Differentiation In Small Cell Lung Cancer Research Articles

P66.08 Differential Expression of INSM1 Between Pure SCLC and LCNEC After Surgical Resection and Its Clinicopathological Significance

According to WHO diagnostic criteria, both Small cell lung cancer (SCLC) and Large cell neuroendocrine (LCNEC) are categorized into high grade neuroendocrine carcinoma, based on morphological features and immunostaining of neuroendocrine markers including CD56, chromogranin A (CgA) and synaptophysin (Syn). However, the above markers have limited satisfactory significance of specificity and sensitivity. Insulinoma-associated protein 1 (INSM1) is highly expressed in SCLC cells as a zinc finger protein, and numerous studies have shown that INSM1 positive staining has a strong correlation with NE differentiation in SCLC and an alternative diagnostic biomarker for SCLC, but few studies are involved in LCNEC and its significance remains unclear in LCNEC. The aim of this study was to investigate the potential of INSM1 as a neuroendocrine marker by comparing the expression of INSM1 in SCLC and LCNEC and its clinicopathological significance. 347 surgically resected lung cancer including 290 pure SCLC and 57 pure LCNEC samples were retrieved in this retrospective study. INSM1 expression was detected by immunohistochemistry staining in SCLC and LCNEC tissue microarrays. The different expression of INSM1 between SCLC and LCNEC as well as the correlation with clinicopathological features including gender, age, smoking history, tumor location, AJCC 7th stage, reginal lymph node metastasis, pleural invasion and tumor thrombosis were analyzed by chi-square test. Of the included patients, male/female ratio was 2.33 (203/87) in SCLC and 10.4 (52/5) in LCNEC, the smoker rate was 64.5% (187/290) in SCLC and 43.9% (25/57) in NSCLC. According to the AJCC Cancer Staging Manual (7th edition), the SCLC patients were 29.0% (84/290) in stage I, 27.2% (79/290) in stage II, 40.7% (118/290) in stage III, and 3.1% (9/290) in stage IV, while the NSCLC patients were 49.1% (28/57) in stage I, 26.3% (15/57) in stage II, and 24.6% (14/57) in stage III, respectively. INSM1 was localized in the nucleus of lung cancer cells, a significant higher positive rate was detected in SCLC than that in LCNEC (271/290,93.4% in SCLC; 32/57,56.1% in LCNEC; p<0.05). INSM1 protein expression significantly correlated with smoking history (p=0.023), pleural invasion (p=0.010), and a positive trend of regional lymph node metastasis (p=0.052) in SCLC, but did not correlate with any of the clinicopathological features in LCNEC (p＞0.05). INSM1 protein was found no prognostic significance neither in SCLC nor in LCNEC (p＞0.05). INSM1 protein is highly expressed in SCLC and positively correlated with pleural invasion and smoking history. In contrast, INSM1 expression in LCNEC is significant lower and does not correlated with such clinicopathological features. Further research needs to be done on exploring the potential different underlying mechanisms and diagnostic significance of INSM1 in SCLC and LCNEC.

Differentiation of brain metastases from small and non-small lung cancers using apparent diffusion coefficient (ADC) maps

BackgroundBrain metastases are particularly common in patients with small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), with NSCLC showing a less aggressive clinical course and lower chemo- and radio sensitivity compared to SCLC. Early adequate therapy is highly desirable and depends on a reliable classification of tumor type. The apparent diffusion coefficient is a noninvasive neuroimaging marker with the potential to differentiate between major histological subtypes. Here we determine the sensitivity and specificity of the apparent diffusion coefficient to distinguish between NSCLC and SCLC.MethodsWe enrolled all NSCLC and SCLC patients diagnosed between 2008 and 2019 at the University Medical Center Göttingen. Cranial MR scans were visually inspected for brain metastases and the ratio of the apparent diffusion coefficient (ADC) was calculated by dividing the ADC measured within the solid part of a metastasis by a reference ADC extracted from an equivalent region in unaffected tissue on the contralateral hemisphere.ResultsOut of 411 enrolled patients, we detected 129 patients (83 NSCLC, 46 SCLC) with sufficiently large brain metastases with histologically classified lung cancer and no hemorrhage. We analyzed 185 brain metastases, 84 of SCLC and 101 of NSCLC. SCLC brain metastases showed an ADC ratio of 0.68 ± 0.12 SD, and NSCLC brain metastases showed an ADC ratio of 1.47 ± 0.31 SD. Receiver operating curve statistics differentiated brain metastases of NSCLC from SCLC with an area under the curve of 0.99 and a 95% CI of 0.98 to 1, p < 0.001. Youden's J cut-point is 0.97 at a sensitivity of 0.989 and a specificity of 0.988.ConclusionsIn patients with lung cancer and brain metastases with solid tumor parts, ADC ratio enables an ad hoc differentiation of SCLC and NSCLC, easily achieved during routine neuroradiological examination. Non-invasive MR imaging enables an early-individualized management of brain metastases from lung cancer.Trial registration: The study was registered in the German Clinical Trials Register (DRKS00023016).

Phase 1 study of AMG 119, a chimeric antigen receptor (CAR) T cell therapy targeting DLL3, in patients with relapsed/refractory small cell lung cancer (SCLC).

TPS8576 Background: SCLC is an aggressive neuroendocrine tumor, with initial sensitivity to chemotherapy and radiotherapy often followed by chemoresistant disease progression. Notch signaling is a key regulator of neuroendocrine differentiation in SCLC, and delta-like ligand 3 (DLL3) is an inhibitory ligand of Notch receptors. DLL3 is expressed in most SCLC tumors but minimally expressed in normal tissues, suggesting that it may be a promising target for cancer immunotherapy. AMG 119 is an adoptive cellular therapy that consists of a patient’s autologous T cells that have been genetically modified ex vivo to express a transmembrane CAR that targets DLL3 and redirects cytotoxic T cell specificity to DLL3-positive cells. AMG 119 CAR T cells show potent killing of SCLC cells expressing DLL3 in vitro and inhibit tumor growth in an SCLC xenograft model in vivo. Methods: This phase 1 study will evaluate the safety and tolerability of AMG 119 administered as a single infusion in adult patients with relapsed/refractory SCLC who have progressed after at least 1 platinum-based chemotherapy regimen. The primary objectives are to evaluate safety and tolerability and determine the maximum tolerated cell dose (MTCD) or recommended phase 2 cell dose (RP2CD). Secondary objectives are to evaluate preliminary evidence of antitumor activity, expansion and persistence of AMG 119, and trafficking of AMG 119 to the tumor in post-treatment biopsy. Key inclusion criteria include histologically confirmed SCLC with radiographically documented disease progression or recurrence after at least 1 platinum-based regimen, ECOG performance status 0–1, at least 2 measurable lesions per modified RECIST 1.1, no untreated or symptomatic brain metastases, and adequate organ function. In the cell dose exploration phase, 3–4 patients will receive a single IV infusion of AMG 119 at each cell dose. Cell dose escalation/de-escalation decisions will be guided by a modified toxicity probability interval design. The dose expansion phase will seek to confirm the MTCD or RP2CD and obtain further safety and efficacy data. Clinical trial information: NCT03392064.

Notch signaling and Tp53/RB1 pathway in pulmonary neuroendocrine tumorigenesis

Small cell lung cancer (SCLC) is a unique histological type of lung cancers, characterized by high grade malignant biological behavior and neuroendocrine differentiation. SCLC is subdivided into pure and combined types, and in the latter type, non-small cell lung cancer (NSCLC) features co-exist alongside with SCLC features. It has been reported that double mutations in Tp53 and RB1 are essential in small cell carcinogenesis, and that neuroendocrine differentiation is regulated by proneural transcription factors, such as Achaete-scute homolog 1 (ASCL1) and its signaling regulator; Notch signaling pathway. According to a recent article reported by Meder et al ., secondary SCLC is derived from NSCLC, with loss of Notch activity, accompanying with increased ASCL1 activity, and with further additional genetic changes in Tp53 and RB1 . They analyzed combined type SCLC cases, from the view point of the Notch-ASCL1-p53-RB axis, and it was the first to address comprehensively the molecular mechanisms of small cell carcinogenesis, regarding these four molecules. It is thus an urgent issue to clarify the molecular mechanisms of small cell carcinogenesis, progression, metastasis and acquisition of resistance to chemo-radiotherapy, for proper identification of a novel therapeutic target. But, in this perspective, we discussed the molecular mechanisms of small cell carcinogenesis, from the point of neuroendocrine differentiation in SCLC.

Insulinoma-Associated Protein 1 Is a Crucial Regulator of Neuroendocrine Differentiation in Lung Cancer

Insulinoma-associated protein 1 (INSM1) is expressed exclusively in embryonic developing neuroendocrine (NE) tissues. INSM1 gene expression is specific for small-cell lung cancer (SCLC), along with achaete-scute homolog-like 1 (ASCL1) and several NE molecules, such as chromogranin A, synaptophysin, and neural cell adhesion molecule 1. However, the underlying biological role of INSM1 in lung cancer remains largely unknown. We first showed that surgically resected SCLC samples specifically expressed INSM1. Forced expression of the INSM1 gene in adenocarcinoma cell lines (H358 and H1975) induced the expression of ASCL1, brain-2 (BRN2), chromogranin A, synaptophysin, and neural cell adhesion molecule 1; in contrast, knockdown of the INSM1 gene by siRNA in SCLC (H69 and H889) decreased their expression. However, forced/knockdown expression of ASCL1 and BRN2 did not affect INSM1 expression. A chromatin immunoprecipitation study revealed that INSM1 bound to the promoter region of the ASCL1 gene. A xenotransplantation assay using tet-on INSM1 gene-transfected adenocarcinoma cell lines demonstrated that INSM1 induced NE differentiation and growth inhibition. Furthermore, we found that INSM1 was not expressed in non-small-cell lung cancer and some SCLC cell lines expressing Notch1-Hes1. By forced/knockdown expression of Notch1 or Hes1 genes, we revealed that Notch1-Hes1 signaling suppressed INSM1, as well as ASCL1 and BRN2. INSM1, expressed exclusively in SCLC, is a crucial regulator of NE differentiation in SCLCs, and is regulated by the Notch1-Hes1 signaling pathway.

In vitro chemo- and radio-resistance in small cell lung cancer correlates with cell adhesion and constitutive activation of AKT and MAP kinase pathways.

Most small cell lung cancer (SCLC) patients relapse within 12 months of starting combination chemotherapy plus radio-therapy, due to the development of acquired chemo- and radio-resistance. This phenomenon relates to the induction of tumour differentiation, resulting in apoptosis-resistant, morphologically variant (v-SCLC) cells, which lack the neuroendocrine expression of classic (c-) SCLC cells. In this study spontaneously adherent SCLC sublines were shown by differential gene expression analysis to provide an in vitro model of variant differentiation in SCLC, with down-regulation of neuroendocrine markers and up-regulation of epithelial differentiation markers cyclin D1, endothelin, the cell adhesion molecules CD 44 and integrin subunits alpha2, beta3 and beta4. The sensitivity of adherent SCLC sublines to etoposide, cyclophosphamide and gamma radiation was significantly diminished relative to parent suspension cell lines. Western blot analysis using phosphorylation-specific antibodies to Akt and MAP kinase revealed markedly elevated activation in adherent SCLC sublines, paralleled by increased levels of phosphorylated Bad protein and activated NF-kappaB. Subcultivation of the adherent sublines on uncoated surfaces reversed their adherent phenotype immediately and under these conditions Akt activity reverted to low levels. These results suggest that c-SCLC cells can differentiate spontaneously to v-SCLC and that the associated cellular adhesion may trigger Akt-dependent inhibition of apoptosis in SCLC cells, thus leading to acquired chemo- and radio-resistance.

Induction of phenotypic changes in SCLC cell lines in vitro by hexamethylene bisacetamide, sodium butyrate, and cyclic AMP

Hexamethylene bisacetamide (HMBA), sodium butyrate (NaBt), and cyclic AMP (cAMP) have been shown to induce differentiation, which may regulate tumour growth differently from conventional cytotoxic drugs. It was the intention in the present study to determine whether alterations could be induced in the phenotype of small cell lung cancer (SCLC) cell lines with HMBA, NaBt and cAMP, and whether these alterations would correlate with reduced growth in vivo, implying a phenotypic shift from malignancy towards differentiation. The cell lines were NCI-H69, H187 and H128. The activity of dopa decarboxylase (DDC), the BB isozyme of creatine kinase (CK-BB), the synthesis of bombesin-like peptide (BLI), and the presence of neurone specific enolase (NSE) and chromogranin were used as markers of the small cell phenotype. Clonogenicity in suspension in agar, and growth as xenografts in nude mice, were used as malignancy-associated properties. Cell proliferation in vitro was determined by cell counting and growth curve analysis. HMBA, NaBt and cAMP were found to be reversibly cytostatic in liquid culture and pre-exposure reduced the cloning efficiency in agar by 60%-80%. Growth as xenografts was inhibited (three- to five-fold increase in the tumour doubling time), most significantly by NaBt. Effects of phenotypic markers were more complex. The most significant were a two-fold reduction in DDC with NaBt and HMBA, a 50% increase in CK-BB with cAMP, and a 70%-100% increase in secreted BLI with HMBA and cAMP, in NCI-H69 cells. No significant effects were seen on NSE and chromogranin. There was little sign of an interaction with adriamycin and vincristine, although a slight increase was observed in the ID50 of VP-16 following treatment with cAMP. NaBt, HMBA and cAMP were cytostatic and inhibited tumour growth, but there was no coordinated response in marker expression that would confirm phenotypic alteration indicative of differentiation. The problem of defining differentiation in SCLC further complicated the analysis. The possibility remains of combining these agents with conventional cytotoxics as there appears to be little antagonistic effect, and other studies have suggested synergism may be possible with correct scheduling.

Cytotoxic chemotherapy induces cell differentiation in small-cell lung carcinoma.

Despite the high response rates resulting from chemotherapy, the majority of small-cell lung cancer (SCLC) patients relapse with chemoresistant tumors. To analyze the phenotypic changes that are precursors of chemoresistant status, and to investigate the role of chemotherapy in these changes, tumor samples from 20 patients, taken before chemotherapy (etoposide, doxorubicin, and cyclophosphamide) and again at the onset of chemoresistance (after at least three courses of chemotherapy), were compared. The histologic changes were minor in 10 of 20 patients, as shown by an increase in cell size; they were major in 10 of 20 patients, with the appearance of mixed composite tumors in which neuroendocrine (NE), epidermoid, and glandular components were mixed. Major changes correlated with a good response to chemotherapy (P = .001). Ultrastructural studies showed an increase in neurosecretory granules and desmosomes, and a high frequency of multidirectional differentiation (45%) when comparison was made with pretherapy samples (10%) (P less than .01). Immunohistochemical (IH) analysis showed an increase in cytokeratin (CK) expression in treated patients, with a different labeling pattern and the expression of higher molecular weight CK. The expression of NE lineage markers (Leu 19, Sy 38, SL 11-14) remained stable, while that of NE differentiation markers (Leu 7, chromogranin) increased in the treated patients. The neuron-specific enolase (NSE) activity remained stable in treated SCLC. Large cells with a more differentiated phenotype and proliferative capacity (as shown by Ki 67 labeling), appeared to be characteristic of treated and secondary chemoresistant SCLC. The acquisition of a more complex phenotype, which correlates with primary response to therapy, implies a drug-induced differentiation in SCLC.