Chromogenic in situ hybridization (CISH) to detect HER2 gene amplification in breast and gastric cancer: Comparison with immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH)

The chromogenic in situ hybridization (CISH) assay, designed to detect the amplification of the HER2 gene in formalin‐fixed, paraffin‐embedded (FFPE) breast cancer (BC) and gastric cancer (GC) tissue specimens, was evaluated in 125 FFPE BC cases and 198 FFPE GC cases for which the HER2 status had been predetermined using immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). In the 125 BC cases and the 198 gastric cases, we found a very good concordance (98.4% and 99.0%, respectively) between CISH and FISH. In particular, we evaluated the polysomy cases, as these cases often have ambiguous treatment options in clinical practice. The polysomy of chromosome 17 was defined as the presence of three or more CEP17 signals in at least 10% of the tumor cells. In the 50 BC cases and 54 GC cases displaying chromosome 17 polysomy, the concordance between FISH and CISH was 98.0% and 98.1%, respectively. These results indicate that CISH could provide an accurate and practical alternative to FISH for the clinical diagnosis of HER2 gene amplification in FFPE BC and FFPE GC samples.     

Anaplastic lymphoma kinase translocation is correlated with anaplastic lymphoma kinase expression and mutually exclusive with epidermal growth factor receptor mutation in Taiwanese non‐small cell lung cancer

The echinoderm microtubule‐associated protein‐like 4‐anaplastic lymphoma kinase (EML4‐ALK) fusion gene is an important biomarker for target therapy. The aim of this study is to better understand the clinical and molecular features of the EML4‐ALK fusion gene in lung cancer patients in Taiwan and therefore to generate an efficient algorithm for the detection of ALK translocation. In the first cohort, ALK translocation was identified in 1 adenocarcinoma from 100 lung cancer patients by using break apart fluorescent in situ hybridization (FISH). Next, we detected 6 ALK translocations in another 40 EGFR wild type adenocarcinomas but not in 40 cases with EGFR mutation. Histological analysis revealed that solid growth with signet‐ring cells or cribriform glands with extracellular mucin were noted in all the 7 ALK translocated cases. One ALK positive cancer with mucinous cribriform pattern had no ALK expression. ALK expression was correlated with ALK translocation (p < 0.001), but not with ALK gene copy number gain (CNG) (P = 0.838). ALK translocation was also mutually exclusive with EGFR mutation in Taiwanese non‐small cell lung cancer (P = 0.033). These results indicate that screening tests for EGFR mutation status and/or ALK expression could help efficiently select ALK translocated patients for target therapy.     

Identification of DJ‐1 as a contributor to multidrug resistance in human small‐cell lung cancer using proteomic analysis

Proteomic approaches have been proven to provide an important tool in identifying drug resistance‐associated proteins. The aim of this study was to investigate the protein profiling of drug resistance‐related proteins in small‐cell lung cancer (SCLC) by proteomic analysis. The proteomic profiling was performed by two‐dimensional fluorescence difference gel electrophoresis (2D‐DIGE) coupled with MALDI‐TOF‐TOF of SCLC in the multidrug‐resistant cell line H69AR and its parental cell line H69. A total of 11 proteins were identified to be >2‐fold up‐or downregulated between the two cell lines. DJ‐1, one of the differently expressed proteins identified by proteomics, was further examined by immunohistochemistry staining in 116 cases of SCLC tissues. Immunohistochemical results demonstrated that DJ‐1 was expressed in 51.7% (60/116) of SCLC. DJ‐1 expression was correlated significantly with survival time of SCLC patients (P < 0.05), but not with other clinical parameters such as gender, age and clinical stage (P > 0.05). Downregulation of DJ‐1 using DJ‐1‐siRNA in H69AR cells sensitized cancer cells to chemotherapeutic drugs through increasing drug‐induced cell apoptosis accompanied with G0‐G1 phase arrest. These findings suggest DJ‐1 may serve as a potential biomarker for chemoresistance and prognostic factor for patients with SCLC.     

The alpha1 subunit of the sodium pump could represent a novel target to combat non-small cell lung cancers

With an overall 5 year survival rate as low as 15% for non-small cell lung cancer (NSCLC), even with surgical intervention and the use of newer molecules in adjuvant chemotherapy, there is an urgent need for new biological targets and associated novel anti-cancer agents. The present study was undertaken to evaluate the potential of the Na(+)/K(+)-ATPase alpha1 subunit as a novel target in NSCLC and revealed that alpha1 expression is markedly higher in a significant proportion of NSCLC clinical samples compared to normal lung tissue. Furthermore, reduction in alpha1 expression in A549 NSCLC cells by anti-alpha1 siRNA resulted in markedly impaired proliferation and migration of these cancer cells. Finally, of three cardenolides investigated, UNBS1450, which is known to bind to Na(+)/K(+)-ATPase and displays potent anti-tumour activity in vivo in experimental models of human NSCLCs, is the most potent inhibitor of Na(+)/K(+)-ATPase isozymes (alpha1beta1, alpha2beta1 and alpha3beta1), most strikingly of alpha1beta1. This was reflected in the compound's more potent anti-proliferative activity in all NSCLC cell lines evaluated (A549, Cal-12T, NCI-H727 and A427); the first three of which over-express alpha1. The marked impairment in A549 NSCLC cell proliferation and migration, and resulting similar morphology following anti-alpha1 siRNA or UNBS1450 treatment, was associated with features of abnormal cytokinesis, mediated in the case of UNBS1450 by disorganization of the actin cytoskeleton. Collectively these data strongly suggest that targeting the Na(+)/K(+)-ATPase alpha1 using specific cardenolides could represent a novel means to combat certain NSCLCs.     

Genetic variants of immunoglobulin γ and κ chains influence humoral immunity to the cancer‐testis antigen XAGE‐1b (GAGED2a) in patients with non‐small cell lung cancer

GM (γ marker) allotypes, genetic variants of immunoglobulin γ chains, have been reported to be associated strongly with susceptibility to lung cancer, but the mechanism(s) underlying this association is not known. One mechanism could involve their contribution to humoral immunity to lung tumour‐associated antigens. In this study, we aimed to determine whether particular GM and KM (κ marker) allotypes were associated with antibody responsiveness to XAGE‐1b, a highly immunogenic lung tumour‐associated cancer‐testis antigen. Sera from 89 patients with non‐small cell lung cancer (NSCLC) were allotyped for eight GM and two KM determinants and characterized for antibodies to a synthetic XAGE‐1b protein. The distribution of various GM phenotypes was significantly different between XAGE‐1b antibody‐positive and ‐negative patients (P = 0·023), as well as in the subgroup of XAGE‐1b antigen‐positive advanced NSCLC (P = 0·007). None of the patients with the GM 1,17 21 phenotype was positive for the XAGE‐1b antibody. In patients with antigen‐positive advanced disease, the prevalence of GM 1,2,17 21 was significantly higher in the antibody‐positive group than in those who lacked the XAGE‐1b antibody (P = 0·026). This phenotype also interacted with a particular KM phenotype: subjects with GM 1,2,17 21 and KM 3,3 phenotypes were almost four times (odds ratio = 3·8) as likely to be positive for the XAGE‐1b antibody as the subjects who lacked these phenotypes. This is the first report presenting evidence for the involvement of immunoglobulin allotypes in immunity to a cancer‐testis antigen, which has important implications for XAGE‐1b‐based immunotherapeutic interventions in lung adenocarcinoma.     

FOXP1 protein overexpression is associated with inferior outcome in nodal diffuse large B‐cell lymphomas with non‐germinal centre phenotype,independent of gains and structural aberrations at 3p14.1

Hoeller S, Schneider A, Haralambieva E, Dirnhofer S & Tzankov A
(2010) Histopathology 57, 73–80
FOXP1 protein overexpression is associated with inferior outcome in nodal diffuse large B‐cell lymphomas with non‐germinal centre phenotype, independent of gains and structural aberrations at 3p14.1 Aims: To determine the molecular epidemiology and prognostic importance of structural and numeric FOXP1 gene aberrations with respect to BCL‐6 gene and to FOXP1 protein expression in 389 diffuse large B‐cell lymphomas (DLBCL) from the pre‐rituximab era on tissue microarrays. Methods and results: By interphase fluorescence in situ hybridization with colour‐labelled bacterial artificial chromosome clones, 12% (27/223) analysable cases showed FOXP1 gains and 1% (2/210) FOXP1 breaks. Seven percent of cases with known BCL‐6 and FOXP1 gene status (n = 159) showed an isolated FOXP1 gain, 19% an isolated BCL‐6 gain and 18% a trisomy 3. FOXP1 gains (isolated and due to trisomy 3) were more frequent in nodal than extranodal DLBCL and in non‐germinal centre B‐cell‐like (non‐GCB) DLBCL than in GCB DLBCL. By immunohistochemistry, FOXP1 protein was more often overexpressed in non‐GCB than in GCB cases. FOXP1 overexpression was associated with poor disease‐specific survival in all DLBCL, particularly in nodal and non‐GCB cases. There was no correlation between FOXP1 gene aberrations and either FOXP1 protein expression or survival. Conclusions: FOXP1 is recurrently targeted by numeric, and rarely by structural, genetic aberrations in DLBCL. Only the presence of FOXP1 protein, irrespective of its gene status, is decisive for prognosis in DLBCL.     

What's new in non-small cell lung cancer for pathologists: the importance of accurate subtyping, EGFR mutations and ALK rearrangements

In the past, the only critical point of distinction in the pathological diagnosis of lung cancer was between small cell and non-small cell lung cancer (NSCLC). The emergence of new targeted therapies and clinical trials demonstrating differing efficacy and toxicity of treatments according to specific histological subtypes of NSCLC, has resulted in an increasing need for improvements in pathological diagnosis. Accurate distinction between adenocarcinoma and squamous cell carcinoma is now critical as histological subtyping has the potential to influence clinical decision making and impact on patient outcome. While morphological criteria remain the most important feature to distinguish NSCLC subtypes, use of mucin and immunohistochemical stains (TTF-1, p63 and CK5/6) can be of assistance in difficult small biopsy cases. With the emergence of selective kinase inhibitors targeting epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK), there is a corresponding need to identify the subset of NSCLCs harbouring specific genetic mutations associated with sensitivity to these agents, almost all of which are found in adenocarcinomas. In this review, the importance of accurately subtyping NSCLC is discussed, along with a suggested approach for distinguishing histological subtypes in small biopsy specimens. The significance of EGFR and ALK mutations in NSCLC and the impact of these genotypes on pathology and clinical practice are also reviewed.