Molecular characterization of intestinal (IS) and diffuse subtypes (DS) of gastric adenocarcinomas.

60 Background: Gastric adenocarcinomas are histologically categorized into IS and DS. TCGA categorization suggests that IS is more common in tumors arising via the chromosomal instability pathway and DS is more common in genomically stable tumors. However, molecular differences between these subtypes are not well understood. Methods: Gastric adenocarcinomas were examined using NextGen sequencing (MiSeq platform on 47 genes or NextSeq on 592 genes), protein expression, and gene amplification techniques. For tumors sequenced with NextSeq, tumor mutational load (TML) was calculated based on somatic nonsynonymous missense mutations, and microsatellite instability (MSI) was evaluated on known MSI loci in target regions. Chi-square and t-tests were used for comparative analyses. Results: In total, 268 gastric adenocarcinomas with annotated histology (DS [n = 144]; IS [n = 124]) were analyzed. Patients with DS were younger than those with IS (mean age: 27y [DS] vs. 65y [IS], p < 0.0001). The majority of patients with DS were female (56% [DS] vs. 35% [IS], p = 0.0004). Most frequently mutated genes in DS were TP53 (44%), ARID1A (37%) , CDH1 (14%), BAP1 (8%), and PIK3CA (5%); whereas the most frequent mutations in IS were ARID1A (57%), TP53 (51%), CDKN2A (13%), MUTYH (13%), and PIK3CA (12%). IS had a higher rate of APC mutations (10% vs. 2%, p = 0.04), whereas DS had a higher rate of CDH1 (14% vs. 2%, p = 0.01). There was no difference in PD-L1 tumor expression (DS: 4%; IS: 8%). IS, when compared to DS, exhibited higher overexpression of TOP2A (90% vs 50%, p < 0.0001), TS (58% vs 28%, p < 0.0001), RRM1 (47% vs 22%, p = 0.0006), and Her2/neu (14% vs 1%, p < 0.0001), and greater Her2 amplification (19% vs 2%, p < 0.0001). Microsatellite instability was not detected in DS, and there was a trend toward a higher frequency of MSI-high in IS (6% [IS] vs. 0% [DS], p = 0.06), as well as a higher mean TML (10.3 vs. 6.6 mutation/megabase, p = 0.01). Conclusions: Significant molecular differences between IS and DS gastric adenocarcinomas were observed, a finding that indicates different carcinogenic pathways and biology, as well as potential differences in response to therapy. Low frequency mutations in several druggable genes may provide therapeutic options.

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Molecular characterization of squamous cell carcinoma of the anal canal (SCCA).

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.4_suppl.538 ◽

2017 ◽

Vol 35 (4_suppl) ◽

pp. 538-538 ◽

Cited By ~ 3

Author(s):

Benjamin Adam Weinberg ◽

Heinz-Josef Lenz ◽

David Arguello ◽

Wafik S. El-Deiry ◽

Joanne Xiu ◽

...

Keyword(s):

Low Frequency ◽

Hpv Infection ◽

Response To Therapy ◽

Molecular Characteristics ◽

Missense Mutations ◽

Chi Square ◽

Gene Testing ◽

Tumor Mutational Burden ◽

N 93

538 Background: Nivolumab has shown promising results in SCCA patients. The majority of SCCA cases have been linked to prior human papillomavirus (HPV) infection. However, HPV negative tumors are frequently TP53 mutated and often resistant to therapy. Molecular characteristics of SCCA are largely undefined. Here we explored the underlying biology of SCCA and the differences between TP53-wild type ( TP53-WT) and TP53-mutated ( TP53-MT) tumors. Methods: SCCA specimens underwent multiplatform testing with protein expression (IHC), gene amplification (ISH), and sequencing (NGS). Tumor mutational burden (TMB) was calculated using only somatic nonsynonymous missense mutations. Chi-square tests were used for comparative analyses. Results: In total, 253 tumors were studied. The most frequently mutated genes included PIK3CA (24%), BRCA2 (14%), FBXW7 (12.4%), TP53 (9.7%), and PTEN (8.9%). In a subset of 23 tumors subjected to Illumina NextSeq (592 gene) testing, the most common mutations were NOTCH2 (30%), NOTCH1 (27.3%), POLE (21.7%) , TSC2 (17.4%), PTEN (14.3%), BRAF (13.6%), BRCA2 (13.0%), PIK3CA (13.0%), and FBXW7 (9.5%). Tumors frequently expressed MRP1 (97.6%), EGFR (92.7%), TOP2A (88.5%), TOPO1 (69.5%), MGMT (67.8%), and RRM1 (59.9%). Expression of PD-1 was seen in 55.8% (24/43) of tumors, and PD-L1 in 15.4% (9/34). HER2 was amplified in 2% (3/147) of samples, which has not been previously described in SCCA. When compared with TP53-WT (n = 93) tumors, T P53-MT (n = 10) had higher rates of BRAF (22% vs. 1%, p < 0.001) and RB1 mutations (44% vs. 0%, p < 0.001), whereas TP53-WT had higher expression of TOPO1 (76% vs. 40%, p = 0.01) and TUBB3 (19% vs. 50%, p = 0.02). There were no differences between the two groups in the frequency of PD-1 or PD-L1 expression. Mean TMB was 8.6 mutations/megabase and, using a TMB cut-off > 17 mutations/megabase to define high vs. low TMB, 6.7% of tumors were TMB-High. High TMB did not correlate with PD-1 (p = 0.50) or PD-L1 status (p = 0.52). Conclusions: Molecular profiling differences between TP53-MT and TP53-WT SCCA indicate different carcinogenic pathways and biology, which may influence response to therapy. Low frequency mutations in several druggable genes may provide therapeutic opportunities for patients with SCCA.

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Molecular variances between rectal and left-sided colon cancers.

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.4_suppl.522 ◽

2017 ◽

Vol 35 (4_suppl) ◽

pp. 522-522 ◽

Cited By ~ 6

Author(s):

John Marshall ◽

Heinz-Josef Lenz ◽

Joanne Xiu ◽

Wafik S. El-Deiry ◽

Jeffrey Swensen ◽

...

Keyword(s):

Tumor Infiltrating Lymphocytes ◽

Genetic Alterations ◽

Recent Analysis ◽

Missense Mutations ◽

Chi Square ◽

Primary Tumors ◽

Colon Cancers ◽

Nextgen Sequencing ◽

Disease Biology ◽

Infiltrating Lymphocytes

522 Background: Recent analysis of CALGB 80405 showed that left sided colon tumors (LT) respond differently to biologics compared with right-sided tumors, likely due to molecular differences. Molecular variations between LT and rectal tumors remain undefined. Herein, we report our exploration of these variations. Methods: Tumors with origins clearly defined as splenic flexure to descending colon (SFT), sigmoid colon (SgT), or rectum (RT) were included. Protein expression, gene amplification and NextGen sequencing was tested. Microsatellite instability (MSI) was measured by PCR. Tumor mutational load (TML) was calculated using only somatic nonsynonymous missense mutations. Chi-square tests were used for comparative analyses. Results: In total, 1,457 primary tumors (SFT 125; SgT 460, and RT 872) were examined. When compared with SFT, RT had a higher frequency of TP53 (71% vs. 57%, p = 0.03) and APC (66% vs. 49%, p = 0.01); a lower frequency of PIK3CA (11% vs. 22%, p = 0.02), BRAF (3% vs. 15% p = 0.0001), GNAS (0.9% vs. 4%, p = 0.04), HNF1A (0.7% vs. 5%, p = 0.01), and CTNNB1(0.3% vs. 4%, p = 0.003); and a higher expression of TOPO1 (52% vs. 31%, p = 0.0001), ERCC1 (29% vs. 15%, p = 0.03), and MGMT (64% vs. 53%, p = 0.048). When compared with SgT, RT had higher expression of TLE3 (33% vs. 23%, p = 0.007), TOPO1 (52% vs. 35%, p < 0.001), TUBB3 (41% vs. 28%, p = 0.003), and MGMT (64% vs. 54%, p = 0.003). There were no differences between SFT, SgT, and RT in the frequency of PD-L1 expression (5%, 2%, and 2%) on tumor cells, PD-1 expression on tumor-infiltrating lymphocytes (54%, 42%, and 42%), or Her-2 expression (1%, 2%, and 3%) and amplification (3%, 3%, and 5%). MSI was seen in 7% of SFT, 4% of SgT, and 0.7% of RT (total LT vs. RT, p = 0.01). Mean TML was 23, 6.5, and 7 mutations (mut)/MB (332 tumors), and the portion of tumors carrying a TML of > 17mut/MB was 9%, 1.6%, and 4% for SFT, SgT, and RT, respectively. In all 3 cohorts, a TML > 17 mut/MB was highly concordant with MSI. There was a correlation between PD-1 and TML in RT (p = 0.04) but not in SFT or SgT. There were no correlations between PD-L1 and TML. Conclusions: Tumors arising in the rectum may carry genetic alterations that are distinct from LT. A better understanding of disease biology may help to identify therapeutic targets and advance precision medicine

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Impact of patient age on molecular alterations in left-sided colorectal tumors.

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.15_suppl.3592 ◽

2017 ◽

Vol 35 (15_suppl) ◽

pp. 3592-3592 ◽

Cited By ~ 4

Author(s):

Benjamin Adam Weinberg ◽

Kelsey Poorman ◽

David Arguello ◽

John Marshall ◽

Mohamed E. Salem

Keyword(s):

Checkpoint Inhibitors ◽

Tumor Biology ◽

Distal Colon ◽

Predictive Biomarker ◽

Missense Mutations ◽

Chi Square ◽

Molecular Alterations ◽

Nextgen Sequencing ◽

Colon And Rectum ◽

The Impact

3592 Background: The incidence of colorectal cancer (CRC) in younger patients (pts) is rising. This increase is most pronounced in tumors arising from the distal colon and rectum. Since tumor sidedness has emerged as an important prognostic and predictive biomarker in CRC, we aim to explore the impact of age on the tumor biology of left-sided colon cancer (LCC). Herein, we compare profiles of LCC from younger (≤ 45 years) and older pts (≥ 65 years). Methods: LCCs (splenic flexure to rectum; n = 1,602) were examined by NextGen sequencing, protein expression, gene amplification, and microsatellite instability fragment analyses. Tumor mutational load (TML) was calculated using only somatic nonsynonymous missense mutations. Chi-square tests were used for comparisons. Results: LCCs from younger (median age 40, range 22-45 years, n = 229) and older (median age 71, range 65-89, n = 503) pts were studied. The most frequently mutated genes included APC, TP53, KRAS, PIK3CA, ARID1A, FBXW7, SMAD4, ATM, BRAF, and NRAS. Comparing younger v. older pts, there were no significant differences in the rates of APC (75.3% v. 82.9%, P = 0.139), TP53 (79.5% v. 73.1%, P = 0.261), KRAS (37.6% v. 43.0%, P = 0.403), PIK3CA (9.4% v. 14.6%, P = 0.234), ARID1A (14.3% v. 13.2%, P = 0.884), FBXW7 (11.4% v. 10.5%, P = 0.830), SMAD4 (13.1% v. 7.4%, P = 0.129), BRAF (4.8% v. 5.7%, P = 0.762), or NRAS (3.5% v. 2.6%, P = 0.680) mutations. Additionally there were no significant differences in protein overexpression. However, there was a trend towards increased HER2 amplification in younger pts (5.7% v. 2.1%, P = 0.05). MSH6 (4.8% v. 0.5%, P = 0.015), MSH2 (2.4% v. 0%, P = 0.032), POLE (2.4% v. 0%, P = 0.032), and NF1 (7.9% v. 0%, P < 0.001) mutations were observed at higher rates in younger pts. High TML (≥ 17 mutations per megabase) was seen more frequently in younger pts (8.2% v. 2.6%, P = 0.02). Conclusions: The molecular differences between LCC in younger and older pts are mostly due to mutations in mismatch repair genes. Higher TML may predict a higher response rate to checkpoint inhibitors in younger pts with LCC. The differences in tumor biology observed here warrant further study and may eventually be used to tailor therapy.

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82 Struggle behavior and vocalizations of male piglets during castration

Journal of Animal Science ◽

10.1093/jas/skaa054.004 ◽

2020 ◽

Vol 98 (Supplement_3) ◽

pp. 3-4

Author(s):

Maria E Lou ◽

Yuzhi Li ◽

Beth Ventura

Keyword(s):

Birth Weight ◽

Acute Pain ◽

High Frequency ◽

Video Recording ◽

Low Frequency ◽

Peak Frequency ◽

Analysis Software ◽

Chi Square ◽

Behavioral Indicators ◽

Treatment Groups

Abstract Castration without the use of analgesia is routinely performed on male piglets. The objective of this study was to assess acute pain during castration through behavioral indicators. Piglets (n=88) were randomly allocated to one of two treatments: castration without the use of analgesia (C) and sham-castration (S). Within 24 hours after birth (birth weight = 1.78kg ±0.71), identical procedures were followed for both treatment groups, except sham piglets were not castrated. Struggle behavior (curl ups, leg kicks, and body flailing) and vocalizations were collected via continuous video recording as piglets received treatment from start (first application of scalpel) to end (application of iodine). Vocalization parameters (duration and peak frequency) were analyzed using the Raven Pro: Interactive Sound Analysis Software (Version 1.5). Peak frequency was defined as low (< 1000 Hz) and high (≥ 1000 Hz). Data were analyzed using the Glimmix Procedure of SAS. For struggle behavior, treatment did not affect curl up frequency. However, castrated piglets kicked more frequently than did sham piglets (C=28.8±0.9 vs. S=21.3±0.9 kicks/min; P=0.02). Additionally, 52% of castrated piglets displayed body flailing, whereas only 4.4% of sham piglets displayed the same behavior (Chi-Square = 24.2; P < 0.0001). For vocalizations, no difference was found for duration and peak frequency of low frequency calls. However, castrated piglets responded with more high frequency calls than sham piglets (C=23.6±0.3 vs. S=18.6±0.3 calls/min; P=0.04). High frequency calls tended to be of longer duration for castrated piglets (C=0.45±0.04 vs. S=0.27±0.04 sec/call; P=0.08). Results indicate that castration without the use of analgesia increased the frequency of leg kicks, body failing, and high frequency calls. This suggests that leg kicks, body flailing, and high frequency calls maybe useful behavioral indicators of acute pain in piglets.

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Characterization of microsatellite instability (dMMR/MSI-H) and mutational landscape in a large contemporary cohort of upper tract urothelial cancer (UTUC) patients.

Journal of Clinical Oncology ◽

10.1200/jco.2021.39.6_suppl.465 ◽

2021 ◽

Vol 39 (6_suppl) ◽

pp. 465-465

Author(s):

Arpit Rao ◽

Julie Elaine McGrath ◽

Joanne Xiu ◽

Andre Luiz De Souza ◽

Shuchi Gulati ◽

...

Keyword(s):

Microsatellite Instability ◽

Chromatin Remodeling ◽

Statistical Significance ◽

Small Sample ◽

Biological Pathways ◽

High Rate ◽

Chi Square ◽

Mutational Landscape ◽

Comparison Results ◽

Small Sample Sizes

465 Background: UTUC is a rare genitourinary malignancy and a number of studies, limited by small sample sizes, have attempted to characterize its mutational landscape. Because immunotherapy is commonly used for this disease type, we evaluated the prevalence of microsatellite instability and characterized the mutational landscapes of UTUC in a large contemporary patient cohort. Methods: UTUC tumor samples were analyzed using next generation sequencing (NGS) (NextSeq, 592 gene panel) or whole exome sequencing (WES) (NovaSeq) (Caris Life Sciences, Phoenix, AZ). Mismatch repair status (deficient [dMMR] or proficient [pMMR]) and microsatellite instability status (MSI-high or stable [MSS]) were detected by immunohistochemistry (IHC), fragment analysis, and NGS. Tumor mutational burden (TMB) was measured by counting all somatic mutations found per tumor (high cutoff ≥ 10 mutations per MB). PD-L1 expression was tested by IHC using PD-L1 antibody clones 22c3 (Agilent; positive cutoff CPS ≥ 10) and SP142 (Ventana; positive cutoff ≥ 5% IC). Pathogenic fusion events were detected using whole transcriptome sequencing (NovaSeq). Statistical significance was determined using the Chi-square test and adjusted for multiple comparison. Results: 538 patients with included – median (range) age 71.5 (30-89) years and 37.5% female/62.5% male. Prevalence of dMMR/MSI-H was 3.9% (21/538) and TMB-high was 22.7% (96/423). Significant molecular differences were not detected in primary vs metastatic disease or in male vs female cases. dMMR/MSI-H tumors had higher frequency of TMB-high compared to MSS tumors (100% vs. 19%, p = 0.00003). dMMR/MSI-H tumors also had a higher frequency than MSS tumors for mutations in genes involved in chromatin remodeling (ASXL 82.4%, CREBBP 60%, SMARCA4 40%, KMT2D 95%, ARIDIA 100%, KMT2A 20%, KMT2C 35.3%, NSD1 20%), DNA-damage repair (FANCG 10%, ATM 45%, ATRX 40%) and other biological pathways (RNF43 10%, PTCH1 21.4%, ERBB3 30%, CDKN2A 25%, TSC2 15%, FLNC 15%, HNF1A 20%, CIC 15%, DNMT3A 17.6%); all adjusted p < 0.05. Pathogenic fusions were detected in 3.8% (17/443) cases, with FGFR3 fusion being the most common, occurring in 2.7% (12/443) cases. PD-L1 positivity was identified in 33.2% (133/400) cases tested by 22c3 antibody and 28.4% (89/313) cases tested by SP142 antibody. No difference was seen in PD-L1 positivity between MSI-H/dMMR vs. MSS tumors. Conclusions: In the largest analysis to date, we found a 3.9% prevalence of dMMR/MSI-high rate in UTUC. All dMMR/MSI-H tumors displayed TMB-high. PD-L1 positivity was comparable between dMMR/MSI-H and MSS tumors. dMMR/MSI-H tumors had a significantly higher rate of mutations in genes involved in chromatin remodeling and DDR biological pathways. These results could inform design of targeted therapy trials in UTUC.

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Characteristics of the mutation spectrum identified by comprehensive investigation of the CFTR gene in the Russian patients

Almanac of Clinical Medicine ◽

10.18786/2072-0505-2019-47-004 ◽

2019 ◽

Vol 47 (1) ◽

pp. 38-46

Author(s):

N. V. Petrova ◽

A. Yu. Marakhonov ◽

T. A. Vasilyeva ◽

N. Yu. Kashirskaya ◽

E. I. Kondratyeva ◽

...

Keyword(s):

Gene Mutations ◽

Hereditary Disease ◽

Mutation Spectrum ◽

Missense Mutations ◽

Nonsense Mutations ◽

Sequencing Method ◽

Novel Variants ◽

Frequent Mutations ◽

Large Rearrangements

Rationale: Cystic fibrosis (CF; OMIM 219700) is a common hereditary disease caused by mutations in the CFTR gene (OMIM 602421). The distribution and frequencies of the CFTR gene mutations vary considerably between countries and ethnic groups. By now about 11% alleles of the CFTR gene remain unidentified after testing for frequent mutations in the Russian patients. A full determination of the mutation spectrum in the CFTR gene is necessary to optimize medical and genetic assistance to the population and to implement the achievements of targeted therapy in the treatment of CF patients.Materials and methods: The sample included 121 Russian CF patients, in whom testing for 34 routinely analyzed mutations did not identify one (n = 107) or both (n = 14) mutant alleles. Assessment of the coding sequence of the CFTR gene, including the regions of exon-intron junctions, 5’- and 3’-untranslated regions was performed by the Sanger sequencing method; in addition, the search for large rearrangements was conducted by the multiplex ligation-dependent probe amplification (MLPA) method.Results: In addition to the previously identified, 88 more variants were determined, including 28 missense mutations, 15 nonsense mutations, 18 frameshift mutations (14 deletions, 4 insertions), 14 splicing mutations, 1 in-frame insertion, 1 in-frame deletion, 1 in/del mutation, and 10 large rearrangements (7 deletions, 3 duplications). Twenty three (23) novel variants were sequenced. Four (4) complex mutant alleles were found. Sixty (60) variants are found once each. One hundred and thirty four (134) of 135 tested mutant alleles were identified.Conclusion: Consequent use of the sequencing and MLPA methods has allowed for identification of a high proportion of the tested mutant alleles in CF patients from Russia (134/135, > 99%), to detect a significant diversity of the CFTR mutation spectrum (88 additional variants, 32 of them novel), a number of repeated mutations (c.2353C>T, c.1240_1244delCAAAA, c.1766+1G>A and c.3929G>A) encountered in 5 or more unrelated patients, which could be included in the panel of routinely analyzed variants in the Russian CF patients; and a high proportion of large rearrangements of the CFTR gene.

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526 MICROSATELLITE INSTABILITY IN GASTRO-ESOPHAGEAL ADENOCARCINOMAS

Diseases of the Esophagus ◽

10.1093/dote/doaa087.140 ◽

2020 ◽

Vol 33 (Supplement_1) ◽

Author(s):

A Katz ◽

G Evaristo ◽

M Issac ◽

J Ramirez-GraciaLuna ◽

M Park ◽

...

Keyword(s):

Overall Survival ◽

Microsatellite Instability ◽

Cox Regression ◽

Significant Proportion ◽

Genomic Analysis ◽

Poor Response ◽

Tissue Microarrays ◽

Braf V600e ◽

Chi Square ◽

Response To Chemotherapy

Abstract Microsatellite instability (MSI) in gastro-esophageal adenocarcinomas (GEAs) is associated with poor response to chemotherapy; however, this association is poorly understood. The aim of this study is to better understand the clinical relevance and mutational landscape of MSI in GEA. Methods In order to assess the status of mismatch repair (MMR) proteins, we performed immunohistochemistry for MLH1, MSH2, MSH6, PMS2 and BRAF V600E on tissue microarrays constructed from 161 patients with esophageal adenocarcinoma and 65 patients with gastric adenocarcinoma, operated between 2011 to 2019. MMR- deficient (MMR-D) status was confirmed using full sections of tumors. Demographics, tumor, and treatment details, operative variable and overall survival were evaluated. Genomic characterization of 3 MSI-H and 10 MSS patients was performed using deep targeted sequencing of 234 potential oncogenic genes. Statistical analysis was performed using Cox regression, logistic regression, ANOVA or Chi-square tests. Results Among the 28 (12%) MMR-D immunophenotype patients identified: 25 showed a combined MLH1/PMS2 loss, one case with MSH2/MSH6 loss and two cases with isolated MSH6 or MSH2 loss. Patients in the MMR-D were older (74 ± 14 vs 67 ± 11 years, p = 0.01) and with more Siewert-III and sub-cardia tumors [64%(18) vs 40%(79), p = 0.002] compared to MMR-Stable patients. All other variables including sex, stage and grade were not significantly different. MMR-D immunophenotype was associated with better overall survival (Median survival of 5.7vs2.3 years, p < 0.04) (Figure 1A). Genomic analysis linked patients with D-MMR with a higher mutational burden with some potentially targetable mutations (Figure 1B). Conclusion The significant proportion of MMR-D immunophenotype identified in this gastroesophageal cohort and the different pattern of overall survival and genetic mutations associated with this phenotype, highlight the importance of further characterization and understanding the mechanisms and role of MMR status in GEAs.

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MSH2-deficient murine lymphomas harbor insertion/deletion mutations in the transforming growth factor beta receptor type 2 gene and display low not high frequency microsatellite instability

Blood ◽

10.1182/blood.v95.5.1767.005k07_1767_1772 ◽

2000 ◽

Vol 95 (5) ◽

pp. 1767-1772 ◽

Cited By ~ 21

Author(s):

Robert Lowsky ◽

Anthony Magliocco ◽

Ryo Ichinohasama ◽

Armin Reitmair ◽

Stuart Scott ◽

...

Keyword(s):

Growth Factor ◽

Microsatellite Instability ◽

Transforming Growth Factor Beta ◽

High Frequency ◽

Transforming Growth Factor ◽

Low Frequency ◽

Hematologic Malignancies ◽

Receptor Type ◽

Beta Receptor ◽

Mmr Deficiency

High-frequency microsatellite instability (MSI), defined as more than 20% unstable loci, is an inconsistent finding in hematologic malignancies; consequently, the significance of deficient DNA mismatch repair (MMR) to their pathogenesis has been questioned. To further investigate the relationship between MMR deficiency and genomic instability in hematologic malignancies, this study evaluatedMSH2−/− murine lymphomas for insertion/deletion (ID) mutations within the transforming growth factor (TGF)-beta receptor type II (TβR-II) gene and MSI at 10 neutral microsatellites. The lymphomas displayed ID mutations within short mononucleotide runs of TβR-II at a high frequency, whereas nonmalignant tissue from corresponding animals lacked mutations. Loss ofTβR-II transcripts and protein was seen in 6 of 7 murine lymphomas harboring acquired TβR-II mutations. In the analysis of paired nonmalignant and tumor DNA samples, low-frequency but not high-frequency MSI was found. Low-frequency MSI occurred in 8 of 20 lymphomas and 12 displayed microsatellite stability. MSI was even less frequent in nonmalignant tissue as only 3 of 20 samples displayed low-frequency MSI and 17 displayed stability. Evaluation of 20 single cell clones from the MSH2−/− lymphoma cell lines R25 and L15 identified high-frequency MSI in 4 and 2 clones, respectively. The remaining clones showed low-frequency MSI or stability. These findings suggest that acquired TβR-IImutations represent important inactivating events in tumor pathogenesis following MSH2 deficiency. Furthermore, for some hematolymphoid malignancies, the evaluation of cancer-associated genes for ID mutations may represent a more sensitive marker of MMR deficiency than evaluation of neutral microsatellites for high-frequency MSI.

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