Analysis of MicroRNA in Bile Cytologic Samples Is Useful for Detection and Diagnosis of Extrahepatic Cholangiocarcinoma.

OBJECTIVES: This study aimed to develop reliable biomarkers that improve the ability of bile cytology to diagnose cholangiocarcinoma vs benign biliary lesions. METHODS: Many studies indicate that microRNAs (miRNAs) are potential candidates for the early diagnosis of cancer. We analyzed the expression of five tumor-associated miRNAs (miR-31-5p, miR-122-5p, miR-378d, miR-182-5p, and miR-92a-3p) in cytology samples using quantitative reverse transcription polymerase chain reaction. We collected 52 surgically resected tissue samples, 84 cytologic specimens from smears (53 cases of cancer and 31 cases of noncancer), and 40 residual sediments after smearing for routine cytology at Hiroshima University Hospital. RESULTS: The expression of miR-31-5p, miR-378d, and miR-122-5p was significantly higher in cancer tissues than those in normal tissues, while miR-182-5p expression was lower. The expression of miR-31-5p, miR-378d, miR-182-5p, and miR-92a-3p was significantly higher in detached cell samples from smears of cholangiocarcinoma cases than in those from noncancer cases. CONCLUSIONS: These results suggest that the analysis of miRNAs in bile cytologic specimens is a promising auxiliary tool for distinguishing cholangiocarcinoma from benign biliary lesions.

KRAS fluorescence in situ hybridisation testing for the detection and diagnosis of pancreatic adenocarcinoma.

AIMS: The aim of our study was to analyse correlations between KRAS mutation status, chromosomal changes that affect KRAS status in cells from pancreatic tumours. METHODS: We collected 69 cases of surgically resected pancreatic ductal adenocarcinoma (PDA) and seven cases of chronic pancreatitis (CP). Chromosomal abnormalities of KRAS and CEP12 were detected using fluorescence in situ hybridisation (FISH). RESULTS: The number of CEP12 signals per cell ranged from 1.78 to 2.04 and 1.46 to 4.88 in CP and PDA samples, respectively, while the number of KRAS signals per cell ranged from 1.94 to 2.06 and 1.88 to 8.18 in CP and PDA samples, respectively. The 'chromosomal instability index', which was defined as the percentage of cells with any chromosomal abnormality, was over 5.7 times greater in PDA than in CP. We performed KRAS mutation analysis by direct sequencing and found that tumours with KRAS mutations have a significantly higher mean KRAS signal per cell from PDA samples compared with tumours with wild-type KRAS. KRAS amplification was noted in 10% of cases. Although we found that lymph node metastasis and distal metastasis of PDA were more frequent in cases with KRAS amplification, this was not correlated with overall survival. Using a threshold of 40%, we found that the chromosomal instability index robustly discriminated PDA cells from CP cells. CONCLUSIONS: Based on these findings, we concluded that FISH testing of KRAS using cytology samples may represent an accurate approach for the diagnosis of PDA.

Utility of cytopathological specimens and an automated image analysis for the evaluation of HER2 status and intratumor heterogeneity in breast carcinoma.

OBJECTIVES: Although updated HER2 testing guidelines have been improved by a collaboration between the American Society of Clinical Oncology (ASCO) and the College of American Pathologists (CAP) in 2013, HER2 evaluation is still problematic because of issues involving CEP17 polysomy, heterogeneity, and HER2 score 2+ cases. The aim of this retrospective study was to evaluate the relationship between HER2 gene heterogeneity, or so called CEP17 polysomy, using breast carcinoma cells sampled by scraping and the IHC score graded by automated image analysis using whole slide image. MATERIAL AND METHODS: We randomly selected 23 breast carcinoma cases with a HER2 score 0, 24 cases with a HER2 score 1+, 24 cases with HER2 score 2+, and 23 cases with HER2 score 3+ from the records of patients with breast cancer at Hiroshima University Hospital. We compared the results of fluorescent in situ hybridization (FISH) using formalin-fixed, paraffin-embedded (FFPE) tissues and cytological samples and compared the HER2 score calculated using an automated image analysis using wholly scanned slide images and visual counting. RESULTS: We successfully performed the FISH assay in 78 of 94 cases (83%) using FFPE tissues and in all 94 (100%) cases using cytological samples. Frequency of both HER2 amplification and CEP17 polysomy was higher when cytological samples were used than when FFPE tissue was used. Frequency of HER2 heterogeneity using cytological samples was higher that than using FFPE tissue, except for the IHC score 3+ cases. CONCLUSIONS: When assessment of HER2 status based on FISH using FFPE tissue cannot be accomplished, FISH using cytological samples should be considered. When intensity of HER2 is heterogeneous in the tumor tissue, particularly in cases regarded as score 2+, they should be evaluated by automated image analysis using the whole slide image.

Comparison of visual assessment and image analysis in the evaluation of Ki-67 expression and their prognostic significance in immunohistochemically defined luminal breast carcinoma.

OBJECTIVES: To compare the Ki-67 labeling index value obtained through immunohistochemistry analysis by human examiners to that obtained from computer-assisted image analysis, and to establish a cut-off value for Ki-67 labeling index for each method in luminal B breast carcinoma. METHODS: Immunohistochemistry analysis for Ki-67 was performed on the formalin-fixed, paraffin-embedded tissue samples from 403 patients with primary luminal breast cancers. Whole slide images were obtained using the NanoZoomer (Hamamatsu Photonics, Hamamatsu, Japan) and thoroughly analyzed using the Definiens Tissue Studio version 1.1 (Definiens AG, Munich, Germany) to detect the percentage of positively-stained nuclei of carcinoma cells. RESULTS: Although a significant correlation was found between the Ki-67 labeling index obtained by manual assessment and computer-assisted image analysis (Spearman rank correlation coefficient, P < 0.01), the Ki-67 labeling index value obtained by manual assessment was significantly higher than that obtained by computer-assisted image analysis (Wilcoxon signed rank test, P < 0.0001). Disease-free survival was significantly lower in 403 patients with tumors having high Ki-67 labeling index values determined by automated analysis (cut-off value: 11.5%; P < 0.00001) and visual counting (cut-off value: 28.5%; P < 0.00001). Disease-free survival was also significantly lower in 288 patients who received adjuvant endocrine therapy alone having high Ki-67 labeling index values determined by automated analysis (cut-off value: 11.5%; P < 0.0001) and visual counting (cut-off value: 19.7%, P < 0. 0001). CONCLUSIONS: The Ki-67 labeling index values determined by automated analysis and visual counting could equally predict disease-free survival in patients with luminal B breast carcinoma, including those who received endocrine therapy.

Clinical significance of sIL-2R levels in B-cell lymphomas.

Elevated soluble interleukin-2 receptor (sIL-2R) in sera is observed in patients with malignant lymphoma (ML). Therefore, sIL-2R is commonly used as a diagnostic and prognostic marker for ML, but the mechanisms responsible for the increase in sIL-2R levels in patients with B-cell lymphomas have not yet been elucidated. We first hypothesized that lymphoma cells expressing IL-2R and some proteinases such as matrix metalloproteinases (MMPs) in the tumor microenvironment can give rise to increased sIL-2R in sera. However, flow cytometric studies revealed that few lymphoma cells expressed IL-2R α chain (CD25) in diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL), and most CD25-expressing cells in the tumor were T-cells. Distinct correlations between CD25 expression on B-lymphoma cells and sIL-2R levels were not observed. We then confirmed that MMP-9 plays an important role in producing sIL-2R in functional studies. Immunohistochemical (IHC) analysis also revealed that MMP-9 is mainly derived from tumor-associated macrophages (TAMs). We therefore evaluated the number of CD68 and CD163 positive macrophages in the tumor microenvironment using IHC analysis. A positive correlation between the levels of sIL-2R in sera and the numbers of CD68 positive macrophages in the tumor microenvironment was confirmed in FL and extranodal DLBCL. These results may be useful in understanding the pathophysiology of B-cell lymphomas.

Discordant HER2 status between primary breast carcinoma and recurrent/metastatic tumors using fluorescence in situ hybridization on cytological samples.

OBJECTIVE: The aim of this study was to show the usefulness of examining HER2 status on fluorescence in situ hybridization using cytological samples taken from recurrent/metastatic tumors. METHODS: One hundred freshly aspirated or scraped cytological samples were obtained from locoregional recurrences or distant metastases. Fluorescence in situ hybridization assay for HER2 amplification was performed on both these samples and the formalin-fixed, paraffin-embedded tissues of the paired primary tumors of breast cancer, and the relationships between various clinico-pathological factors and HER2 amplification of both tumors were examined. RESULTS: A change in HER2 status was observed in nine cases (9%): six cases (6%) underwent a positive-to-negative conversion in HER2 status and three cases (3%) underwent a negative-to-positive conversion in HER2 status. A positive-to-negative conversion of HER2 status was noted in 4 (36%) of 11 'luminal-B' cases. The change in HER2 status in recurrent or metastatic tumor was noted in more cases treated with drug therapy than in those with no drug therapy (P < 0.05; Fisher's exact probability). Although the time to relapse was 3 years or more in three cases showing a negative-to-positive conversion in HER2 status, the time to relapse was less than 3 years in six cases showing a positive-to-negative conversion (P < 0.05; Fisher's exact probability). CONCLUSIONS: HER2 examination on fluorescence in situ hybridization using fine-needle aspiration cytology samples of tumors in recurrent/metastatic sites or disseminated tumor cells in effusion is beneficial, particularly when the primary tumor is not suitable for the testing of HER2 status or negative for HER2 amplification, because aspiration using a needle is technically feasible and not as traumatic as biopsy.

A case of HER-2-positive recurrent breast cancer showing a clinically complete response to trastuzumab-containing chemotherapy after primary treatment of triple-negative breast cancer.

We report a case of HER-2-positive recurrent breast cancer showing a clinically complete response to trastuzumab-containing chemotherapy 6 years after primary treatment of triple-negative breast cancer. The primary tumor was negative for HER-2 as determined by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) (1+, and ratio, 1.1), but examination of the recurrent lymph node metastasis showed positivity for HER-2 by FISH (ratio, 5.2). No lesions were detected in either her left breast or in other organs, and the patient was diagnosed as having HER-2-positive recurrent disease. Combination chemotherapy using weekly paclitaxel and trastuzumab was initiated, and a clinically complete response was achieved. This report suggests the benefit of routine evaluation of HER-2 status in recurrent breast cancer with the introduction of HER-2-targeting agents.

Establishment of an HS23 stromal cell-dependent myeloma cell line: fibronectin and IL-6 are critical.

A multiple myeloma (MM) cell line, MSG1, which depends on HS23 stromal cells for its survival, was established from the pleural effusion of a patient with MM who expressed the M-protein of IgA-λ in his serum. During the first 2 months of culture, the myeloma cells survived on adhesive cells from the pleural effusion and, subsequently, they continued to proliferate on HS23 stromal cells. The phenotype of the established MSG1 cell line was: CD138(+), CD38(++), CD19⁻, CD56⁻, VLA-4(+), VEGFR1(+) and VEGFR2(+). Immunohistochemical staining also demonstrated expression of the IgA and λ chain in MSG1 cytoplasm. Karyotype analysis indicated complex chromosomal abnormalities; hypertriploidy, including the deletion of chromosomes 13 and 17, and c-myc translocation. MSG1 cells continued to proliferate, not only when co-cultured with HS23 cells, but also when cultured only on fibronectin-coated plates with the supernatant of HS23 cells or with control medium containing IL-6. Tocilizumab, an anti-IL-6 receptor antibody, inhibited MSG1 survival under these conditions. Therefore, MSG1 may be a unique myeloma cell line that is useful for the study of cell adhesion-mediated drug resistance induced by adhesion molecules and IL-6 stimulation of myeloma cells.

Comparison of immunohistochemistry assays and real-time reverse transcription-polymerase chain reaction for analyzing hormone receptor status in human breast carcinoma.

The estrogen receptor (ER) and progesterone receptor (PgR) status of 163 surgical breast cancer specimens determined on real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) using frozen tumor tissue were compared with that determined using three automated immunohistochemistry (IHC) assays including Dako (Glostrup, Denmark), Ventana (Tucson, AZ, USA) and BioGenex (San Ramon, CA, USA) assay. All specimens were semiquantified according to the Allred score and J-score. The cut-offs for ER determined by log (ER/glyceraldehyde-3-phosphate dehydrogenase (GAPDH)) were -3.6 and -3.2 based on the Allred score and J-score, respectively, and those for PgR determined by log (PgR/GAPDH) were -3.2 and -2.8, respectively. The Allred total score (TS) and the J-score for ER and PgR on IHC were significantly correlated with the result on RT-PCR (P < 0.00001). There was a high degree of concordance among ER and PgR status on IHC and those on RT-PCR, suggesting that RT-PCR is a useful method for evaluation of ER and PgR status. Some discrepancies between the IHC and RT-PCR results were identified, however. Accordingly, further studies of RT-PCR assays for hormone receptor (HR) are necessary with regard to biological behavior and responsiveness to hormone therapy.

Risk factors for severity of pneumothorax after CT-guided percutaneous lung biopsy using the single-needle method.

The purpose of this study is to evaluate the risk factors for the severity of pneumothorax after computed tomography (CT)-guided percutaneous lung biopsy using the single-needle method. We reviewed 91 biopsy procedures for 90 intrapulmonary lesions in 89 patients. Patient factors were age, sex, history of ipsilateral lung surgery and grade of emphysema. Lesion factors were size, location and pleural contact. Procedure factors were position, needle type, needle size, number of pleural punctures, pleural angle, length of needle passes in the aerated lung and number of harvesting samples. The severity of pneumothorax after biopsy was classified into 4 groups: "none", "mild", "moderate" and "severe". The risk factors for the severity of pneumothorax were determined by multivariate analyzing of the factors derived from univariate analysis. Pneumothorax occurred in 39 (43%) of the 91 procedures. Mild, moderate, and severe pneumothorax occurred in 24 (26%), 8 (9%) and 7 (8%) of all procedures, respectively. Multivariate analysis showed that location, pleural contact, number of pleural punctures and number of harvesting samples were significantly associated with the severity of pneumothorax (p < 0.05). In conclusion, lower locations and non-pleural contact lesions, increased number of pleural punctures and increased number of harvesting samples presented a higher severity of pneumothorax.

Comparison of evaluations of hormone receptors in breast carcinoma by image-analysis using three automated immunohistochemical stainings.

The aim of this study was to compare the results of immunohistochemistry (IHC) assays evaluated by human examiners with the results evaluated by computerized image analysis, and to compare the computerized image analysis results among three automated IHC assays, namely the BioGenex, Dako and Ventana assays. All slides were semiquantitatively evaluated according to the Allred score and J-score by human examiners. The images were analyzed using MacSCOPE version 2.6 for Macintosh according to the H-score and the percentage of positive-stained nuclei per area of carcinoma cells (PP) irrespective of the intensity of the stained nuclei. The H-score for the estrogen receptor (ER) was significantly correlated with the Allred score (P<0.0001) and the PP for the ER was significantly correlated with the J-score (P<0.0001), suggesting that the image analysis used in the present study is a useful method for the evaluation of ER status. Several discrepancies were identified between the Allred score and H-score and between the PP and J-score due to the positive-stained cytoplasm area of carcinoma cells and/ or the positive-stained nuclei area of non-carcinoma cells, including benign epithelial cells, lymphocytes and stromal cells. Accordingly, advances in the algorithm of the digitized analyzing system is necessary.

Usefulness of human telomerase reverse transcriptase in pancreatic juice as a biomarker of pancreatic malignancy.

OBJECTIVES: Human telomerase reverse transcriptase (hTERT), one of the subunits of telomerase, is a promising diagnostic marker for pancreatic cancer. In the present study, we did a large-scale analysis of 115 preoperative pancreatic juice specimens to evaluate the feasibility of detection of hTERT expression by immunohistochemistry for preoperative diagnosis of pancreatic malignancy. METHODS: The expression of hTERT was examined by immunohistochemistry in preoperative pancreatic juice samples. RESULTS: In pancreatic juice samples, hTERT expression was detectable in 84% of pancreatic ductal adenocarcinomas (PDACs), whereas 62% of PDACs were positive by cytology. In intraductal papillary mucinous neoplasms (IPMNs), hTERT expression was detectable in 88% of malignant IPMNs, whereas only 22% were positive by cytology. The sensitivity, specificity, and overall accuracy of hTERT expression for differentiation between carcinoma and other benign diseases were 85.1%, 82.1%, and 84.3%, respectively, whereas the same values for cytologic accuracy were 47.1%, 89.3%, and 57.4%, respectively. When the results of cytology and hTERT expression were combined, the sensitivity and overall accuracy increased to 92.0% and 87.8%, respectively. CONCLUSIONS: Our results suggested that the assessment of hTERT expression in preoperative pancreatic juice increased the sensitivity and accuracy of diagnosis of PDACs and malignant IPMNs without using special techniques.