Malignant pleural effusions due to small cell carcinoma of the lung. An immunocytochemical cell-surface analysis of lymphocytes and tumor cells

Thirteen malignant pleural effusions due to small cell carcinoma (SCC) of the lung were immunocytochemically studied using the peroxidase-antiperoxidase adhesive slide assay for the determination of cell surface antigens. A panel of monoclonal antibodies (MAbs) was used to determine the lymphocyte subpopulations and the reactivity of the tumor cells. Of the lymphocytes, 87 +/- 1% were CD3+ T cells, with 72 +/- 10% CD4+ helper/inducer T cells and 20 +/- 5% CD8+ suppressor/cytotoxic T cells. Only a minority of T lymphocytes were activated in terms of expressing the surface markers CD38 and HLA-DR. The distribution of the lymphocyte subpopulations was not significantly different from the distribution in other malignant and nonmalignant pleural diseases previously studied, indicating that the reaction pattern of the lymphocytes in the pleural cavity is similar in different diseases. The tumor cells from all cases were positive for LeuM1, CD16 and HLA-DR; 10 of 11 cases were positive for HEA-125, Sam 2 and Sam 10. Positivity for epithelial membrane antigen was observed in 11 cases, for OKT9 in 8 cases and for carcinoembryonic antigen in 6 cases. A total or partial loss of the reactivity with HLA-1 was found in nine cases. The reactivity pattern of the tumor cells with the MAbs used in this study is not specific for SCC of the lung because other carcinoma cells also reacted with these markers. Additional morphologic criteria, such as cell size and cell configuration, are needed to recognize the immunocytochemically positive-reacting cells as tumor cells from SCC of the lung. However, the immunostaining allows a better identification of the tumor cells, especially in cases with a small quantity of tumor cells.     

Vimentin and leukocyte common antigen-negative molding cells in pleural effusions of patients with small cell lung carcinoma

OBJECTIVE: To determine whether the presence of vimentin and leukocyte common antigen (LCA)-negative molding cells (VLNMC) could help in identifying rare small cell lung carcinoma (SCLC) cells. STUDY DESIGN: Thirty-four cell blocks of pleural effusions (PEs) from 26 patients with confirmed SCLC were stained immunohistochemically with vimentin and LCA antibodies and compared with hematoxylin and eosin-stained preparations. RESULTS: VLNMC were present in 22/22 PEs originally diagnosed as positive or atypical/suspicious for SCLC. Focal vimentin staining was seen in SCLC in 10/22 cases, and 1 case showed many vimentin-positive SCLC cells. One of 11 PEs originally interpreted as negative showed rare groups of VLNMC. This was supported by a subsequent PE obviously positive for SCLC. CONCLUSION: Immunoperoxidase stains for vimentin and LCA highlight SCLC in PEs as VLNMC; however, morphologic criteria must prevail in making the final diagnosis.     

Detection of cytoskeletal proteins in small cell lung carcinoma

Small cell lung carcinoma (SCLC) is the most aggressive of lung tumors, metastasize widely and are virtually incurable by surgical means. Therefore, the classification of lung cancer into SCLC and non-small cell lung carcinoma is essential for disease prognosis and treatment. For this purpose we have compared the immunohistochemical distribution of different cytoskeletal proteins as tumor markers. Analysis was performed by using of monoclonal antibodies directed against cytokeratins, neurofilaments, betaIII-tubulin, epithelial membrane antigen and neuron-specific enolase. Our results indicate that keratin and epithelial membrane antigen are reliable epithelial markers for SCLC. In addition, the positive staining with monoclonal antibodies TU-20 against betaIII-tubulin and neuron-specific enolase was found in some cases of SCLC. We suggest, that these antibodies could be a useful tool for complex immunohistochemical diagnosis of SCLC.     

Implications of nuclear diameter in small cell lung carcinoma

The nuclear diameter of 5,117 malignant cells from 42 small cell lung carcinoma (SCLC) patients was assessed either on pretreatment tissue sections (35 cases) or cytologic smears (7 cases) by ocular micrometry. The SCLCs were subtyped as 30 oat cell carcinomas and 12 intermediate cell carcinomas according to the World Health Organization classification, based on the predominant histology of the tumor. The median number of nuclei measured from each patient was 110. All patients were treated identically by sequential hemibody and local irradiation combined with chemotherapy and had a median follow-up time of 310 days. The mean nuclear diameter (+/- standard error) obtained from tissue sections was 8.2 +/- 0.03 microns (median = 8.0), including 7.3 +/- 0.03 microns (median = 7.0) for oat cell cases and 9.5 +/- 0.06 microns (median = 9.0) for intermediate cell cases (P less than .001). In 28.6% of these patients, the nuclear diameter overlapped in the range of 8 microns to 9 microns between both subtypes. Comparisons between the nuclear diameter of primary and metastatic SCLC cells revealed no statistically significant differences. The nuclear diameter of malignant cells correlated with the mitotic index and stage of disease, but did not correlate with the other nuclear morphologic variables or with survival. The only identified prognostic factor was the stage of disease; these results indicate that the nuclear diameter of malignant cells should not be considered a prognosticator or a guide for therapy in SCLC patients.     

Bombesin production by human small cell carcinoma of the lung

A series of continuous cell lines of human small cell carcinoma of the lung (SCCL) have been evaluated for the production of bombesin (BN). In early established cultures BN was detected in the medium of 9 out of 11 cell lines and in 6 out of 7 cell homogenates examined. Levels in the medium were frequently higher in cultures of later passages compared to earlier passages of the same line and low levels developed in the two previously negative cell lines. Plasma concentrations were greater than 80 pmol/l in 2 out of 27 (7%) randomly selected patients with SCCL. A culture (DMS 406) established from the tumor of a patient with the highest plasma level (1240 pmol/l) was the highest producer in vitro. The results indicate that BN, which has been demonstrated immunocytochemically to be present in normal bronchial mucosal cells, is frequently produced by SCCL in vitro but elevated plasma levels are infrequently found in patients with this neoplasm.     

Bombesin-like peptides in human small cell carcinoma of the lung

The nature of bombesin-like immunoreactive peptides was studied in extracts of small cell carcinoma of the human lung. Three peaks, I, II and III, designated by their increasing retention times, were separated by reversed-phase high performance liquid chromatography (HPLC) with trifluoroacetic acid (TFA) as counter ion. None of the peaks corresponded to bombesin. Peak III was eluted at the same position as porcine gastrin releasing peptide (GRP) but was separated from it in another reversed-phase system using heptafluorobutyric acid (HFBA). Peak II material eluted in the position of bombesin in the HFBA system but not in the TFA system. The elution position of Peak I corresponded to that of the carboxyl terminal fragments of GRP, i.e. GRP18-27 and GRP19-27. This correspondence was observed in each of the reversed-phase and gel filtration systems used. The Peak III peptide was converted to peak I after incubation with trypsin. It was reasoned that this conversion could be one of the steps in the processing of bombesin-like peptides in human small cell carcinoma.     

Biochemical and immunochemical analysis of gangliosides of human small cell lung carcinoma: production of monoclonal antibodies against a unique marker of small cell lung carcinoma, ganglioside Fuc GM1

Immunization of mice with a pure preparation of the ganglioside adsorbed on Salmonella typhimurium and hybridization of splenocytes with myeloma P3-X63-Ag 8.653 have resulted in hybridomas producing monoclonal antibodies against ganglioside Fuc GM1, a marker of human small cell lung carcinoma. Characterization of four hybridomal clones and data on the antigenic specificity of the monoclonal antibodies are given. All four monoclonal antibodies reacted only with Fuc GM1 in an enzyme-linked immunosorbent assay. In radioimmunodetection of the antigen on thin-layer plates, two of the four monoclonal antibodies gave cross-reactions with Fuc GD1b. The obtained monoclonal antibodies have revealed the presence of Fuc GM1 in all seven cases of small cell lung carcinoma we have studied and the absence of Fuc GM1 in the normal human lung tissue and in lung adenocarcinomas.     

Immunohistochemical analysis for cell proliferation-related protein expression in small cell carcinoma of the esophagus; a comparative study with small cell carcinoma of the lung and squamous cell carcinoma of the esophagus

Small cell carcinoma is a rare neoplasm in the esophagus. To evaluate cell proliferation activity and its underlying mechanisms in this tumor, we examined immunohistochemically 5 cases of small cell carcinoma of the esophagus (SCCE) for expressions of tumor suppressor proteins, oncoproteins and cell proliferation markers including p53, p21WAF1/CIP1, retinoblastoma (Rb) protein, bcl-2, Ki-67 and PCNA, and compared the results with those of 5 cases of small cell carcinoma of the lung (SCCL) and 10 cases of squamous cell carcinoma of the esophagus (SQCE). The prevalence and labeling index of p53-immunoreactivity tended to be higher in SCCE (4/5; 56.6%) and SCCL (4/5; 79.9%) than in SQCE (6/10; 48.8%). Expression of p21WAF1/CIP1 was observed in 2 of 10 cases of SQCE. In contrast, its expression could not be detected in any cases of SCCE and SCCL examined. Expression of Rb protein was observed in 9 out of 10 cases of SQCE, but not in any cases of SCCE and SCCL. SCCE and SCCL showed more frequent and intense immunoreactivity for bcl-2 than SQCE. In expression of cell proliferation markers (Ki-67 and PCNA), no remarkable difference was observed among SCCE, SCCL and SQCE. These results suggest that SCCE and SCCL could share some genetic alternations including mutation of p53, loss of Rb gene and overexpression of bcl-2, and these may be related to the similar biological potentials between the two. Furthermore, SCCE was different from SQCE in expression of Rb protein and bcl-2, and these two types of esophageal carcinoma could arise through different molecular mechanisms.     

Predicting ROR1/BCL2 combination targeted therapy of small cell carcinoma of the lung

Small cell lung cancer (SCLC) remains a deadly form of cancer, with a 5-year survival rate of less than 10 percent, necessitating novel therapies. Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncofetal protein that is emerging as a therapeutic target and is co-expressed with BCL2 in multiple tumor types due to microRNA coregulation. We hypothesize that ROR1-targeted therapy is effective in small cell lung cancer and synergizes with therapeutic BCL2 inhibition. Tissue microarrays (TMAs) and formalin-fixed paraffin-embedded (FFPE) SCLC patient samples were utilized to determine the prevalence of ROR1 and BCL2 expression in SCLC. Eight SCLC-derived cell lines were used to determine the antitumor activity of a small molecule ROR1 inhibitor (KAN0441571C) alone and in combination with the BCL2 inhibitor venetoclax. The Chou-Talalay method was utilized to determine synergy with the drug combination. ROR1 and BCL2 protein expression was identified in 93% (52/56) and 86% (48/56) of SCLC patient samples, respectively. Similarly, ROR1 and BCL2 were shown by qRT-PCR to have elevated expression in 79% (22/28) and 100% (28/28) of SCLC patient samples, respectively. KAN0441571C displayed efficacy in 8 SCLC cell lines, with an IC50 of 500 nM or less. Synergy as defined by a combination index of <1 via the Chou-Talalay method between KAN0441571C and venetoclax was demonstrated in 8 SCLC cell lines. We have shown that ROR1 inhibition is synergistic with BCL2 inhibition in SCLC models and shows promise as a novel therapeutic target in SCLC.Subject terms: Cancer genetics, Targeted therapies, Small-cell lung cancer     

Patterns of relapse following hippocampal avoidance prophylactic cranial irradiation for small cell lung carcinoma

BackgroundHippocampal avoidance techniques are an evolving standard of care for patients undergoing cranial irradiation. Our aim was to assess the oncological outcomes and patterns of failure following hippocampal avoidance prophylactic cranial irradiation (HA-PCI) as a standard of care in unselected patients with both limited and extensive stage small cell lung carcinoma.Materials and methodsConsecutive patients with small cell lung carcinoma with a complete (limited stage) or good partial (extensive stage) response following chemotherapy were eligible to receive HA-PCI, with a total dose of 25 Gray in 10 fractions. All patients had a negative baseline MRI brain scan with gadolinium prior to HA-PCI. Patients had baseline and follow up Common Toxicity Criteria Adverse Event assessments. Following completion of HA-PCI, all patients had three-monthly MRI brain scans with gadolinium until confirmation of intracranial relapse, as well as three-monthly CT of the chest, abdomen and pelvis. Overall and progression-free survival were calculated using the Kaplan-Meier method.ResultsA total of 17 consecutive patients, 9 men and 8 women, with a mean age of 70 years received HA-PCI between May 2016 and June 2020 after completion of their initial chemotherapy. There were no Grade 4 or greater adverse events. No patient had an isolated hippocampal avoidance zone relapse alone; three of 17 patients had multifocal relapses that included the hippocampal avoidance zone.ConclusionIn our series, there were no hippocampal only relapses and we conclude that HA-PCI is a safe alternative to standard PCI in the setting of small cell lung cancer.     

Differences in expression of pro-caspases in small cell and non-small cell lung carcinoma.

Expression of several molecular determinants of apoptosis was analyzed in 10 untreated small cell (SCLC) and 6 untreated non-small cell (NSCLC) lung carcinoma cell lines. Although SCLC lines were more prone to spontaneous apoptosis compared with NSCLC lines, the former showed higher Bcl-2 expression and a higher Bcl-2/Bax ratio. In order to understand this apparent contradiction, the expression of pro-caspases as well as calpain was analyzed in these cell lines at the protein and mRNA levels. No differences in protein level of pro-caspases-2, -3, -7, and -9 and of calpain were detected between the SCLC and the NSCLC lines, but a striking difference in pro-caspase-8 expression was noted. All 6 NSCLC, but only 2 of the 10 SCLC lines, expressed pro-caspase-8 protein. Further experiments using the RNase protection assay indicated that the lack of pro-caspase-8 expression at the mRNA level was characteristic for SCLC. Using the same experimental approach, we found that SCLC cell lines in addition to pro-caspase-8 were deficient in mRNA expression of pro-caspases-1, -4, and -10, suggesting a different caspase-activating cascade in SCLC compared with NSCLC. This first systematic characterization of pro-caspase expression in lung cancer surprisingly showed that SCLC, which are more prone to undergo spontaneous apoptosis, are deficient in several pro-caspases and have a high Bcl-2/Bax ratio. Thus, the propensity of SCLC cells to undergo apoptosis cannot be explained only by the expression of factors involved in regulation or execution of apoptosis.