Epidermal growth factor receptor (EGFR) gene mutations and increased copy numbers are considered as predictors of response to EGFR tyrosine kinase inhibitors (EGFR-TKI) in non-small-cell lung cancer (NSCLC). Lung cancer diagnosis is often based on cytology alone. However, almost all published data on EGFR gene analyses were obtained from biopsies. This study tested the feasibility of EGFR gene analyses on cytological specimens. Eighty-four cytological specimens from NSCLCs were prospectively analysed for EGFR gene mutation in exons 18 -21 and EGFR gene copy numbers were evaluated by fluorescence in situ hybridisation (FISH). A FISHpositive result was defined according to the criteria by Cappuzzo et al established for biopsies of NSCLCs. Fluorescence in situ hybridisation results of cytological specimens were compared to the FISH results on matching biopsies (n ¼ 33). Initial diagnosis of NSCLC was solely based on cytology in 37 out of 84 (44.0%) patients. Out of 80 NSCLCs, 6 (7.5%) showed EGFR gene mutations. Out of 67 cancers, 45 (67.2%) were FISH positive on cytological specimens. Comparison of FISH showed a FISH-positive result in 21 out of 33 (63.6%) cytological specimens but in only 8 out of 33 (24.2%) matched biopsies. Epidermal growth factor receptor gene analyses are well applicable to cytological specimens. The high FISH-positive rate of NSCLC on cytological specimens contrasts with the low rate on biopsies when previously suggested criteria are used. New criteria for a positive EGFR FISH status to predict response to therapy with EGFR-TKI need to be defined for cytological specimens.

Confounding effects of specific KRAS gene alterations on colorectal cancer (CRC) prognosis stratified by microsatellite instability (MSI) and BRAF V600E have not yet been investigated. The aim of our study was to evaluate the combined effects of MSI, BRAF V600E and specific KRAS mutation (Gly fi Asp; G12D, Gly fi Asp, G13D; Gly fi Val; G12V) on prognosis in 404 sporadic and 94 hereditary CRC patients. MSI status was determined according to the Bethesda guidelines. Mutational status of KRAS and BRAF V600E was assessed by direct DNA sequencing. In sporadic CRC, KRAS G12D mutations had a negative prognostic effect compared to G13D and wild-type cancers (p 5 0.038). With MSI, specific KRAS and BRAF V600E mutations, 3 distinct prognostic subgroups were observed in univariate (p 5 0.006) and multivariable (p 5 0.051) analysis: patients with (i) KRAS mutation G12D, G12V or BRAF V600E mutation, (ii) KRAS/BRAF V600E wild-type or KRAS G13D mutations in MSS/MSI-L and (iii) MSI-H and KRAS G13D mutations. Moreover, none of the sporadic MSI-H or hereditary patients with KRAS G13 mutations had a fatal outcome. Specific KRAS mutation is an informative prognostic factor in both sporadic and hereditary CRC and applied in an algorithm with BRAF V600E and MSI may identify sporadic CRC patients with poor clinical outcome.Sporadic colorectal cancer (CRC) is a heterogeneous disease arising from at least 2 different and seemingly independent pathways, namely the chromosomal instability (CIN, or microsatellite stable, MSS) and microsatellite instability (MSI) pathways. 1 CRCs from the MSS pathway are thought to originate from a relatively uniform and linear accumulation of genetic changes in APC, KRAS and TP53. 2 However, in practice, only 10% of these tumors are characterized by this classic genotype. 3,4 Sporadic MSI-high (MSI-H) cancers occur through promoter hypermethylation of the MLH1 gene, more frequently found in females, and tend to be poorly differentiated tumors, of mucinous subtype, associated with a Crohn's like reaction at the invasive tumor front and often harboring somatic mutations in BRAF V600E . 5-7 MSI-H CRCs can also arise in the context of Lynch syndrome/hereditary nonpolyposis CRC (HNPCC), an autosomal dominant disorder, accounting for 3% of all CRC cases, characterized by germline mutations of mismatch-repair genes. 8 Both sporadic and Lynch syndrome-associated MSI-H tumors are characterized by dense tumor-infiltrating lymphocyte populations and less frequent metastases suggesting enhanced immunogenicity irrespective of the mechanism of DNA mismatch repair inactivation. 9,10 Recently, MSI status, BRAF V600E and KRAS mutation status have attracted significant attention due to their potential prognostic and predictive role in CRC. 11 On one hand, patients with MSI-H cancers are specifically thought to have a favorable outcome; yet on the other hand, they are generally nonresponsive to certain chemotherapeutic agents such as 5-flurouracil (5-FU). 6,12 The prognostic effect of somatic KRAS mutations, described in 30...

CpG island methylator phenotype (CIMP) is being investigated for its role in the molecular and prognostic classification of colorectal cancer patients but is also emerging as a factor with the potential to influence clinical decision-making. We report a comprehensive analysis of clinico-pathological and molecular features (KRAS, BRAF and microsatellite instability, MSI) as well as of selected tumour- and host-related protein markers characterizing CIMP-high (CIMP-H), -low, and -negative colorectal cancers. Immunohistochemical analysis for 48 protein markers and molecular analysis of CIMP (CIMP-H: ≥ 4/5 methylated genes), MSI (MSI-H: ≥ 2 instable genes), KRAS, and BRAF were performed on 337 colorectal cancers. Simple and multiple regression analysis and receiver operating characteristic (ROC) curve analysis were performed. CIMP-H was found in 24 cases (7.1%) and linked (p < 0.0001) to more proximal tumour location, BRAF mutation, MSI-H, MGMT methylation (p = 0.022), advanced pT classification (p = 0.03), mucinous histology (p = 0.069), and less frequent KRAS mutation (p = 0.067) compared to CIMP-low or -negative cases. Of the 48 protein markers, decreased levels of RKIP (p = 0.0056), EphB2 (p = 0.0045), CK20 (p = 0.002), and Cdx2 (p < 0.0001) and increased numbers of CD8+ intra-epithelial lymphocytes (p < 0.0001) were related to CIMP-H, independently of MSI status. In addition to the expected clinico-pathological and molecular associations, CIMP-H colorectal cancers are characterized by a loss of protein markers associated with differentiation, and metastasis suppression, and have increased CD8+ T-lymphocytes regardless of MSI status. In particular, Cdx2 loss seems to strongly predict CIMP-H in both microsatellite-stable (MSS) and MSI-H colorectal cancers. Cdx2 is proposed as a surrogate marker for CIMP-H.

Purpose: KIT (CD117) is a transmembrane tyrosine kinase representing a target for STI571 (Glivec) therapy. Some KIT-overexpressing solid tumors have responded favorably to STI571, potentially because of the presence of KIT-activating mutations.Experimental Design: To investigate the epidemiology of KIT overexpression and mutations, we investigated a series of 1654 breast cancers. All tumors were analyzed by immunohistochemistry in a tissue microarray format.Results: KIT expression was always present in normal breast epithelium. However, cancer analysis revealed the only 43 of 1654 (2.6%) tumors were KIT-positive. KIT expression was more frequent in medullary cancer (9 of 47 positive; 19.1%) than in any other histological tumor subtype (P < 0.001). KIT expression was significantly associated with high tumor grade (P < 0.0001) but unrelated to pT and pN categories or patient survival. Mutation analysis of exons 2, 8, 9, 11, 13, and 17 was negative in 10 KIT-positive tumors.Conclusions: Overall, our data show that a high level of KIT expression occurs infrequently in breast cancer. KITpositive breast cancers may not reflect "KIT up-regulation" because KIT is also expressed in normal breast epithelium. The lack of KIT mutations also argues against the therapeutic efficacy of STI571 in breast cancer.

We aimed to immunohistochemically examine the expression of KIT (CD 117) in human posterior uveal melanoma and to analyze KIT-positive tumors for gene mutations. Brought into a tissue microarray (TMA) format were 101 formalin-fixed, paraffin-embedded posterior uveal melanomas. Immunohistochemistry was performed using the polyclonal anti-CD117 antibody from Dako (A4502). In ten selected KIT-positive tumors, exons 2, 8, 9, 11, 13 and 17 were sequenced. Of the 101 cases, 89 (88%) could be evaluated on the TMAs. Immunohistochemistry for CD 117 was weakly positive in 5 cases (6%), moderately positive in 10 cases (12%) and strongly positive in 57 cases (69%). No KIT mutations were detected in the analyzed exons. In conclusion, human posterior uveal melanoma frequently expresses CD117 at high levels. Although KIT mutations could not be found, it appears justified to investigate the utility of imatinib mesylate in the treatment of these patients.