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. Cancer (Cancer Cytopathol) 2015;123:620-8. V C 2015 American Cancer Society.

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. Ó2016 AACR.

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% neutralbuffered 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.

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.

A combined test using both cell sediment DNA and supernatant ccfDNA samples increases the concordance rate of EGFR mutations between primary tumour and corresponding metastases. Our findings indicate that supernatant ccfDNA is useful even in cases where the cytological diagnosis is negative.

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 × 10 /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.

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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. Cancer Cytopathol 2019;127:598-605. © 2019 American Cancer Society.KEY WORDS: bronchial and pleural cytology; cell block; insulinoma-associated protein 1 (INSM1); lung cancer; small cell lung carcinoma.