Ji-Young Choe

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.

Indoleamine 2,3-dioxygenase (IDO) is frequently expressed in stromal cells of Hodgkin lymphoma and is associated with adverse clinical features: a retrospective cohort study

BackgroundRegulation of tumor microenvironment is closely involved in the prognosis of Hodgkin lymphoma (HL). Indoleamine 2,3-dioxygenase (IDO) is an enzyme acting as immune modulator through suppression of T-cell immunity. This study aims to investigate role of IDO in the microenvironment of HL.MethodsA total of 121 cases of HL were enrolled to do immunohistochemistry for IDO, CD163, CD68, CD4, CD8, and FoxP3. Positivity was evaluated from area fractions or numbers of positive cells using automated image analyzer. Correlations between IDO expression and various cellular infiltrates and clinicopathologic parameters were examined and survival analyses were performed.ResultsIDO was expressed in histiocytes, dendritic cells and some endothelial cells with variable degrees, but not in tumor cells. IDO positive cells were more frequently found in mixed cellularity type than other histologic types, and in cases with EBV+, high Ann Arbor stages, B symptoms, and high IPS (all p < 0.05). High IDO expression was associated with inferior survival (p < 0.001) and reflects an independent prognostic factor in nodular sclerosis HL.ConclusionsThis is the first study suggesting that IDO is the principle immunomodulator and is involved to adverse clinical outcomes of HL.

Inflammatory pseudotumor‐like follicular dendritic cell sarcoma of the spleen: A report of six cases with increased IgG4‐positive plasma cells

Inflammatory pseudotumor (IPT)‐like follicular dendritic cell (FDC) sarcoma is a rare neoplasm typically occurring in the spleen or liver. We present six cases of EBV+ IPT‐like FDC sarcoma of the spleen among Koreans along with their clinicopathologic features and IHC results. Most patients presented with an asymptomatic, incidentally detected single splenic mass and were successfully managed by splenectomy alone. Concomitant disease was found in one case, showing EBV+ gastric carcinoma with lymphoid‐rich stroma. Histologic features showed fibro‐inflammatory lesions that were often accompanied by necrosis and epithelioid histiocytic collection, which are barely distinguishable from IPT. Tumor cells did not frequently express conventional FDC markers, including CD21 (3/6 positive cases), clusterin (4/6), and D2‐40 (2/6), but showed uniform positivity for smooth muscle actin (SMA). Noticeably, significant numbers of IgG4+ plasma cells were found within all six tumors. We suggest that the diagnosis of IPT‐like FDC sarcoma should be made by the application of a panel of FDC markers, and CD21 negativity or SMA positivity cannot be the criterion for exclusion of IPT‐like FDC sarcoma. Relationship of IPT‐like FDC sarcoma of the spleen and IgG4‐related sclerosing disease should be investigated in further studies.

Expression of c-Met Is Different along the Location and Associated with Lymph Node Metastasis of Head and Neck Carcinoma

Background Activation of the c-Met pathway is involved in cancer progression and the prognosis. We aimed to identify any association of c-Met protein expression with a number of clinicopathologic variables including infection of human papillomavirus and Epstein-Barr virus (EBV) in head and neck carcinomas (HNCa). Methods Eighty-two cases were enrolled in this study. Expression of c-Met and p16 was investigated immunohistochemically. EBV was detected by in situ hybridization and amplification of the c-Met gene by fluorescence in situ hybridization. Results The c-Met protein was expressed in 41.5% (34/82), and gene amplification was found in 1.4% (1/71). High expression of c-Met was associated with the primary location of the tumor; the hypopharynx showed the highest expression, followed by the oral cavity, larynx, and nasal cavity. Squamous cell carcinoma expressed c-Met more frequently than undifferentiated carcinoma. Also, p16 immunoreactivity or EBV infection was associated with the tumor location and well-differentiated histologic type, but were not linked to c-Met expression. The patients with positive c-Met expression showed frequent lymph node metastasis. Conclusions Activation of the c-Met pathway might be involved in a subset of HNCa. Cases showing positive c-Met expression should be carefully monitored because of the high probability of lymph node metastasis.

MYC overexpression correlates with MYC amplification or translocation, and is associated with poor prognosis in mantle cell lymphoma.

We aimed to investigate MYC expression and chromosomal aberration in mantle cell lymphoma (MCL), and the clinical significance of these factors.

Sonic hedgehog signalling proteins are frequently expressed in retinoblastoma and are associated with aggressive clinicopathological features

Aims This study aimed to examine the expression of Sonic hedgehog (SHH) signalling proteins in retinoblastoma and to evaluate its clinical significance. Methods Seventy-nine enucleated retinoblastoma tumours were investigated immunohistochemically using antibodies against SHH pathway proteins, such as SHH, glioma-associated oncogene homologue (GLI) 1, GLI2, GLI3 and ABC binding cassette G2 (ABCG2). Western blotting of SHH signalling proteins was performed in two retinoblastoma cell lines. Results SHH was expressed in most retinoblastoma cases (78 of 79, 98.7%), with 21 cases (26.6%) showing strong expression. GLI1 and GLI2 were also frequently expressed: 67 of 78 cases (85.9%) and 71 of 77 cases (92.2%), respectively. GLI3, a transcriptional repressor, was expressed at low levels in 23 of the 78 cases (29.5%). High ABCG2 expression was found in 23 of the 78 cases (29.5%). High expression levels of these proteins in retinoblastoma cell lines were confirmed by western blotting. The expression of SHH was associated with advanced stages, local invasion and metastasis (all p<0.05). Conclusions SHH signalling molecules were frequently expressed in retinoblastoma tumour cells, and high SHH expression was closely related to an advanced disease status. Our results suggest that the SHH signalling pathway may play a role in the progression of retinoblastoma.

Distinctive role of SIRT1 expression on tumor invasion and metastasis in breast cancer by molecular subtype

The aim of this study was to evaluate silent mating type information regulation 2 homolog 1 (SIRT1) expression levels by subtype and evaluate its predictive power of axillary lymph node metastasis (LNM) and its association with clinical outcome. A total of 427 patients diagnosed with invasive ductal carcinoma were chosen, immunohistochemical staining for SIRT1 expression was performed on tissue microarrays, and in vitro experiments with each intrinsic subtype of human breast cancer cell line were carried out. Increased expression of SIRT1 in hormone receptor-positive breast cancer and HER2 breast cancer subtype significantly correlated with lower risks of LNM. On the contrary, in triple-negative breast cancer, increased SIRT1 expression was more frequently observed in LNM-positive subgroup than LNM-negative subgroup. Combination of statistically significant, independent parameters including SIRT1 revealed predictive performance for LNM with area under the curve of 0.602, 0.587, and 0.726 for hormone receptor-positive breast cancer, HER2 breast cancer, and triple-negative breast cancer subtype, respectively. Inhibition of SIRT1 expression with small interfering RNA suppressed tumor invasion in MDA-MB-231, specifically. This is the first study to examine SIRT1 expression in breast cancer by subtype, and we have observed the potentially different role of SIRT1 gene having tumor-suppressive or tumor-promoting influence depending on the subtype; thus, different associations between SIRT1 expression and prognosis by subtype should be considered in its target therapy.

Clinicopathological categorization of Epstein–Barr virus-positive T/NK-cell lymphoproliferative disease: an analysis of 42 cases with an emphasis on prognostic implications

Abstract Epstein–Barr virus-positive T/NK-cell lymphoproliferative diseases (EBV-T/NK-LPDs) include several overlapping EBV-related conditions with variably aggressive courses. For prognostic categorization, we retrospectively analyzed 42 EBV-T/NK-LPD cases. Male (79% [33/42]), young (≤40 years; 83% [35/42]) patients and T-cell lineage (81% [34/42]; CD8/CD4 = 1.8) were predominant. Clinicopathologically, three systemic and one cutaneous category were developed: hemophagocytic lymphohistiocytosis (HLH; 26% [11/42]), chronic active EBV infection (CAEBV; 31% [13/42]), systemic unclassifiable disease (24% [10/42]), and hydroa vacciniforme/hydroa vacciniforme-like lymphoma (HV/HVL; 19% [8/42]). Prognostically, cutaneous disease (HV/HVL) was better than systemic disease (p = 0.014; median, 285 vs. 10 months). In systemic diseases, HLH was worst (p = 0.002; 3[HLH] vs. 4[unclassifiable] vs. not reached [CAEBV]). Univariate survival analysis (n = 42) revealed cytopenia (≥one lineage; p < 0.001), onset age (>40 years; p = 0.001), T-cell lineage (p = 0.041), hemophagocytic histiocytes (p = 0.031), elevated lactate dehydrogenase (p = 0.020), and liver dysfunction (p = 0.023) predicted shorter survival. In multivariate analysis, T-cell lineage (p = 0.025 [HR =11.3]) and cytopenia (p = 0.028 [HR =5.4]) were independent prognostic factors. Therefore, EBV-T/NK-LPD could be classified into four prognostic categories.