determine if the second tumor represents an independent primary tumor or recurrence/metastasis, because it will significantly change the management and prognosis. Microsatellite instability (MSI) and loss of heterozygosity (LOH) represents molecular disorders acquired by the cell during neoplastic transformation. Both are associated with genetic instability. Functional silencing of tumour suppressor genes may be the consequence of genomic instability, particularly of the globally occurring LOH phenomenon. Numerous studies have confirmed the role of MSI/LOH at both the early and the late stages of multiple primary lung cancer. This paper reviews the published literatures focused on the role of MSI/LOH significance in multiple primary lung cancer. Additionally, a new method based on the allelic variations at polymorphic microsatellite markers was offered that it does not rely on collection of normal tissue, performed with minimal tumor sample, and will complement clinical criteria for diagnostic discrimination between multiple primary cancers versus solitary metastatic diseases.

Abstract 1896: Clinicopathologic characteristics for elevated microsatellite alterations at selected tetranucleotie repeats (EMAST) in colorectal cancer of Korean patients

Abstract Microsatellite instability (MSI) is a hallmark of tumors with mismatch repair deficiency. Recently, a different type of instability, elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) has been reported in colorectal cancers. EMAST was reported to be associated with various clinicopathologic characteristics, including MSI status, race, tumor progression, and morphology and differentiation of tumor. However, contrary to MSI status which is well known prognostic and predictive marker in CRC, clinical implication of EMAST in CRC is still unclear. We evaluated EMAST and MSI status in 88 sporadic CRC cases using five markers (MYCL1, D9S242, D20S85, D8S321, and D20S82) for EMAST and Bethesda panel for MSI status, respectively. Clinicopathologic data of the patients were collected and analyzed. Tumors showing frameshifts in at least two or more EMAST markers were classified as EMAST-positive. Among 88 tumors, EMAST-positive tumors were 22 (25.0%) and MSI-H tumors were 32 (36.4%). EMAST-positive tumors showed proximal location of tumor (90.9% vs. 66.7%, p=0.029), less frequent loss of thymidylate synthase expression (4.5% vs. 34.8%, p=0.005), MSI-H (81.8% vs. 21.2%, p&amp;lt;0.001), nuclear heterogeneity of hMSH3 expression (77.3% vs. 51.5%, p=0.046) when compared with non-EMAST tumor. The other variables including age, gender, gross type and differentiation of tumor, a presence of stromal reaction, and tumor stage were not associated with EMAST status. In addition, overall and disease-free survival rate were similar between EMAST and non-EMAST tumor. In this study, MSI-H tumors showed very high frequency of EMAST-positive and clinical characteristics of EMAST and non-EMAST tumor were definitely different. Citation Format: Myong Hoon Ihn, Duck-Woo Kim, Hye Seung Lee, Kyoung Un Park, Soo Young Lee, Heung-Kwon Oh, Sung-Bum Kang. Clinicopathologic characteristics for elevated microsatellite alterations at selected tetranucleotie repeats (EMAST) in colorectal cancer of Korean patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1896. doi:10.1158/1538-7445.AM2014-1896

Exhaled HPV Infection in Lung Cancer: Role of MA at 3p

Lung cancer has recently been associated with human papilloma virus (HPV) infection. The most important event associated with HPV infection in cancer foresees HPV DNA integration into the host genome. Sites of integration such as the fragile site FRA3B adjacent to the FHIT frequently undergo microsatellite alterations (MAs). In this study we aim to verify the role of MAs at 3p in non-small cell lung cancer (NSCLC) with HPV positivity and eventual correlation with sex, histotype, TNM stage and cigarette smoking. We enrolled 26 NSCLC patients previously investigated for the presence of HPV in their airways (11 HPV+ and 15 HPV-). All subjects had allelotyping analysis of DNA from exhaled breath condensate (EBC), blood and bronchial brushing of microsatellite D3S1300 located in the chromosomal region 3p. For the first time we described the presence of MAs at 3p in EBC of NSCLC patients with HPV positivity. MAs in EBC corresponded to those in paired brushing. The number of patients with 3p MAs was higher in the group of NSCLC with HPV positivity than with HPV negativity. No relationship between the presence and type of MAs in EBC-brushing/DNA and sex, histotype or tumor stage was found. Our results suggested that MAs at 3p are present in caucasic NSLC HPV+ patients and might be involved in lung carcinogenesis. In consideration of the possible clinical usefulness of the analysis of MAS at 3p in the EBC of HPV+ patients in the non-invasive screening for lung cancer, these results merit further studies.