A novel five-antibody immunohistochemical test for subclassification of lung carcinoma.

Malignant epithelial lung carcinoma can be subclassified by histology into several tumor types, including adenocarcinoma and squamous cell carcinoma. The need for a uniform method of classifying lung carcinomas is growing as clinical trials reveal treatment and side effect differences associated with histological subtypes. Diagnosis is primarily performed by morphological assessment. However, the increased use of needle biopsy has diminished the amount of tissue available for interpretation. These changes in how lung carcinomas are diagnosed and treated suggest that the development of improved molecular-based classification tools could improve patient management. We used a 551-patient surgical specimen lung carcinoma retrospective cohort from a regional hospital to assess the association of a large number of proteins with histological type by immunohistochemistry. Five of these antibodies, targeting the proteins TRIM29, CEACAM5, SLC7A5, MUC1, and CK5/6, were combined into one test using a weighted algorithm trained to discriminate adenocarcinoma from squamous cell carcinoma. Antibody-based classification on 600 muM tissue array cores with the five-antibody test was compared to standard histological evaluation on surgical specimens in three independent lung carcinoma cohorts (combined population of 1111 patients). In addition, the five-antibody test was tested against the two-marker panel thyroid transcription factor-1 (TTF-1) and TP63. Both the five-antibody test and TTF-1/TP63 panel had similarly low misclassification rates on the validation cohorts compared to morphological-based diagnosis (4.1 vs 3.5%). However the percentage of patients remaining unclassifiable by TTF-1/TP63 (22%, 95% CI: 20-25%) was twice that of the five-antibody test (11%, 95% CI: 8-13%). The results of this study suggest the five-antibody test may have an immediate function in the clinic for helping pathologists distinguish lung carcinoma histological types. The results also suggest that if validated in prospectively defined clinical trials this classifier might identify candidates for targeted therapy that are overlooked with current diagnostic approaches.

Prognostic significance of epithelial-mesenchymal and mesenchymal-epithelial transition protein expression in non-small cell lung cancer.

PURPOSE: In carcinomas, invasive tumor growth is accompanied by desmoplastic stroma reaction and facilitated by epithelial-mesenchymal transition (EMT) of cancer cells. We investigated the prognostic significance of the EMT indicator proteins periostin and vimentin in comparison with versican, a putative indicator of the opposite mechanism mesenchymal-epithelial transition (MET), and to the desmoplasia proteins collagen and elastin in non-small cell lung cancer (NSCLC). EXPERIMENTAL DESIGN: Tumor of 533 patients with surgically resected NSCLC was used for analysis of stromal and epithelial protein expression by immunohistochemistry (EMT-MET proteins) and Elastica van Gieson histochemical staining (collagen and elastin). A semiquantitative sum scoring system was done on three tissue microarrays. RESULTS: Of the 533 patients, 48% had squamous cell carcinoma, 47% adenocarcinoma, and 5% adenosquamous carcinoma. High expression of periostin in either stroma or tumor epithelia, independently scored by two pathologists, correlated with male gender, higher stage, higher pT category, and larger tumor size, and in only stroma with tumor relapse. High expression of versican in either stroma or epithelia as well as of stromal collagen had fewer but concordant associations with advanced tumor and periostin, respectively. High expression of elastin was oppositely associated with less advanced disease. Associations of high vimentin were inconsistent (all P values < 0.05). High stromal periostin was found to be a prognostic factor for decreased progression-free survival on univariate analysis (P = 0.007). CONCLUSIONS: Because up-regulation is frequently observed in the stromal and epithelial tumor compartment, EMT-MET indicator proteins may be integrated in progression models of NSCLC.

Automated ERCC1 immunohistochemistry in non-small cell lung cancer: comparison of anti-ERCC1 antibodies 8F1, D-10, and FL-297.

INTRODUCTION: The excision repair cross-complementation group 1 (ERCC1) protein is the key enzyme of the nucleotide excision repair (NER) pathway and thus a potential predictor for platinum-based chemotherapy response. We aimed for evaluating different anti-ERCC1 antibodies on formalin-fixed tumor tissue of non-small cell lung cancer patients by automated immunohistochemistry (IHC). METHODS: ERCC1 protein expression was assessed on a tissue microarray of 491 NSCLC's using 2 monoclonal mouse (Mab 8F1, Mab D-10) and 1 polyclonal rabbit (Rab FL-297) antibody. Two automated IHC platforms with different detection systems and immunofluorescence were used. Protein expression levels were independently scored by 2 pathologists for both intensity and intensity multiplied by percentage of positive cells (H-score). RESULTS: On both platforms, the 8F1 ab showed best nuclear staining quality. D-10 had additional unspecific background at the plasma membrane and in goblet cells. FL-297 could not be scored owing to high cytoplasmic background. Both 8F1 and D-10 antibodies produced a speckled granular pattern over the whole nuclear compartment. No intranuclear compartmentalization was observed, apart from omission of the nucleolus. Interobserver κ value was good to very good for 8F1 and D-10. Using 8F1, low ERCC1 was correlated with the adenocarcinoma histotype, increased tumor size and clinical stage, high pT and pN category and the presence of metastasis. No relation to progression-free or overall survival was observed. CONCLUSIONS: In terms of staining quality and restriction to the nuclear compartment, the antibody 8F1 is superior to D-10 or FL-297 on automated IHC platforms.

EML4-ALK fusion lung cancer: a rare acquired event.

A recurrent gene fusion between EML4 and ALK in 6.7% of non-small cell lung cancers (NSCLCs) and NKX2-1 (TTF1, TITF1) high-level amplifications in 12% of adenocarcinomas of the lung were independently reported recently. Because the EML4-ALK fusion was only shown by a reverse transcription-polymerase chain reaction approach, we developed fluorescent in situ hybridization assays to interrogate more than 600 NSCLCs using break-apart probes for EML4 and ALK. We found that EML4-ALK fusions occur in less than 3% of NSCLC samples and that EML4 and/or ALK amplifications also occur. We also observed that, in most cases in which an EML4/ALK alteration is detected, not all of the tumor cells harbor the lesion. By using a detailed multi-fluorescent in situ hybridization probe assay and reverse transcription-polymerase chain reaction, we have evidence that other, more common mechanisms besides gene inversion exist including the possibility of other fusion partners for ALK and EML4. Furthermore, we confirmed the NKX2-1 high-level amplification in a significant subset of NSCLC and found this amplification to be mutually exclusive to ALK and EML4 rearrangements.