Optimal Volume Assessment for Serous Fluid Cytology.

OBJECTIVE: This study aimed to investigate the optimal volume of serous fluid needed for accurate diagnosis using The International System for Reporting Serous Fluid Cytopathology (TIS), as well as to provide information on the distribution of serous effusion cases in the TIS categories (ND: non-diagnostic, NFM: negative for malignancy, AUS: atypia of undetermined significance, SFM: suspicious for malignancy, MAL: malignant) and relevant epidemiological data. METHODS: A retrospective analysis of 2340 serous effusion cases (pleural, peritoneal, and pericardial) from two hospitals between 2018 and 2020 was conducted. TIS categories were assigned to each case, and for 1181 cases, these were correlated with the volume of the analyzed fluid. RESULTS: Our study found statistically significant differences in volume distributions between certain TIS categories. Statistically lower volumes were observed in NFM compared to MAL, in UNCERTAIN (ND, AUS, SFM) compared to both MAL and NFM, and in NOT MAL (ND, NFM, AUS, SFM) compared to MAL. However, these differences were not substantial enough to hold any clinical relevance. CONCLUSIONS: This study suggests that while fluid volume may slightly influence the TIS category, it does not impact the diagnostic accuracy of serous effusion cytology. Therefore, the ideal serous effusion specimen volume can be defined solely by practical parameters.

Excessive Leukocytosis Leading to a Diagnosis of Aggressive Thyroid Anaplastic Carcinoma: A Case Report and Relevant Review.

INTRODUCTION: Leukocytosis and particularly neutrophilia are usually caused by acute infection, inflammation, and myeloproliferative neoplasms. However, leukocytosis can also occur in patients with malignancy either due to bone marrow metastases or in the context of a paraneoplastic syndrome. CASE PRESENTATION: An 86-year-old female was admitted to our hospital due to marked leukocytosis (white blood cells [WBC] >40,000/µL), neutrophilia, and monocytosis. She was afebrile and reported hoarseness and mild difficulty swallowing. Upon physical examination, lung auscultation revealed inspiratory wheezing and a non-tender mass was observed in the anterior midline of the neck. Blasts and immature WBC were not found, and polymerase chain reaction for the detection of BCR/ABL gene was negative. A mass (5.4 cm in diameter) of abnormal parenchymal composition with calcifications occupying the right lobe, was seen on thyroid ultrasound. Cytology, after fine-needle aspiration, showed an anaplastic thyroid carcinoma (ATC). The cervical and chest computed tomography scan revealed a low-density lesion with calcifications that shifts and presses the trachea and multiple lung nodular lesions bilaterally. Since the case was inoperable and the airway was severely obstructed, a DUMON stent was placed. Biopsy of specimens from the trachea lesion revealed a tumor with significant atypical cells and focal squamoid features. The patient's WBC increased to 72,470/µL. Additionally, interleukin-6 (IL-6) was markedly elevated (20.2 pg/mL). The patient passed away due to respiratory arrest 55 days after her initial admission. DISCUSSION: Excessive leukocytosis in a patient, having excluded infectious disease and myelodysplastic syndrome, could represent a manifestation of a paraneoplastic syndrome due to various cytokines secretion from the tumor. In our case, ATC synthesized and secreted IL-6, which seems to be the cause of severe leukocytosis.

Expression of γ-tubulin in non-small cell lung cancer and effect on patient survival.

INTRODUCTION: It has been reported that overexpression and altered compartmentalization of γ-tubulin may contribute to tumorigenesis and tumor aggressiveness in a variety of human malignancies. We have shown that γ-tubulin expression and cellular distribution pattern is also altered in non-small cell lung cancer (NSCLC) (Histol. Histopathol. 2012; 27: 1183-1194). In the present study we examined the relationship between γ-tubulin expression and patient overall survival (OS). MATERIAL AND METHODS: Immunohistochemistry was performed, with well-characterized anti-γ-tubulin antibodies, on 109 formalin-fixed, paraffin-embedded NSCLC specimens (p-TNM stage I-III). γ-Tubulin labeling indexes (LIs) were determined, and the association of γ-tubulin expression with clinicopathological parameters was evaluated. To analyze OS rates according to γ-tubulin LIs, patients were categorized into three groups: those with low (0-30%), intermediate (31-69%) or high (70-100%) γ-tubulin LI. Association of clinicopathological parameters and γ-tubulin with survival were examined using univariate and multivariate Cox regression analysis. RESULTS: No statistically significant association was seen between γ-tubulin overexpression and histological type, tumor differentiation, p-TNM stage and adenocarcinoma subtyping. Longer survival was observed in the high γ-tubulin LI group of patients with p-TNM stages II+III when compared to intermediate or low γ-tubulin LI groups, but the difference was not statistically significant (p=0.066). On the other hand, when combined low and intermediate γ-tubulin LI groups (p-TNM stages II+III) where compared to high γ-tubulin LI group, statistically significant longer survival was observed in high γ-tubulin group (p=0.021). CONCLUSION: Our findings suggest that level of γ-tubulin expression may have an impact on patient survival at more advanced NSCLC stages.

Quantitative INTERSPECIMEN variability during biphasic pleural fluid cytological sampling in adenocarcinoma effusions.

OBJECTIVES: Given that cytology of adenocarcinoma-induced pleural effusions has a high diagnostic yield, we have comparatively evaluated the cytological information of smears during biphasic sampling of pleural fluid in patients with metastatic pleural adenocarcinoma from various primary sites. METHODS: We studied 25 male and 21 female patients, aged 59.4 ±17.2 years (mean ± SD) with unilateral malignant effusion of varying magnitude due to confirmed adenocarcinoma from various primary sites. At thoracocentesis we collected two 30-ml samples of pleural fluid, the first at the very beginning of fluid aspiration (S1) and the second just before termination of fluid removal (S2) and recorded the volume of fluid aspirated between the 2 samples. Cytological smears were examined under light microscopy by 3 independent cytologists after Papanicolaou stain. Quantitative assessment of cell types was averaged among 50 visual fields for each smear. RESULTS: In S1 versus S2 the mean number of mesothelial cells was 7.8 ± 4.8 versus 12.1 ± 5.4 (mean ± SD), of lymphocytes 64.6 ± 12.9 versus 85.9 ± 17.4, of neutrophils 8.5 ± 4.4 versus 11.7 ± 5.2, of eosinophils 1.5 ± 0.3 versus 1.7 ± 0.8, and the number of malignant cell aggregates(NMCA) was 11.9 ±4.9 versus 20.7 ± 5.1. The differences in numbers of all cell types including NMCA were statistically significant between S1 and S2 (P < .01). A strong significant linear association between S2/S1 ratio of NMCA and the volume of fluid aspirated between samples was also found (95% confidence interval [CI], 0.209-0.236, P-value < .001). CONCLUSION: Specimens aspirated before completion of fluid drainage are shown to contain significantly more diagnostic information than those aspirated at the beginning of fluid removal.

Overexpression and Nucleolar Localization of γ-Tubulin Small Complex Proteins GCP2 and GCP3 in Glioblastoma.

The expression, cellular distribution, and subcellular sorting of the microtubule (MT)-nucleating γ-tubulin small complex (γTuSC) proteins, GCP2 and GCP3, were studied in human glioblastoma cell lines and in clinical tissue samples representing all histologic grades of adult diffuse astrocytic gliomas (n = 54). Quantitative real-time polymerase chain reaction revealed a significant increase in the expression of GCP2 and GCP3 transcripts in glioblastoma cells versus normal human astrocytes; these were associated with higher amounts of both γTuSC proteins. GCP2 and GCP3 were concentrated in the centrosomes in interphase glioblastoma cells, but punctate and diffuse localizations were also detected in the cytosol and nuclei/nucleoli. Nucleolar localization was fixation dependent. GCP2 and GCP3 formed complexes with γ-tubulin in the nucleoli as confirmed by reciprocal immunoprecipitation experiments and immunoelectron microscopy. GCP2 and GCP3 depletion caused accumulation of cells in G2/M and mitotic delay but did not affect nucleolar integrity. Overexpression of GCP2 antagonized the inhibitory effect of the CDK5 regulatory subunit-associated tumor suppressor protein 3 (C53) on DNA damage G2/M checkpoint activity. Tumor cell GCP2 and GCP3 immunoreactivity was significantly increased over that in normal brains in glioblastoma samples; it was also associated with microvascular proliferation. These findings suggest that γTuSC protein dysregulation in glioblastomas may be linked to altered transcriptional checkpoint activity or interaction with signaling pathways associated with a malignant phenotype.

Overexpression of γ-tubulin in non-small cell lung cancer.

We and others have previously shown that increased expression and altered compartmentalization of γ-tubulin may contribute to tumorigenesis and tumor progression (J. Cell Physiol. 2009;223:519-529; Cancer Biol. Ther. 2010;9:66-76). Here we have determined by immunohistochemistry the localization and cellular distribution of γ-tubulin in clinical tissue samples from 109 non-small cell lung cancer (NSCLC) cases. The expression and distribution of γ-tubulin protein and transcripts was also determined in the NSCLC tumor cell lines NCI-H460 (HTB-177) and NCI-H69 (HTB-119) by immunocytochemistry, quantitative immunoblotting and reverse transcription quantitative real-time PCR (RT-qPCR). Polyclonal and monoclonal anti-peptide antibodies recognizing epitopes in the C- or N-terminal domains of γ-tubulins and human gene-specific primers for γ-tubulins 1 (TUBG1) and 2 (TUBG2) were used. In non-neoplastic cells of the airway epithelium in situ, γ-tubulin exhibited predominantly apical surface and pericentriolar localizations. In contrast, markedly increased, albeit heterogeneous and variously prominent γ-tubulin immunoreactivity was detected in clinical tumor specimens and in the NCI-H460 and NCI-H69 cell lines, where tumor cells exhibited overlapping multi-punctate and diffuse patterns of localization. Co-expression of γ-tubulin and Ki-67 (MIB-1) was detected in a population of proliferating tumor cells. A statistically significant increase of γ-tubulin expression was found in Stage III compared to lesser stage tumors (p<0.001 v. Stages I/II) regardless of histological subtype or grade. By quantitative immunoblotting NCI-H460 and NCI-H69 cells expressed higher levels of γ-tubulin protein compared to small airway epithelial cells (SAEC). In both tumor cell lines increase in TUBG1 and TUBG2 transcripts was detected by RT-qPCR. Our results reveal for the first time an increased expression of γ-tubulin in lung cancer.

Metastasis to the breast from an adenocarcinoma of the lung with extensive micropapillary component: a case report and review of the literature.

Breast metastasis from extra-mammary malignancy is rare. Based on the literature an incidence of 0.4-1.3% is reported. The primary malignancies most commonly metastasizing to the breast are leukemia-lymphoma, and malignant melanoma. We present a case of metastasis to the breast from a pulmonary adenocarcinoma, with extensive micropapillary component, diagnosed concomitantly with the primary tumor. A 73-year-old female presented with dyspnea and dry cough of 4 weeks duration and a massive pleural effusion was found on a chest radiograph. Additionally, on physical examination a poorly defined mass was noted in the upper outer quadrant of the left breast. The patient underwent bronchoscopy, excisional breast biopsy and medical thoracoscopy. By cytology, histology and immunohistochemistry primary lung adenocarcinoma with metastasis to the breast and parietal pleura was diagnosed. Both the primary and metastatic anatomic sites demonstrated histologically extensive micropapillary component, which is recently recognized as an important prognostic factor. The patient received chemotherapy but passed away within 7 months. Accurate differentiation of metastatic from primary carcinoma is of crucial importance because the treatment and prognosis differ significantly.

Fine needle aspiration cytology in primary breast angiosarcoma: a case report.

BACKGROUND: Angiosarcoma of the breast is an uncommon, aggressive, vascular tumor. The cytomorphologic features of angiosarcomas have rarely been reported. CASE: The present study describes a case of breast angiosarcoma initially diagnosed by fine needle aspiration cytology. Angiosarcoma appeared in the left breast of a 58-year-old woman after 12 years of a mastectomy (without radiotherapy) of the contralateral breast for invasive ductal carcinoma. Fine needle aspiration cytology yielded very bloody material with moderate cellularity. Microscopically, two types of cells were observed: spindle cells and epithelial-like cells with nuclear atypia. The latter were arranged in tight clusters with papillary configuration. Both cell types exhibited immunoreactivity for endothelial markers. The diagnosis of angiosarcoma was confirmed by histopathology of the surgically excised tumor. CONCLUSION: Angiosarcoma rarely occurs in the breast, and a definitive diagnosis is extremely difficult relying exclusively on cytologic features. Predominance of epithelioid cells may suggest an epithelial tumor, especially in patients with a history of breast carcinoma, whereas predominance of spindle cells can be misinterpreted as phyllodes tumor or another type of sarcoma. Cell block immunocytochemistry and tumor cell labeling with endothelial markers are necessary for accurate diagnosis.

Phosphorylation of caspase-9 in the cytosolic fraction of the cerebral cortex of newborn piglets following hypoxia.

We have previously shown that hypoxia leads to increased expression and increased activity of caspase-9 in the cerebral cortex of newborn piglets. Previous studies have demonstrated the importance of caspase-9 in the initiation of the apoptotic cascade, however, the mechanism of caspase-9 activation is not well understood. Experiments were conducted on newborn piglets 2-3 days of age that were anesthetized and mechanically ventilated. Hypoxia was induced by lowering the FiO(2) to 0.05-0.07 x 1h, and was confirmed biochemically by demonstrating decreased levels of ATP and PCr in the hypoxic groups in comparison with the normoxic group. The ATP level was 1.99+/-0.66 in the hypoxic group versus 4.10+/-0.19 in the normoxic group, P<0.05, and the PCr value was 0.68+/-0.14 in the hypoxic group, compared to 2.98+/-0.39 in the normoxic group, P<0.05. The cytosol of the neuronal nuclei from the cerebral cortex was probed with anti-phosphorylated Ser(196) caspase-9 antibody, using Western blot analysis. Protein bands were analyzed using image densitometry. In both the hypoxic and normoxic samples, protein bands were demonstrated just above the 50 kDa marker. Phosphorylated caspase-9 expression in OD x mm(2) was 43.85+/-8.4 in the normoxic group and 67.6+/-9.88 in the hypoxic group, P<0.05. The results of this study demonstrate that caspase-9, a key protein in hypoxia induced apoptosis, is phosphorylated at the Ser(196) site during hypoxia. The results demonstrate that hypoxia results in a post-translational modification of caspase-9 at Ser(196), which may alter the activity of caspase-9 in the hypoxic newborn brain.

Simultaneous cytological diagnosis of herpes simplex virus infection and primary lung cancer: report of two cases.

Herpes simplex is an uncommon cause of lower respiratory tract infection that requires prompt diagnosis and treatment to prevent late complications. We report two cases with simultaneous herpes simplex virus infection of the lower respiratory tract and lung carcinoma. Cytology of bronchial brushing and washing fluids and postbronchoscopic sputum established the diagnosis, which was further corroborated by real-time polymerase chain reaction.

Prognostic impact of Deoxyribonucleic acid (DNA) image analysis cytometry and immunohistochemical expression of Ki67 in surgically resected non-small cell lung cancers.

BACKGROUND: The aim of the present study was to evaluate the prognostic significance of DNA ploidy and Ki67 expression in non-small cell lung carcinoma (NSCLC). METHODS: This prospective study included 96 patients with stages I-IIIA NSCLC who underwent surgical excision. DNA image analysis cytometry was applied on imprints. Calculation of the DNA index (DI) and the 5c exceeding rate (5cER) was performed and the histograms were classified as peridiploid, peritetraploid, and x-ploid-multiploid. The Ki67 immunoreactivity was determined according to the avidin-biotin complex immunoperoxidase method. RESULTS: DNA histogram classification disclosed 30 peridiploid cases, 15 peritetraploid and 51 x-ploid-multiploid. Forty-eight cases (50%) had 5cER > 5%. The Ki67 immunoreactivity was below 25% in 53 tumors (62.4%) and above 25% in 32 (32.6%). Our results revealed the existence of a statistically significant relationship of DNA ploidy with nodal status (p = 0.042) and grade (p = 0.005). Adenocarcinomas and large cell carcinomas were more frequently encountered in x-ploid-multiploid tumors as compared to squamous cell carcinomas, which were more frequently peridiploid (p = 0.003). 5cER showed statistically significant association with nodal status (p = 0.037). Univariate analysis with respect to survival revealed significant association with stage (p < 0.001), nodal status (p < 0.001), tumor status (p < 0.001), DNA ploidy (p = 0.008) and 5cER (p = 0.0124). Multivariate analysis revealed stage and ploidy status as independent factors: peridiploid tumors were associated with better survival as compared to x-ploid-multiploid tumors (p = 0.022). CONCLUSION: Our results suggest that DNA ploidy, as determined by image analysis, provides an independent prognostic parameter for patients with NSCLC and thus, could be used to identify a subset of patients with more aggressive tumors.