Maria Planck

Microsatellite instability and expression of MLH1 and MSH2 in carcinomas of the small intestine

Carcinomas of the small intestine are rare, but the risk is greatly increased in patients with hereditary nonpolyposis colorectal cancer (HNPCC) due to an inherited mismatch repair (MMR) gene mutation, most commonly affecting the genes MLH1 or MSH2. Defective MMR is characterized by microsatellite instability (MSI) and loss of MMR protein expression in the tumor tissue. However, a subset of several sporadic tumor types, including about 15% of colon cancers, also evolve through defective MMR.

Microsatellite instability is rare in rectal carcinomas and signifies hereditary cancer.

We analysed microsatellite instability (MSI) in a consecutive series of 165 rectal carcinomas. Data on a personal and/or family history of cancer were collected from all patients and revealed metachronous cancer in 9 patients, 2 of whom had developed colorectal cancer, and a suspected familial aggregation of colorectal cancer in three families. Only three of the 165 (2%) rectal cancers showed MSI. The patients whose tumours displayed MSI had clinical histories suggesting hereditary cancer--a family history of colorectal cancer and/or synchronous colorectal cancers. Denaturing gradient gel (DGGE) analysis was used to screen the MSI+ patients for mutations in the hMLH1 and hMSH2 genes and revealed two new germline mutations; a 1 bp deletion in exon 10 of hMSH2 creating a premature stop-codon and a splice donor site mutation in intron 16 of hMLH1. Considering colorectal carcinomas as a group, MSI has been reported to occur in approximately 10-20% of the tumours and thus can not, per se be used for clinical detection of hereditary tumours. This study shows, however, that MSI is rare in rectal carcinomas and when present strongly suggests a hereditary predisposition for colorectal cancer development.

CD99 is a novel prognostic stromal marker in non-small cell lung cancer

The complex interaction between cancer cells and the microenvironment plays an essential role in all stages of tumourigenesis. Despite the significance of this interplay, alterations in protein composition underlying tumour–stroma interactions are largely unknown. The aim of this study was to identify stromal proteins with clinical relevance in non‐small cell lung cancer (NSCLC). A list encompassing 203 stromal candidate genes was compiled based on gene expression array data and available literature. The protein expression of these genes in human NSCLC was screened using the Human Protein Atlas. Twelve proteins were selected that showed a differential stromal staining pattern (BGN, CD99, DCN, EMILIN1, FBN1, PDGFRB, PDLIM5, POSTN, SPARC, TAGLN, TNC and VCAN). The corresponding antibodies were applied on tissue microarrays, including 190 NSCLC samples, and stromal staining was correlated with clinical parameters. Higher stromal expression of CD99 was associated with better prognosis in the univariate (p = 0.037) and multivariate (p = 0.039) analysis. The association was independent from the proportion of tumour stroma, the fraction of inflammatory cells and clinical and pathological parameters like stage, performance status and tumour histology. The prognostic impact of stromal CD99 protein expression was confirmed in an independent cohort of 240 NSCLC patients (p = 0.008). Furthermore, double‐staining confocal fluorescence microscopy showed that CD99 was expressed in stromal lymphocytes as well as in cancer‐associated fibroblasts. Based on a comprehensive screening strategy the membrane protein CD99 was identified as a novel stromal factor with clinical relevance. The results support the concept that stromal properties have an important impact on tumour progression.

Immunohistochemistry in the differential diagnostics of primary lung cancer: an investigation within the southern Swedish lung cancer study.

OBJECTIVES To assess immunohistochemical (IHC) stains differentially expressed between different types of lung cancer. METHODS We evaluated 16 different IHC stains in 209 prospectively included, surgically treated primary lung cancers, including 121 adenocarcinomas, 65 squamous cell carcinomas, 15 large-cell carcinomas, 5 adenosquamous carcinomas, 2 sarcomatoid carcinomas, and 1 small-cell carcinoma, using the tissue microarray technique. RESULTS Cytokeratin 5 (CK5) and P63 were both positive in 10% or more of the cells in 97% of the squamous cell carcinomas, with the former being positive (<10% of the cells) in only 2 non-squamous cell carcinomas. Thyroid transcription factor 1 (TTF1) and napsin A were positive in 10% or more of the cells in 88% and 87% of the adenocarcinomas, respectively, with 94% of the adenocarcinomas being positive in at least 1 marker. Fifteen percent of the adenocarcinomas were positive for estrogen receptor. CONCLUSIONS CK5, TTF1, and napsin A are sensitive markers for squamous cell carcinoma and adenocarcinoma of the lung.

Somatic frameshift alterations in mononucleotide repeat-containing genes in different tumor types from an HNPCC family with germline MSH2 mutation

Hereditary nonpolyposis colorectal cancer (HNPCC) is caused by a germline mutation in one of several DNA repair genes, which in the tumors is reflected as microsatellite instability (MSI). MSI+ tumors have been found to carry somatic frameshift mutations in mononucleotide repeats within the coding regions of several genes involved in growth control, apoptosis, and DNA repair, e.g., TGFBRII, BAX, IGFIIR, TCF4, MSH3, and MSH6. We have studied the occurrence of somatic frameshift alterations in these mononucleotide repeat‐containing genes in 24 tumors (15 colorectal cancers, 1 colon adenoma, 4 endometrial cancers, 1 ovarian cancer, 1 gastric cancer, 1 urothelial cancer, and 1 duodenal cancer) from 14 individuals in an HNPCC family with germline hMSH2 mutation. Such somatic frameshift mutations occurred at a variable frequency; the long mononucleotide repeats that characterize intronic MSI markers were mutated in the majority of tumors, 13 of the tumors displayed alterations in the (A)10 tract of TGFBII, eight tumors (all of gastrointestinal origin) had alterations in the (A)9 repeat of TCF4, and one to five tumors had somatic frameshift alterations in the shorter mononucleotide repeats of IGFIIR, BAX, MSH3, and MSH6. Thus, longer mononucleotide repeats were more frequently affected by somatic frameshift mutations. The pattern of alterations varied between the tumors from different family members as well as between different tumors from the same individual. To what extent this variable pattern depends on the widespread mismatch repair deficiency induced by the underlying MSH2 mutation, or represents alternative ways whereby the tumors can achieve a tumorigenic phenotype, is unknown. We suggest, however, that the accumulation of somatic frameshifts, rather than the specific loci in which these occur, drives the development of the tumorigenic phenotype in HNPCC. Genes Chromosomes Cancer 29:33–39, 2000.

High frequency of microsatellite instability and loss of mismatch-repair protein expression in patients with double primary tumors of the endometrium and colorectum

Patients with the familial syndrome hereditary nonpolyposis colorectal carcinoma (HNPCC) exhibit an increased risk for several tumor types, of which the greatest lifetime risk is for colorectal and endometrial carcinoma. HNPCC is caused by a germline mutation in one of several identified mismatch repair (MMR) genes and typically presents with microsatellite instability (MSI) and frequent loss of MMR protein expression in the tumor tissue. The objective of this study was to estimate the proportion of double primary tumors of the endometrium and colorectum that displays tumor characteristics suggestive of MMR deficiency.

hMLH1, hMSH2 and hMSH6 mutations in hereditary non-polyposis colorectal cancer families from southern Sweden

We have screened 17 Southern Sweden individuals/families with suspected hereditary non‐polyposis colorectal cancer (HNPCC) for mutations in the DNA‐mismatch repair genes hMLH1, hMSH2 and hMSH6 using denaturing gradient gel electrophoresis, protein truncation test and direct DNA sequencing. The families were selected on the basis of a family history of HNPCC‐related tumors or the occurrence of metachronous colorectal cancer/endometrial cancer at young age in an individual with a weak family history of cancer. Furthermore, we required that tumor tissue from at least one individual in the family had to display microsatellite instability. We identified germ‐line mutations in 9 individuals from 8 families. Five families had mutations in hMLH1, 4 of which were splice site mutations, 2 had frameshift mutations in hMSH2 and 1 patient with metachronous endometrial and rectal cancer but with a weak family history of cancer had a nonsense mutation in hMSH6. Our results present novel germ‐line DNA‐repair gene mutations, one of these in hMSH6, and demonstrate the diversified mutation spectrum in Sweden, where no founder mutation has so far been identified. Int. J. Cancer 83:197–202, 1999.

Genome-wide DNA Methylation Analysis of Lung Carcinoma Reveals One Neuroendocrine and Four Adenocarcinoma Epitypes Associated with Patient Outcome

Purpose: Lung cancer is the worldwide leading cause of death from cancer. DNA methylation in gene promoter regions is a major mechanism of gene expression regulation that may promote tumorigenesis. However, whether clinically relevant subgroups based on DNA methylation patterns exist in lung cancer remains unclear. Experimental Design: Whole-genome DNA methylation analysis using 450K Illumina BeadArrays was performed on 12 normal lung tissues and 124 tumors, including 83 adenocarcinomas, 23 squamous cell carcinomas (SqCC), 1 adenosquamous cancer, 5 large cell carcinomas, 9 large cell neuroendocrine carcinomas (LCNEC), and 3 small-cell carcinomas (SCLC). Unsupervised bootstrap clustering was performed to identify DNA methylation subgroups, which were validated in 695 adenocarcinomas and 122 SqCCs. Subgroups were characterized by clinicopathologic factors, whole-exome sequencing data, and gene expression profiles. Results: Unsupervised analysis identified five DNA methylation subgroups (epitypes). One epitype was distinctly associated with neuroendocrine tumors (LCNEC and SCLC). For adenocarcinoma, remaining four epitypes were associated with unsupervised and supervised gene expression phenotypes, and differences in molecular features, including global hypomethylation, promoter hypermethylation, genomic instability, expression of proliferation-associated genes, and mutations in KRAS, TP53, KEAP1, SMARCA4, and STK11. Furthermore, these epitypes were associated with clinicopathologic features such as smoking history and patient outcome. Conclusions: Our findings highlight one neuroendocrine and four adenocarcinoma epitypes associated with molecular and clinicopathologic characteristics, including patient outcome. This study demonstrates the possibility to further subgroup lung cancer, and more specifically adenocarcinomas, based on epigenetic/molecular classification that could lead to more accurate tumor classification, prognostication, and tailored patient therapy. Clin Cancer Res; 20(23); 6127–40. ©2014 AACR.

Landscape of somatic allelic imbalances and copy number alterations in human lung carcinoma

Lung cancer is the worldwide leading cause of death from cancer and has been shown to be a heterogeneous disease at the genomic level. To delineate the genomic landscape of copy number alterations, amplifications, loss‐of‐heterozygosity (LOH), tumor ploidy and copy‐neutral allelic imbalance in lung cancer, microarray‐based genomic profiles from 2,141 tumors and cell lines including adenocarcinomas (AC, n = 1,206), squamous cell carcinomas (SqCC, n = 467), large cell carcinomas (n = 37) and small cell lung carcinomas (SCLC, n = 88) were assembled from different repositories. Copy number alteration differences between lung cancer histologies were confirmed in 285 unrelated tumors analyzed by BAC array comparative genomic hybridization. Tumor ploidy patterns were validated by DNA flow cytometry analysis of 129 unrelated cases. Eighty‐nine recurrent copy number alterations (55 gains, 34 losses) were identified harboring genes with gene expression putatively driven by gene dosage through integration with gene expression data for 496 cases. Thirteen and 26 of identified regions discriminated AC/SqCC and AC/SqCC/SCLC, respectively, while 48 regions harbored recurrent (n > 15) high‐level amplifications comprising established and putative oncogenes, differing in frequency and coamplification patterns between histologies. Lung cancer histologies displayed differences in patterns/frequency of copy number alterations, genomic architecture, LOH, copy‐neutral allelic imbalance and tumor ploidy, with AC generally displaying less copy number alterations and allelic imbalance. Moreover, a strong association was demonstrated between different types of copy number alterations and allelic imbalances with tumor aneuploidy. In summary, these analyses provide a comprehensive overview of the landscape of genomic alterations in lung cancer, highlighting differences but also similarities between subgroups of the disease.

Defective mismatch-repair in patients with multiple primary tumours including colorectal cancer

Individuals with an inherited predisposition to cancer development are at an increased risk of developing multiple tumours. Hereditary non-polyposis colorectal cancer (HNPCC) is one of the most common hereditary cancer syndromes and is estimated to account for approximately 2% of colorectal cancers. However, HNPCC individuals are at an increased risk of developing other tumour types such as cancers of the endometrium, urothelium and small intestine. We have utilised a population-based regional cancer registry to identify all patients with double primary colorectal cancers and at least one additional malignancy and characterised the tumour spectrum in this patient group. We subsequently selected those 47 individuals who had developed at least four malignancies, including two colorectal cancers, for studies of the tumour characteristics associated with HNPCC. In total, these individuals developed 209 tumours, 156 of which were successfully retrieved. Microsatellite instability (MSI), a phenomenon caused by defective mismatch-repair (MMR), was identified in 63/154 (41%) evaluable tumours with a MSI-high pattern in 59 and a MSI-low pattern in four tumours. All tumours were immunohistochemically stained for the MMR proteins MLH1 and MSH2, with loss of expression in 55/63 (87%) MSI tumours and in 2/89 (2%) microsatellite stable (MSS) tumours. This loss affected MLH1 in 24 tumours and MSH2 in 33 tumours. A concordant loss of expression for the same MMR protein in several tumours from the same individual, a pattern that strongly suggests an underlying germline MMR gene mutation, was found in 17/45 (38%) patients and affected MLH1 in 8 patients and MSH2 in 9 patients. We conclude that the development of multiple primary tumours, including synchronous or metachronous colorectal cancers, is associated with an increased frequency of MSI and loss of immunohistochemical expression of MLH1 and MSH2.