CXCR4 Expression and Cancer-associated Fibroblasts May Play an Important Role in the Invasion of Low-grade Endometrioid Carcinoma.

Well-differentiated endometrioid carcinoma (EC) is a low-grade cancer with relatively indolent behavior. However, even with well-differentiated histology, it sometimes tends to invade extensively and shows metastatic potential, suggesting that this is a group of cancers with heterogeneous behavior. In contrast, due to its tendency for younger onset, the treatment strategy for EC frequently considers fertility preservation, highlighting the need for a more accurate evaluation of myometrial invasion through biopsy and imaging diagnostics. We previously reported the involvement of the CXCR4-CXCL12 and CXCL14 axes in EC invasion. Accordingly, we investigated whether CXCR4 expression could reflect invasive potential and explored its interaction with cancer-associated fibroblasts that produce chemokines in the tumor microenvironment. Immunohistochemical expression of CXCR4 was assessed in 71 cases of EC (14 of EC confined to the endometrium and 57 of myoinvasive EC), 6 cases of endometrial intraepithelial neoplasia, and 42 cases of noncarcinomatous conditions. CXCR4 expression was significantly higher in myoinvasive EC than in noncancerous conditions, endometrial intraepithelial neoplasia, and endometrium-confined EC. By univariate and multivariate analysis, CXCR4 expression significantly reflected myometrial invasion. CXCR4 expression in the biopsied and resected specimens correlated weakly positively. Invasion and wound-healing assays were performed culturing an EC cell line in a cancer-associated fibroblast-conditioned medium. The invasion and wound-healing potentials were dependent on CXCR4 and cancer-associated fibroblast. Our study demonstrated that CXCR4 expression is an independent factor in myometrial invasion and can support diagnostic evaluation before treatment in the biopsy sample.

Insulinoma-associated protein 1 is a novel diagnostic marker of small cell lung cancer in bronchial brushing and cell block cytology from pleural effusions: Validity and reliability with cutoff value.

BACKGROUND: In recent years, insulinoma-associated protein 1 (INSM1) has been shown to be a key regulator of neuroendocrine development, and it has been evaluated for diagnostic use in some organs. METHODS: To evaluate the relationship between INSM1 and synaptophysin, and to confirm the cutoff value using receiver operating characteristic curve (ROC) analysis, the authors performed INSM1 immunocytochemistry using cell block (CB) samples from 53 cases of bronchial brushings and 32 cases of pleural effusions (29 small cell lung cancer [SCLC] specimens and 56 non-small cell lung cancer specimens). The marker expression ratio was calculated by counting the positive tumor cells, and the tumor proportion score (TPS) was applied. RESULTS: INSM1 was expressed in all SCLC specimens, but generally was expressed in <10% of tumor cells in adenocarcinomas. In bronchial brushing samples of SCLC, the INSM1 TPS was 37.5% (staining of >50%), 25.0% (staining of 25%-50%), 29.5% (staining of 10%-25%), and 8.3% (staining of 1%-10%). There were 3 cases (12.5%) with no detectable synaptophysin expression, although the correlation between nuclear INSM1 expression and cytoplasmic synaptophysin expression was statistically significant (P < .001). Receiver operating characteristic curve analysis indicated that 8.68% was the best cutoff value for INSM1, and the sensitivity and specificity between SCLC and non-small cell lung cancer for expression of INSM1 were 95.8% and 100.0%, respectively, in bronchial brushing samples at that cutoff value. CONCLUSIONS: INSM1 is a novel diagnostic marker for SCLC, and is useful in both bronchial brushing and pleural effusion cytology specimens. Because INSM1 generally is expressed in <10% of tumor cells in adenocarcinomas, determining an accurate cutoff value for INSM1 is important in the diagnosis of SCLC.

Granulocyte colony-stimulating factor-producing pancreatic anaplastic carcinoma in ascitic fluid at initial diagnosis: A case report.

Granulocyte colony-stimulating factor (G-CSF)-producing pancreatic tumors are extremely rare. These tumors have an aggressive clinical course and no established treatment. Here, we report an autopsy case of G-CSF-production in pancreatic anaplastic carcinoma (PAC). A 72-year-old woman presented with a large pancreatic head mass and multiple liver metastases. Laboratory data showed leukocytosis (leukocyte count 113.3 × 103 /µL) and high serum G-CSF levels (441 pg/mL; normal range: <39.0 pg/mL). The ascitic fluid was submitted to our pathology laboratory at initial diagnosis. Cytopathology showed that smears from the ascitic fluid were highly cellular and contained numerous malignant cells, mainly in loose groupings. Occasional pseudoglandular formations and giant cells were also present. The malignant cells were round, and no spindle-shaped cells were visible. The nuclei were round to ovoid with coarsely granular chromatin and large prominent nucleoli. Upon immunocytochemistry, tumor cells were positive for G-CSF and vimentin; there was no E-cadherin expression. Histopathological examination of the tumor showed a mixed composition of adenocarcinomatous and sarcomatous regions. Upon immunohistochemistry, both components were positive for G-CSF. Few CD34-positive myeloblasts were observed in the bone marrow. Thus, we diagnosed this as a case of G-CSF production in PAC with leukocytosis. To the best of our knowledge, this is the first report on G-CSF expression immunocytochemically confirmed in PAC. Diagn. Cytopathol. 2017;45:463-467. © 2017 Wiley Periodicals, Inc.

Breast Cancer Resistance to Antiestrogens Is Enhanced by Increased ER Degradation and ERBB2 Expression.

Endocrine therapies effectively improve the outcomes of patients with estrogen receptor (ER)-positive breast cancer. However, the emergence of drug-resistant tumors creates a core clinical challenge. In breast cancer cells rendered resistant to the antiestrogen fulvestrant, we defined causative mechanistic roles for the transcription factor YBX1 and the levels of ER and the ERBB2 receptor. Enforced expression of YBX1 in parental cells conferred resistance against tamoxifen and fulvestrant in vitro and in vivo Furthermore, YBX1 overexpression was associated with decreased and increased levels of ER and ERBB2 expression, respectively. In antiestrogen-resistant cells, increased YBX1 phosphorylation was associated with a 4-fold higher degradation rate of ER. Notably, YBX1 bound the ER, leading to its accelerated proteasomal degradation, and induced the transcriptional activation of ERBB2. In parallel fashion, tamoxifen treatment also augmented YBX1 binding to the ERBB2 promoter to induce increased ERBB2 expression. Together, these findings define a mechanism of drug resistance through which YBX1 contributes to antiestrogen bypass in breast cancer cells. Cancer Res; 77(2); 545-56. ©2016 AACR.

Epidermal growth factor receptor mutation status in cell-free DNA supernatant of bronchial washings and brushings.

BACKGROUND: The aim of the current study was to examine whether it would be possible to detect epidermal growth factor receptor (EGFR) mutations in cytology cell-free DNA (ccfDNA) from the supernatant fluids of bronchial cytology samples. METHODS: This study investigated cell damage via immunostaining with a cleaved caspase 3 antibody and the quantity of cell-free DNA in supernatant fluid from 2 cancer cell lines, and the EGFR mutation status was evaluated via polymerase chain reaction (PCR) analysis. EGFR mutations were also evaluated via PCR analysis in 74 clinical samples of ccfDNA from bronchial washing samples with physiological saline or from bronchial brushing liquid-based cytology samples with CytoRich Red. RESULTS: The quantity and fragmentation of cell-free DNA in the supernatant fluid and the cell damage and cleaved caspase 3 expression in the sediment gradually increased in a time-dependent manner in the cell lines. In the 74 clinical samples, the quantity of ccfDNA extracted from the supernatant was adequate to perform the PCR assay, whereas the quality of ccfDNA in physiological saline was often decreased. The detection of EGFR mutations with ccfDNA showed a sensitivity of 88.0%, a specificity of 100%, a positive predictive value of 100%, a negative predictive value of 89.7%, and an accuracy of 94.1% in samples with malignant or atypical cells. CONCLUSIONS: These results suggest that activating EGFR mutations can be detected with ccfDNA extracted from the supernatant fluid of liquid-based samples via a PCR assay. This could be a rapid and sensitive method for achieving a parallel diagnosis by both morphology and DNA analysis in non-small cell lung cancer patients.

Heterogeneity of anaplastic lymphoma kinase gene rearrangement in non-small-cell lung carcinomas: a comparative study between small biopsy and excision samples.

INTRODUCTION: The standard diagnostic method for echinoderm microtubule-associated protein-like 4-anaplastic lymphoma receptor tyrosine kinase translocation is fluorescence in situ hybridization (FISH). Recently, immunohistochemistry (IHC) has been reported as a potential method in screening for anaplastic lymphoma kinase (ALK)-positive non-small-cell lung carcinomas (NSCLC), whereas several authors have reported a discordance between FISH and IHC results. We investigated the heterogeneity of ALK gene rearrangement in excision specimens by FISH and also examined whether the FISH score of ALK gene rearrangement corresponded in excision and biopsy samples from the same patient. METHODS: Twenty ALK IHC-positive patients including six patients treated with crizotinib therapy were evaluated for the presence of ALK FISH. For evaluation of heterogeneity of ALK gene rearrangement in excision specimens, we defined six to 10 observation areas in each case, and the number of ALK FISH positive observation areas (≥15% rearrangement detected) was investigated. ALK FISH score in small biopsy samples was classified as positive (≥15% rearrangement detected), equivocal (5-14% rearrangement detected), or negative (<4% rearrangement detected). RESULTS: Of a total of 64 tumor observation areas from nine excision specimens, 50 areas were positive for ALK gene rearrangement (81.8%). In the comparison of excision and small biopsy samples, all excision specimens were ALK FISH-positive (100%; 6 of 6), whereas only three of the small biopsy samples in these patients were positive (50%; 3 of 6), two were equivocal (33%; 2 of 6), and one was negative (17%; 1 of 6). The two equivocal patients received crizotinib and showed a response. CONCLUSION: ALK gene rearrangement heterogeneity was observed in NSCLC specimens by FISH. Our findings suggested that IHC-positive/FISH-equivocal cases should not be considered true "false-negatives" when a small biopsy sample was used for ALK analysis.

Evaluation of immunohistochemistry using two different antibodies and procedures for primary lung adenocarcinoma harboring anaplastic lymphoma kinase rearrangement.

Rearrangements of anaplastic lymphoma kinase (ALK) have been recently identified in non-small cell lung carcinomas. Previous studies have revealed characteristic features, including adenocarcinoma histology and mucin production, in ALK-positive lung carcinoma. The present study evaluated immunohistochemistry (IHC) in ALK-positive lung carcinoma using two different antibodies, clone 5A4 and D5F3, and compared the results. On the basis of the aforementioned characteristic features, out of 359 primary lung carcinomas, the ALK status of 14 adenocarcinomas was screened using the intercalated antibody-enhanced polymer (iAEP) method with antibody 5A4, and this was compared with the ALK status obtained using rabbit monoclonal antibody D5F3 and fluorescence in situ hybridization for ALK. Eight cases were demonstrated to be ALK-positive by IHC. Seven cases exhibited ALK rearrangement, which was demonstrated by fluorescence in situ hybridization. The IHC for ALK obtained using D5F3 was comparable with that of the iAEP and exhibited low heterogeneity. This finding suggests that IHC for ALK could be useful in limited tissue samples, such as biopsy specimens or cytology, for the screening of ALK-positive lung carcinoma. In the present study, it was demonstrated that IHC with ALK monoclonal antibody D5F3 was useful for screening lung adenocarcinoma harboring ALK rearrangement.

Fixation effect of SurePath preservative fluids using epidermal growth factor receptor mutation-specific antibodies for immunocytochemistry.

BACKGROUND: Cytological diagnosis of respiratory disease has become important, not only for histological typing using immunocytochemistry (ICC) but also for molecular DNA analysis of cytological material. The aim of this study was to investigate the fixation effect of SurePath preservative fluids. METHODS: Human lung cancer PC9 and 11-18 cell lines, and lung adenocarcinoma cells in pleural effusion, were fixed in CytoRich Blue, CytoRich Red, 15% neutral-buffered formalin, and 95% ethanol, respectively. PC9 and 11-18 cell lines were examined by ICC with epidermal growth factor receptor (EGFR) mutation-specific antibodies, the EGFR mutation DNA assay, and fluorescence in situ hybridization. The effect of antigenic storage time was investigated in lung adenocarcinoma cells in pleural effusion by ICC using the lung cancer detection markers. RESULTS: PC9 and 11-18 cell lines in formalin-based fixatives showed strong staining of EGFR mutation-specific antibodies and lung cancer detection markers by ICC as compared with ethanol-based fixatives. DNA preservation with CytoRich Blue and CytoRich Red was superior to that achieved with 95% ethanol and 15% neutral-buffered formalin fixatives, whereas EGFR mutations by DNA assay and EGFR gene amplification by fluorescence in situ hybridization were successfully identified in all fixative samples. Although cytoplasmic antigens maintained high expression levels, expression levels in nuclear antigens fell as storage time increased. CONCLUSIONS: These results indicate that CytoRich Red is not only suitable for ICC with EGFR mutation-specific antibodies, but also for DNA analysis of cytological material, and is useful in molecular testing of lung cancer, for which various types of analyses will be needed in future.