Sanghoon Jheon

Screening of Anaplastic Lymphoma Kinase Rearrangement by Immunohistochemistry in Non-small Cell Lung Cancer: Correlation with Fluorescence In Situ Hybridization

Background: The use of a standard immunohistochemistry (IHC) assay to detect the anaplastic lymphoma kinase (ALK) protein in lung cancer is challenging. There are no universally accepted, evidence-based guidelines on identifying patients with ALK-rearranged lung cancer using IHC. Methods: We retrospectively reviewed 465 resected specimens of non-small cell lung cancer using a tissue microarray as a test set. ALK protein expression using IHC with 5A4 monoclonal antibody (Novocastra) and ALK gene rearrangement using fluorescence in situ hybridization (FISH) with dual-color break-apart probes (Abbott molecular) were examined. Immunoreactivity was scored as 0, 1, 2, or 3, and the results were compared with the FISH results. A diagnostic algorithm was derived from the correlation of the IHC and FISH results and applied to an additional 187 adenocarcinoma samples used as a validation set. Results: In the test set, ALK protein expression was detected in 40 patients (40/465, 8.6%), consisting of IHC scores of 1 (n = 14), 2 (n = 10), and 3 (n = 16), whereas 19 patients (19/453, 4.2%) were FISH-positive. All the FISH-positive patients were assigned IHC scores of 2 or 3. All the patients with ALK IHC scores of 3 were FISH-positive, those with scores of 0 or 1 were FISH-negative, and those with scores of 2 were FISH variable. In the validation set, ALK protein expression was detected in 14 patients (scores of 1, n = 2; scores of 2, n = 6; and scores of 3, n = 6), of which nine patients (9/187, 4.8%) were FISH-positive. All the patients with IHC scores of 0 or 1 were FISH-negative, and those with scores of 3 were FISH- positive. Among the patients with IHC scores of 2, three (3/6, 50%) were FISH-positive. Conclusions: The sensitivity and specificity of IHC was 100% and 95.8%, respectively. These data supported an IHC scoring algorithm in which ALK IHC scores of 0, 1, or 3 were highly compatible with FISH results, and IHC scores of 2 were variable. Based on these findings, the IHC assay using the 5A4 antibody reliably detected non-small cell lung cancer with ALK rearrangement and may be useful as a screening method to identify these tumors.

Detection of ALK Gene Rearrangement in Non-small Cell Lung Cancer A Comparison of Fluorescence In Situ Hybridization and Chromogenic In Situ Hybridization with Correlation of ALK Protein Expression

Introduction: Accurate determination of ALK rearrangement is important in lung cancer patients, especially in determining their eligibility for crizotinib therapy. Fluorescence in situ hybridization (FISH) has been regarded as the gold standard method for detecting ALK rearrangement. However, FISH requires a fluorescence microscope, and the signals are labile and rapidly fade over time. This study evaluates the concordance between ALK gene rearrangement in non-small cell lung cancer assessed by ALK FISH and a newly developed ALK chromogenic in situ hybridization (CISH) and correlates the results with ALK protein expression assessed by immunohistochemistry. Methods: A total of 465 formalin-fixed, paraffin-embedded non-small cell lung cancer samples were analyzed by ALK FISH (PathVysion, Vysis, Abbott) and ALK CISH. For comparison, all specimens were stained by immunohistochemistry (clone 5A4, Novocastra) and interobserver reproducibility was assessed. Results: We found that agreement between the pathologists on the CISH-determined ALK status was achieved in 449 patients (96.6%), and ALK rearrangement was identified in 18 patients (4.0%) in CISH method. Among these cases, 443 cases (95.3%) had results matching the corresponding FISH results: 17 rearranged, 425 wild types, and 1 discordant case. There was high concordance in the assessment of ALK gene rearrangement between FISH and CISH techniques (&kgr; = 0.92) and between observers (&kgr; = 0.97). In addition, there was high concordance in the ALK gene status and ALK protein expression between CISH and IHC tests (&kgr; = 0.82). Conclusions: CISH is a highly reproducible and practical method to detect ALK gene rearrangement and correlated well with ALK protein expression. Here, we present a diagnostic algorithm (Chungs SNUBH ALK protocol) to detect lung cancer with ALK rearrangements using IHC, FISH and CISH. Because CISH allows a concurrent analysis of histological features of the tumors and gene rearrangement, it appears to be a useful method in determining ALK gene rearrangement.

Clinicopathologic implication of ALK rearrangement in surgically resected lung cancer: A proposal of diagnostic algorithm for ALK-rearranged adenocarcinoma

BACKGROUND To characterize the clinicopathologic features of ALK-rearranged lung cancer, and suggest a molecular test protocol for lung adenocarcinoma in the small biopsy specimen. METHODS In 735 NSCLC surgical specimens, clinicopathologic features, ALK protein over-expression by immunohistochemistry (IHC), and ALK rearrangement by fluorescence in situ hybridization (FISH) as well as EGFR and KRAS mutation studies were analyzed. RESULTS Of the 735 NSCLC cases, 28 (3.8%) were ALK FISH-positive. ALK rearrangement, EGFR and KRAS mutation were mutually exclusive. ALK rearrangement was significantly higher in adenocarcinomas (6.8%, p<0.001), younger age (p<0.0007), women (7.6%, p<0.001), and never-smokers (8.9%, p<0.001) with no gender difference in the adenocarcinoma or never-smoker subgroup. ALK FISH-positivity was not associated with disease recurrence (HR, 0.79; 95% CI, 0.42-1.49) or overall survival (HR, 0.61; 95% CI, 0.24-1.55). However, ALK-rearranged lung cancer tended to show more frequent lymph node metastasis despite its lower T stage. Similar to EGFR-mutated lung cancer, ALK-rearranged lung cancer was enriched in adenocarcinoma, women, and never-smokers. The results of ALK IHC and FISH obtained from tissue microarray (TMA)/biopsy specimens and whole sections after resection were concordant. CONCLUSION ALK rearrangement was not a significant prognostic factor in surgically resectable NSCLC. The clinical profiles of ALK-rearranged lung cancer patients overlapped with those of EGFR-mutated patients. Therefore, we suggest that simultaneous tests for ALK IHC and EGFR mutation (Chungs SNUBH molecular test protocol), which has important implications for the storage and use of small biopsy or cytology samples for genetic analysis.

High incidence of EGFR mutations in Korean men smokers with no intratumoral heterogeneity of lung adenocarcinomas: correlation with histologic subtypes, EGFR/TTF-1 expressions, and clinical features.

Introduction: Epidermal growth factor receptor (EGFR) mutation has been known to be associated with adenocarcinoma with bronchioloalveolar carcinoma (BAC; lepidic) feature. This study was aimed to characterize the frequency of EGFR mutations and their association with histologic subtypes in Korean nonsmall cell lung cancer (NSCLC) patients. Methods: Three hundred eighty-two (88 biopsies and 294 resections) NSCLC patients were investigated for EGFR mutations (exons 18–21) by polymerase chain reaction and direct sequencing method. For the resected adenocarcinoma specimens, histologic subtypes were classified according to both 2004 World Health Organization classification and 2011 International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society classification. The results were correlated with EGFR mutation and clinicopathologic features. Results: EGFR mutations were detected in 196 of 382 NSCLCs (51.3%) and were more frequent in women than in men (65.7% versus 34.3%, p < 0.001) and in nonsmokers than in smokers (63.4% versus 32.0%, p < 0.001). Regarding histologic subtypes of adenocarcinoma, mixed acinar and BAC pattern showed the most frequent EGFR mutation (67.6%), followed by mixed papillary and acinar (65.2%), mixed solid and acinar (38.2%), micropapillary and acinar (30.4%), and acinar and mucinous BAC (13.3%). In addition, EGFR mutations were more frequently observed in tumors with BAC or papillary components than those with mucinous BAC or solid components. Identical EGFR mutations were detected in a single tumor showing mixed histological features. EGFR protein expression was seen more frequently in tumors with EGFR mutations than those without EGFR mutations (75.3% versus 24.7%, p=0.003). EGFR mutations were significantly more common in tumors with thyroid transcription factor-1 (TTF-1) expression than those without TTF-1 (p < 0.001), and almost all (92.7%) mutated adenocarcinomas were TTF-1 positive. Conclusions: The incidence of EGFR mutations is variable according to histologic subtypes, gender, and smoking history. The mixed acinar and BAC and papillary and acinar subtypes, the presence of BAC (lepidic) or papillary components, EGFR, and TTF-1 protein expression can predict higher EGFR mutation in lung adenocarcinoma. However, intratumoral heterogeneity of EGFR mutation was not found. In addition, relatively high incidence of EGFR mutations in Korean men who smoked with adenocarcinoma histology suggests that these patients should not be left behind EGFR mutation test.

Epidermal Growth Factor Receptor Mutation and Pathologic-Radiologic Correlation Between Multiple Lung Nodules with Ground-Glass Opacity Differentiates Multicentric Origin from Intrapulmonary Spread

Introduction: No standard guidelines detailing recommendations for the selection and treatment for multiple lung nodules with ground-glass opacity (GGO) have been established. For treatment decision, we analyzed epidermal growth factor receptor (EGFR)/K-ras somatic aberrations and pathologic-radiologic correlation in multiple lung nodules presented as GGO to differentiate multifocal lesions from intrapulmonary spread. Methods: Twenty-four patients with multiple lung nodules presented as GGO were identified to investigate somatic mutations of EGFR (exon 18–21) and K-ras (codons 2, 13, and 61). This series included 18 atypical adenomatous hyperplasias (AAH), 15 bronchioloalveolar carcinomas (BAC), and 23 adenocarcinomas (ADC) obtained from 24 patients. Results: High frequency of discordant EGFR mutations (17 of 24, 70.8%) could discriminate tumor clonality (18 of 24, 75%) of multiple lung neoplastic nodules presented as GGO. EGFR mutations were common in AAH (38.9%), BAC (46.7%), and ADC (39.1%). In case 4, AAH and BAC had different mutational changes, and in case 10, the BAC lesion contains EGFR mutation that is not in the invasive ADC. In case 17, the BAC had more mutational changes than the carcinoma. The pure GGO appearance in the radiologic examination corresponded preinvasive pathologic change. Conclusions: This study showed that synchronous BAC and/or ADC can have different EGFR or K-ras mutational profiles suggesting these lesions arise as independent events rather than intrapulmonary spread or systemic metastasis. This has significant implication in staging and treatment. These findings might be a clue to establish guidelines of the multiple neoplastic lung nodules with GGO.

The prognostic significance of ERCC1, BRCA1, XRCC1, and βIII-tubulin expression in patients with non-small cell lung cancer treated by platinum- and taxane-based neoadjuvant chemotherapy and surgical resection

OBJECTIVES The DNA repair pathway and isotype composition of beta-tubulin are known to be associated with resistance to platinum- and taxane-based chemotherapy, respectively. The aim of this study was to identify the clinical significance of excision repair cross-complementation 1 (ERCC1), breast cancer susceptibility gene 1 (BRCA1), X-ray repair cross-complementation 1 (XRCC1) and betaIII-tubulin on the chemotherapy response and overall survival in patients with non-small cell lung cancer (NSCLC) who received neoadjuvant chemotherapy. METHODS Protein expression profiles were evaluated by immunohistochemistry on surgical specimens of 82 NSCLC patients who underwent platinum- and taxane-based neoadjuvant chemotherapy. The expression levels of proteins were measured semi-quantitatively and the correlation with tumor responses, pathologic cell death rate and survival were evaluated. RESULTS There were 73 (89.0%) clinical stage III patients. Lobectomy, bilobectomy, and pneumonectomy were performed in 54 (65.0%), 11 (13.4%), and 17 (20.7%) patients, respectively. There was no correlation between clinical response and protein expression. The expression levels of ERCC1, BRCA1, and XRCC1 increased proportionally to the cell death rate (p<0.05); however, betaIII-tubulin expression did not correlate with cell viability. Multivariate analysis demonstrated that early pathologic stage, adjuvant chemotherapy, high ERCC1 and low betaIII-tubulin expression were good prognostic factors for overall survival (p<0.05). CONCLUSIONS The inverse correlation between DNA repair proteins and cell viability suggests that these protein expression levels can be markers for chemotherapy responsiveness. However, only ERCC1 and betaIII-tubulin were prognostic factors after platinum- and taxane-based neoadjuvant chemotherapy following surgical resection.

Protein overexpression and gene amplification of epidermal growth factor receptor in nonsmall cell lung carcinomas: Comparison of four commercially available antibodies by immunohistochemistry and fluorescence in situ hybridization study.

Epidermal growth factor receptor (EGFR) overexpression in nonsmall cell lung carcinomas (NSCLC) is variable ranging from 19% to 89% and its prognostic value remains controversial. We tried to investigate (1) EGFR protein expression using four different antibodies, (2) the correlation between protein overexpression and EGFR gene amplification, and (3) the correlation between EGFR genetic status and clinicopathologic features in NSCLC. We examined EGFR protein expression using four different antibodies including Zymed EGFR kit (Clone 31G7), Dako EGFR pharmDx kit (Clone 2-18C9), Dako (Clone H11) and Novocastra (Clone EGFR 113) by immunohistochemical analysis. The protein overexpression was compared to gene amplification status by fluorescence in situ hybridization (FISH). EGFR protein overexpression was observed in 56% of tumors with Zymed EGFR kit, in 51% with Dako EGFR pharmDx kit, in 5% with Dako and in 18% with Novocastra (p=0.010). Both Zymed and Dako pharmDx kit were more sensitive than the Dako test (clone H11) and Novocastra clone EGFR 113. EGFR overexpression was more prominent in squamous cell carcinomas (SCC) than adenocarcinomas (ADC) (71% vs. 48%, p=0.001 with Zymed, 61% vs. 45%, p=0.011 with Dako pharmDx kit; respectively). EGFR FISH-positivity as represented by high polysomy and gene amplification was observed in 45% of the NSCLC patients. Protein expression levels significantly correlated with the gene copy number per tumor cell (p<0.001). Our data showed a higher percentage of positive cells detected by Zymed and Dako pharmDx tests. The EGFR protein overexpression rate varied from 4% to 72% according to different antibody clones and histologic types. EGFR protein expression detected by Zymed and Dako pharmDx was significantly associated with a high EGFR gene copy number.

CD24, a Novel Cancer Biomarker, Predicting Disease-Free Survival of Non-small Cell Lung Carcinomas: A Retrospective Study of Prognostic Factor Analysis from the Viewpoint of Forthcoming (Seventh) New TNM Classification

Introduction: Metastasis-associated protein CD24 has been identified as a new prognostic factor and stem cell marker in the human neoplasm. However, the importance of the CD24 in non-small cell lung carcinomas (NSCLCs) has not been elucidated well. Methods: We evaluated CD24 expression in 267 consecutive cases of NSCLC by immunohistochemistry using a tissue microarray technique and correlated with clinicopathologic parameters including forthcoming (seventh) new tumor node metastasis classification. Results: CD24-high expression was demonstrated in 87 of 267 (33%) and was associated with adenocarcinoma (ADC) histology than in squamous cell carcinoma histology (64 of 165 [39%] vs. 20 of 88 [23%]; p = 0.023). Patients with CD24-high tumors tended to have a higher risk of disease progression (p < 0.001) and cancer-related death (p = 0.002). Multivariate analysis proved CD24-high expression as independent prognostic factors of disease progression and cancer-related death (p = 0.002, hazard ratio = 1.78, 95% confidence interval = 1.23–2.58 and p = 0.017, hazard ratio = 1.93, 95% confidence interval =1.13–3.31). CD24-high expression had a tendency to correlate with new pathologic stage (p-stage) (p = 0.089) rather than old p-stage (p = 0.253). Performance status and new p-stage, regardless of the tumor histology, were identified as consistent independent prognostic factors of disease progression and cancer-related death. However, age was related to a significantly shorter cancer-specific survival in ADC only. Conclusions: CD24 expression in NSCLC is associated with ADC histology and disease progression and cancer-related death, indicative of aggressive tumor behavior. Performance status and new p-stage, to a lesser extent, age correlated with progression-free survival and cancer-specific survival, regardless of tumor histology.

Genome-wide combination profiling of DNA copy number and methylation for deciphering biomarkers in non-small cell lung cancer patients

Early detection of lung cancer provides the highest potential for saving lives. To date, no routine screening method enabling early detection is available, which is a key factor in the diseases high mortality rate. Copy number changes and DNA methylation alterations are good indicators of carcinogenesis and cancer prognosis. In this study, we attempted to combine profiles of DNA copy number and methylation patterns in 20 paired cancerous and noncancerous tissue samples from non-small cell lung cancer (NSCLC) patients, and we detected several clinically important genes with genetic and epigenetic relationships. Using array comparative genomic hybridization (aCGH), statistically significant differences were observed across the histological subtypes for gains at 1p31.1, 3q26.1, and 3q26.31-3q29 as well as for losses at 1p21.1, 2q33.3, 2q37.3, 3p12.3, 4q35.2, and 13q34 in squamous cell carcinoma (SQ) patients, and losses at 12q24.33 were measured in adenocarcinoma (AD) patients (p < 0.05). In an analysis of DNA methylation at 1505 autosomal CpG loci that are associated with 807 cancer-related genes, we identified six and nine loci with higher and lower DNA methylation levels, respectively, in tumor tissue compared to non-tumor lung tissues from AD patients. In addition, three loci with higher and seven loci with lower DNA methylation levels were identified in tumor tissue from SQ patients compared to non-tumor lung tissue. Subsequently, we searched for regions exhibiting concomitant hypermethylation and genomic loss in both ADs and SQs. One clone representing 7p15.2 (which includes candidate genes such as HOXA9 and HOXA11) and one target ID representing HOXA9_E252_R were detected. Quantitative real-time PCR identified the potential candidate gene HOXA9 as being down-regulated in the majority of NSCLC patients. Moreover, following HOXA9 over-expression, the invasion of representative cell lines, A549 and HCC95, were significantly inhibited. Taken together, our results show that the combined profiling analysis technique is a useful tool for identifying biomarkers in lung cancer and that HOXA9 might be a potential candidate gene for the pathogenesis and diagnosis of NSCLC patients.

Aberrant Wnt1/β-catenin expression is an independent poor prognostic marker of non-small cell lung cancer after surgery.

Introduction: The Wnt signaling pathway plays a major role in cancer development and progression. As a novel anticancer drug can be developed using inhibitors of this pathway, we investigated the clinical significance of the Wnt signaling pathway molecules in non-small cell lung cancer (NSCLC). Methods: Immunohistochemical analysis of a tissue microarray with 262 resected NSCLC specimens was performed to study the expression and subcellular localization of Wnt1 in relation to downstream molecules, including GSK-3&bgr;, &bgr;-catenin, c-Myc, cyclin D1, and p53. These results were correlated with other clinicopathologic features. Results: Cytoplasmic Wnt1 overexpression was detected in 36.6% (96 of 262) NSCLCs, and aberrant &bgr;-catenin staining was identified in 76% (189 of 262) of NSCLCs. There were significant associations between Wnt1 expression and altered expression of &bgr;-catenin (p = 0.034), overexpression of c-Myc (p < 0.001), or overexpression of cyclin D1 (p = 0.018). While there was no significant association between Wnt1 or &bgr;-catenin and stage, the 5-year survival was significantly lower in patients with Wnt1- and &bgr;-catenin-positive NSCLCs than negative NSCLCs (p < 0.05, respectively). In multivariate analysis, stage and Wnt1+/&bgr;-catenin+ expression were independent prognostic factors of overall survival (p < 0.05). Conclusion: These findings show that Wnt1 expression may be one of the possible mechanisms of the activation of the canonical Wnt/&bgr;-catenin signaling pathway in NSCLC, and Wnt1 and altered &bgr;-catenin expression are poor prognostic markers, independent of stage.