Arndt Hartmann

Towards the introduction of the ‘Immunoscore’ in the classification of malignant tumours

The American Joint Committee on Cancer/Union Internationale Contre le Cancer (AJCC/UICC) TNM staging system provides the most reliable guidelines for the routine prognostication and treatment of colorectal carcinoma. This traditional tumour staging summarizes data on tumour burden (T), the presence of cancer cells in draining and regional lymph nodes (N) and evidence for distant metastases (M). However, it is now recognized that the clinical outcome can vary significantly among patients within the same stage. The current classification provides limited prognostic information and does not predict response to therapy. Multiple ways to classify cancer and to distinguish different subtypes of colorectal cancer have been proposed, including morphology, cell origin, molecular pathways, mutation status and gene expression‐based stratification. These parameters rely on tumour‐cell characteristics. Extensive literature has investigated the host immune response against cancer and demonstrated the prognostic impact of the in situ immune cell infiltrate in tumours. A methodology named ‘Immunoscore’ has been defined to quantify the in situ immune infiltrate. In colorectal cancer, the Immunoscore may add to the significance of the current AJCC/UICC TNM classification, since it has been demonstrated to be a prognostic factor superior to the AJCC/UICC TNM classification. An international consortium has been initiated to validate and promote the Immunoscore in routine clinical settings. The results of this international consortium may result in the implementation of the Immunoscore as a new component for the classification of cancer, designated TNM‐I (TNM‐Immune).

Patterns of p53 mutations separate ovarian serous borderline tumors and low- and high-grade carcinomas and provide support for a new model of ovarian carcinogenesis: a mutational analysis with immunohistochemical correlation.

The infrequent association of serous borderline tumors (SBTs) with invasive serous carcinoma has led to the view that SBTs are unrelated to invasive serous carcinoma. Nonetheless, mortality associated with SBTs is generally attributed to malignant transformation, and traditionally these tumors have been designated as “carcinomas of low malignant potential.” Previous immunohistochemical studies evaluating p53 expression and molecular genetic studies evaluating mutational status have reported that p53 overexpression and mutations are infrequent in SBTs and occur in as many as 50% to 80% of invasive serous carcinomas. The different methodologies for determining p53 status and the failure to correlate the findings with tumor grade make these studies difficult to interpret. The current study was undertaken to overcome these deficiencies and to reconcile the relationship of SBTs to invasive serous carcinoma by performing a morphologic, immunohistochemical, and molecular genetic analysis comparing SBTs with low- and high-grade serous carcinoma. The molecular genetic analysis used a highly stringent, carefully designed nucleotide-sequencing method. A total of 96 sporadic serous tumors including 25 SBTs (11 atypical proliferative serous tumors and 14 intraepithelial low-grade serous carcinomas [noninvasive micropapillary serous carcinomas, MPSCs]), 12 low-grade serous carcinomas (invasive MPSCs), and 59 high-grade serous carcinomas were analyzed for their p53 mutational status of exons 5 to 9. Functional mutations, defined as mutations resulting in the alteration of the structure of the encoded protein, were detected in 30 of 59 (50.8%) high-grade serous carcinomas and 1 (8.3%) of 12 low-grade invasive serous carcinomas compared with 2 (8%) of 25 SBTs, both of these in intraepithelial low-grade serous carcinomas (noninvasive MPSCs). The similar frequency of p53 mutations in SBTs and low-grade invasive serous carcinomas in contrast to the significantly higher frequency of p53 mutations in high-grade serous carcinomas (P < 0.0005) suggests a common lineage for SBTs and low-grade invasive serous carcinomas and supports the view that SBTs are unrelated to the usual type of invasive serous carcinoma, which is a high-grade neoplasm. Mutational status was also correlated with p53 immunoreactivity. Although p53 immunoreactivity is generally higher in those specimens containing mutant p53, immunostaining is neither sufficiently specific nor sensitive enough to predict p53 mutations. The molecular genetic findings confirm our hypothesis of dual pathways of serous carcinogenesis based on previous analyses of KRAS and BRAF mutations on the same set of cases in which KRAS and BRAF mutations were found in 60% of SBTs and low-grade serous carcinoma but not in high-grade serous carcinomas. Based on these studies, we have proposed a model of serous carcinogenesis in which SBTs are the precursors of low-grade serous carcinomas whereas the usual type of invasive serous carcinoma is a high-grade neoplasm that develops “de novo” from in situ alterations in epithelial inclusion cysts.

WIF1, a component of the Wnt pathway, is down‐regulated in prostate, breast, lung, and bladder cancer

To detect novel Wnt‐pathway genes involved in tumourigenesis, this study analysed the RNA expression levels of 40 genes of the Wnt pathway by chip hybridization of microdissected matched pairs of 54 primary prostate carcinomas. Eleven genes showed greater than two‐fold differential expression in at least 10% of prostate cancers. Three of these genes encode extracellular components of the Wnt pathway (WNT2, WIF1, SFRP4); two are receptors (FZD4, FZD6); two belong to the intracellular signal cascade (DVL1, PPP2CB); one regulates transcription (TCF4); and three represent genes regulated by this pathway (CCND2, CD44, MYC). While SFRP4, FZD4, FZD6, DVL1, TCF4, and MYC are up‐regulated, WIF1, WNT2, PPP2CB, CCND2, and CD44 are down‐regulated in certain prostate cancer patients. Wnt inhibitory factor 1 (WIF1) and secreted frizzled related protein (SFRP4) showed the most significant aberrant expression at the RNA level. WIF1 was down‐regulated in 64% of primary prostate cancers, while SFRP4 was up‐regulated in 81% of the patients. Immunohistochemical analysis using a polyclonal antibody revealed strong cytoplasmic perinuclear WIF1 expression in normal epithelial cells of the prostate, breast, lung, and urinary bladder. Strong reduction of WIF1 protein expression was found in 23% of prostate carcinomas, but also in 60% of breast, 75% of non‐small cell lung (NSCLC), and 26% of bladder cancers analysed. No significant association between WIF1 down‐regulation and tumour stage or grade was observed for prostate, breast or non‐small cell lung carcinomas, indicating that loss of WIF1 expression may be an early event in tumourigenesis in these tissues. However, down‐regulation of WIF1 correlated with higher tumour stage in urinary bladder tumours (pTa versus pT1–pT4; p = 0.038). Copyright

Multiple Mutation Analyses in Single Tumor Cells with Improved Whole Genome Amplification

Combining whole genome amplification (WGA) methods with novel laser-based microdissection techniques has made it possible to exploit recent progress in molecular knowledge of cancer development and progression. However, WGA of one or a few cells has not yet been optimized and systematically evaluated for samples routinely processed in tumor pathology. We therefore studied the value of established WGA protocols in comparison to an improved PEP (I-PEP) PCR method in defined numbers of flow-sorted and microdissected tumor cells obtained both from frozen as well as formalin-fixed and paraffin-embedded tissue sections. In addition, the feasibility of I-PEP-PCR for mutation analysis was tested using clusters of 50-100 unfixed tumor cells obtained by touch preparation of ten breast carcinomas by conventional sequencing of exon 7 and 8 of the p53 gene. Finally, immunocytochemically stained microdissected single disseminated tumor cells from bone marrow aspirates were investigated with respect to mutations in codon 12 of Ki-ras by restriction fragment length polymorphism (RFLP)-PCR after I-PEP-PCR. The modified I-PEP-PCR protocol was superior to the original PEP-PCR and DOP-PCR protocols concerning amplification of DNA from one cell (efficiency rate I-PEP-PCR 40% versus PEP-PCR 15% and DOP-PCR 30%) and five cells (efficiency rate I-PEP-PCR 100% versus PEP-PCR 33% and DOP-PCR 20%). Preamplification by I-PEP allowed 100% sequence accuracy in > 4000 sequenced base pairs and Ki-ras mutation detection in isolated single disseminated tumor cells. For reliable microsatellite analysis of I-PEP-preamplified DNA, at least 10 unfixed cells from fluorescence-activated cell sorting, 10 cells from frozen tissue, or at least 30 cells from formalin-fixed and paraffin-embedded tissue sections were required. Thus, I-PEP-PCR allowed multiple reliable microsatellite analyses suited for microsatellite instability and losses of heterozygosity and mutation analysis even at the single cell level, rendering this technique a powerful new tool for molecular analyses in diagnostic and experimental tumor pathology.

Minute Gastric Sclerosing Stromal Tumors (GIST Tumorlets) Are Common in Adults and Frequently Show c-KIT Mutations

Multifocal hyperplasia of interstitial cells of Cajal (ICC hyperplasia) is a precursor of hereditary gastrointestinal stromal tumors (GISTs) in patients with germline mutations of c-KIT or PDGFRA, but precursor lesions of sporadic GISTs have not been defined yet. Small hyalinizing stromal tumors of the proximal stomach (referred to in this study as GIST tumorlets) were collected prospectively from 98 consecutive autopsies and additional cases were retrieved from surgical pathology files (total n=57). GIST tumorlets were grossly detectable in 22.5% consecutive autopsies performed in individuals older than 50 years. All lesions were located in the cardia, fundus, or proximal body, and ranged in size from 1 to 10 mm (4 mm). Similar lesions were not detected in the antrum, duodenum, and the remainder of the bowel. Histologically, the spindle cell subtype comprised all cases, with hyalinization and calcification in 57% of cases. The spindle cells were immunohistochemically positive for vimentin, CD117, and CD34. Twenty-four cases yielded sufficient DNA for subsequent molecular analysis, which showed c-KIT mutations in 11 cases (46%) and PDGFRA mutations in 1 case (4%). Sporadic GIST tumorlets of the proximal stomach are common in the general population over the age of 50 years and frequently show somatic c-KIT mutations. GIST tumorlets probably represent the grossly recognizable counterpart of sporadic ICC hyperplasia caused by somatic c-KIT or PDGFRA mutations. Early hyalinization and calcification seems to confer limited growth potential, and complete regression of such lesions is common. GIST tumorlets likely represent preclinical (preneoplastic) lesions that need additional stimuli to evolve into clinical GISTs, raising the possibility of a hyperplasia-neoplasia sequence in the development of sporadic GISTs.

Numerous IgG4-positive plasma cells are ubiquitous in diverse localised non-specific chronic inflammatory conditions and need to be distinguished from IgG4-related systemic disorders

Background IgG4-related systemic fibrosclerosis is a recently defined disorder characterised by a diffuse or tumefactive inflammatory reaction rich in IgG4-positive plasma cells associated with sclerosis and obliterative phlebitis. Although characteristic histopathological features are essential for the diagnosis of these disorders, to date there exists no consensus regarding the cut-off values used to define a ‘significant IgG4-positive plasma cell count,’ and data regarding the distribution of IgG4-positive plasma cells under common (non-specific) inflammatory conditions are lacking. Methods The authors analysed 121 randomly selected histopathological specimens containing prominent lymphoplasmacytic infiltrates (11 obstructive sialadenitis, 27 inflammatory lesions of the oral cavity, 24 inflammatory gastrointestinal lesions, 15 rheumatoid synovitis, 15 non-specific synovitis, eight non-specific dermatitis and 21 primary carcinomas with a peritumoral inflammatory response). For comparison, seven cases of sclerosing sialadenitis (Küttner tumour) were examined. Results High counts of IgG4 plasma cells were found in sclerosing sialadenitis (mean 40/high-power field (hpf)), contrasting sharply with sialadenitis caused by sialolithiasis (mean 3/hpf). Greatly varied but generally high counts of IgG4-positive plasma cells were also seen in several of the other lesions, particularly in rheumatoid synovitis (mean 55/hpf), oral cavity lesions (mean 79/hpf) and carcinoma-associated inflammatory response (mean 24/hpf). The mean IgG4/IgG ratios for all lesions varied between 0 and 0.4. Conclusions The results demonstrate the ubiquitous occurrence of variably high numbers of IgG4-positive plasma cells under diverse non-specific inflammatory conditions, indicating that high IgG4-positive plasma cell counts and high IgG4/IgG ratios per se do not reliably distinguish IgG4-associated systemic disease from non-specific conditions, and that the IgG4 counts must be cautiously interpreted in the context of appropriate clinical and histopathological features.

A novel MCP-1 gene polymorphism is associated with hepatic MCP-1 expression and severity of HCV-related liver disease.

BACKGROUND AND AIMS Factors influencing the progression of chronic hepatitis C virus (HCV) infection are poorly understood. Monocyte chemotactic protein-1 (MCP-1) is a potent chemokine, and its hepatic expression is up-regulated during chronic HCV infection mainly in activated hepatic stellate cells (HSC). In this study, we investigated the correlation of the functional -2518 MCP-1 promoter polymorphism with hepatic MCP-1 expression and the disease outcome in patients with HCV. METHODS MCP-1 genotyping was performed in 206 patients and 139 healthy controls. Hepatic MCP-1 messenger RNA (mRNA) expression was quantified by real-time PCR in 58 HCV patients. Cytokine-induced MCP-1 secretion of activated human HSC (n = 13) was determined by enzyme-linked immunosorbent assay (ELISA). Mobility-shift assays were performed using probes corresponding to the MCP-1 promoter sequence (-2511 to -2528) with or without the A to G mutation at -2518. RESULTS Frequency of MCP-1 genotypes did not differ between HCV patients and controls. However, carriers of the G allele were significantly more frequent in HCV patients with more advanced fibrosis and severe inflammation. In accordance, hepatic MCP-1 mRNA levels were significantly higher in patients with more advanced fibrosis and in patients carrying the G allele. Furthermore, cytokine-induced MCP-1 secretion of HSC isolated from carriers of the G allele was significantly higher, and there was binding activity in nuclear extracts from activated HSC specifically to the G allele, providing a potential mechanism for the differences seen. CONCLUSIONS Inheritance of the -2518 MCP-1 G allele, which appears to affect hepatic MCP-1 expression, may predispose HCV patients to more severe hepatic inflammation and fibrosis.

Mosaicism of activating FGFR3 mutations in human skin causes epidermal nevi

Epidermal nevi are common congenital skin lesions with an incidence of 1 in 1,000 people; however, their genetic basis remains elusive. Germline mutations of the FGF receptor 3 (FGFR3) cause autosomal dominant skeletal disorders such as achondroplasia and thanatophoric dysplasia, which can be associated with acanthosis nigricans of the skin. Acanthosis nigricans and common epidermal nevi of the nonorganoid, nonepidermolytic type share some clinical and histological features. We used a SNaPshot multiplex assay to screen 39 epidermal nevi of this type of 33 patients for 11 activating FGFR3 point mutations. In addition, exon 19 of FGFR3 was directly sequenced. We identified activating FGFR3 mutations, almost exclusively at codon 248 (R248C), in 11 of 33 (33%) patients with nonorganoid, nonepidermolytic epidermal nevi. In 4 of these cases, samples from adjacent histologically normal skin could be analyzed, and FGFR3 mutations were found to be absent. Our results suggest that a large proportion of epidermal nevi are caused by a mosaicism of activating FGFR3 mutations in the human epidermis, secondary to a postzygotic mutation in early embryonic development. The R248C mutation appears to be a hot spot for FGFR3 mutations in epidermal nevi.

The MicroRNA Profile of Prostate Carcinoma Obtained by Deep Sequencing

Prostate cancer is a leading cause of tumor mortality. To characterize the underlying molecular mechanisms, we have compared the microRNA (miRNA) profile of primary prostate cancers and noncancer prostate tissues using deep sequencing. MiRNAs are small noncoding RNAs of 21 to 25 nucleotides that regulate gene expression through the inhibition of protein synthesis. We find that 33 miRNAs were upregulated or downregulated >1.5-fold. The deregulation of selected miRNAs was confirmed by both Northern blotting and quantitative reverse transcription-PCR in established prostate cancer cell lines and clinical tissue samples. A computational search indicated the 3′-untranslated region (UTR) of the mRNA for myosin VI (MYO6) as a potential target for both miR-143 and miR-145, the expression of which was reduced in the tumor tissues. Upregulation of myosin VI in prostate cancer was previously shown by immunohistochemistry. The level of MYO6 mRNA was significantly induced in all primary tumor tissues compared with the nontumor tissue from the same patient. This finding was matched to the upregulation of myosin VI in established prostate cancer cell lines. In luciferase reporter analysis, we find a significant negative regulatory effect on the MYO6 3′UTR by both miR-143 and miR-145. Mutation of the potential binding sites for miR-143 and miR-145 in the MYO6 3′UTR resulted in a loss of responsiveness to the corresponding miRNA. Our data indicate that miR-143 and miR-145 are involved in the regulation of MYO6 expression and possibly in the development of prostate cancer. Mol Cancer Res; 8(4); 529–38. ©2010 AACR.

Circulating micro-RNAs as potential blood-based markers for early stage breast cancer detection.

Introduction MicroRNAs (miRNAs, miRs) are a class of small, non-coding RNA molecules with relevance as regulators of gene expression thereby affecting crucial processes in cancer development. MiRNAs offer great potential as biomarkers for cancer detection due to their remarkable stability in blood and their characteristic expression in many different diseases. We investigated whether microarray-based miRNA profiling on whole blood could discriminate between early stage breast cancer patients and healthy controls. Methods We performed microarray-based miRNA profiling on whole blood of 48 early stage breast cancer patients at diagnosis along with 57 healthy individuals as controls. This was followed by a real-time semi-quantitative Polymerase Chain Reaction (RT-qPCR) validation in a separate cohort of 24 early stage breast cancer patients from a breast cancer screening unit and 24 age matched controls using two differentially expressed miRNAs (miR-202, miR-718). Results Using the significance level of p<0.05, we found that 59 miRNAs were differentially expressed in whole blood of early stage breast cancer patients compared to healthy controls. 13 significantly up-regulated miRNAs and 46 significantly down-regulated miRNAs in our microarray panel of 1100 miRNAs and miRNA star sequences could be detected. A set of 240 miRNAs that was evaluated by radial basis function kernel support vector machines and 10-fold cross validation yielded a specificity of 78.8%, and a sensitivity of 92.5%, as well as an accuracy of 85.6%. Two miRNAs were validated by RT-qPCR in an independent cohort. The relative fold changes of the RT-qPCR validation were in line with the microarray data for both miRNAs, and statistically significant differences in miRNA-expression were found for miR-202. Conclusions MiRNA profiling in whole blood has potential as a novel method for early stage breast cancer detection, but there are still challenges that need to be addressed to establish these new biomarkers in clinical use.