Pulmonary neuroendocrine neoplasms with well differentiated morphology and high proliferative activity: illustrated by a case series and review of the literature.

OBJECTIVES: Pulmonary neuroendocrine neoplasms (NENs) are subdivided in carcinoids and neuroendocrine carcinomas (small cell lung carcinoma and large cell neuroendocrine carcinoma (LCNEC)), based on the presence of necrosis and mitotic index (MI). However, it is unclear if tumors with well differentiated morphology but high proliferation rate should be regarded as LCNEC or as high grade carcinoids. In previous case series, a longer overall survival then expected in LCNEC has been suggested. We describe 7 of those cases analyzed for pRb expression and overall survival. MATERIAL AND METHODS: Cases with well differentiated morphology, but MI > 10/2mm2 and/or Ki-67 proliferation index >20% were selected based on pathology reports of consecutive NENs in our university medical center (Maastricht UMC+, 2007-2018) and confirmed by pathological review. Immunohistochemistry was performed to assess pRb expression. RESULTS: Seven stage IV cases were included in this study. Median overall survival was 8 months (95% confidence interval 5-11 months). Cases with well differentiated morphology and preserved pRb expression (4/7) had a median overall survival of 45 months. CONCLUSION: A subgroup of pulmonary NENs with well differentiated morphology but high proliferation rate likely exists. pRb staining might be helpful to predict prognosis, but clinical relevance remains to be studied.

Exploring imaging features of molecular subtypes of large cell neuroendocrine carcinoma (LCNEC).

OBJECTIVES: Radiological characteristics and radiomics signatures can aid in differentiation between small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC). We investigated whether molecular subtypes of large cell neuroendocrine carcinoma (LCNEC), i.e. SCLC-like (with pRb loss) vs. NSCLC-like (with pRb expression), can be distinguished by imaging based on (1) imaging interpretation, (2) semantic features, and/or (3) a radiomics signature, designed to differentiate between SCLC and NSCLC. MATERIALS AND METHODS: Pulmonary oncologists and chest radiologists assessed chest CT-scans of 44 LCNEC patients for 'small cell-like' or 'non-small cell-like' appearance. The radiologists also scored semantic features of 50 LCNEC scans. Finally, a radiomics signature was trained on a dataset containing 48 SCLC and 76 NSCLC scans and validated on an external set of 58 SCLC and 40 NSCLC scans. This signature was applied on scans of 28 SCLC-like and 8 NSCLC-like LCNEC patients. RESULTS: Pulmonary oncologists and radiologists were unable to differentiate between molecular subtypes of LCNEC and no significant differences in semantic features were found. The area under the receiver operating characteristics curve of the radiomics signature in the validation set (SCLC vs. NSCLC) was 0.84 (95% confidence interval (CI) 0.77-0.92) and 0.58 (95% CI 0.29-0.86) in the LCNEC dataset (SCLC-like vs. NSCLC-like). CONCLUSION: LCNEC appears to have radiological characteristics of both SCLC and NSCLC, irrespective of pRb loss, compatible with the SCLC-like subtype. Imaging interpretation, semantic features and our radiomics signature designed to differentiate between SCLC and NSCLC were unable to separate molecular LCNEC subtypes, which underscores that LCNEC is a unique disease.

Large cell neuroendocrine carcinoma with a solitary brain metastasis and low Ki-67: a unique subtype.

INTRODUCTION: Stage IV large cell neuroendocrine carcinoma (LCNEC) of the lung generally presents as disseminated and aggressive disease with a Ki-67 proliferation index (PI) 40-80%. LCNEC can be subdivided in two main subtypes: the first harboring TP53/RB1 mutations (small-cell lung carcinoma (SCLC)-like), the second with mutations in TP53 and STK11/KEAP1 (non-small-cell lung carcinoma (NSCLC)-like). Here we evaluated 11 LCNEC patients with only a solitary brain metastasis and evaluate phenotype, genotype and follow-up. METHODS: Eleven LCNEC patients with solitary brain metastases were analyzed. Clinical characteristics and survival data were retrieved from medical records. Pathological analysis included histomorphological analysis, immunohistochemistry (pRB and Ki-67 PI) and next-generation sequencing (TP53, RB1, STK11, KEAP1 and MEN1). RESULTS: All patients had N0 or N1 disease. Median overall survival (OS) was 12 months (95% confidence interval (CI) 5.5-18.5 months). Mean Ki-67 PI was 59% (range 15-100%). In 6/11 LCNEC Ki-67 PI was ≤40%. OS was longer for Ki-67 ≤40% compared to >40% (17 months (95% CI 11-23 months) vs 5 months (95% CI 0.7-9 months), P = 0.007). Two patients were still alive at follow-up after 86 and 103 months, both had Ki-67 ≤40%. 8/11 patients could be subclassified, and both SCLC-like (n = 6) and NSCLC-like (n = 2) subtypes were present. No MEN1 mutation was found. CONCLUSION: Stage IV LCNEC with a solitary brain metastasis and N0/N1 disease show in the majority of cases Ki-67 PI ≤40% and prolonged survival, distinguishing them from general LCNEC. This unique subgroup can be both of the SCLC-like and NSCLC-like subtype.

DLL3 expression in large cell neuroendocrine carcinoma (LCNEC) and association with molecular subtypes and neuroendocrine profile.

OBJECTIVES: For stage IV pulmonary large cell neuroendocrine carcinoma (LCNEC), the only therapeutic option is palliative chemotherapy. DLL3 is a new therapeutic target, which seems to be often expressed in SCLC and LCNEC. It has recently been reported that DLL3 mRNA expression is particularly upregulated in the LCNEC subgroup with STK11/KEAP1 and TP53 co-mutations, in contrast to lower expression levels in RB1 and TP53 co-mutated LCNEC. Our aim was to investigate DLL3 protein expression in stage IV LCNEC and correlate data with mutational profiles (i.e.STK11/KEAP1/RB1), immunostaining results (pRb, NE markers) and clinical characteristics. MATERIALS AND METHODS: Immunohistochemical analysis for DLL3 (SC16.65) and ASCL1 (SC72.201) was performed on 94 and 51 FFPE tissue sections, respectively, of pathologically reviewed stage IV LCNEC. DLL3 and ASCL1 were scored positive if ≥1% of the tumor cells showed cytoplasmic/membranous or dotlike (DLL3) or nuclear (ASCL1) immunostaining. Data were correlated with available sequencing (TP53, RB1, STK11, KEAP1), immunostaining (pRb, NE markers) and clinical data. RESULTS: DLL3 was expressed in 70/94 (74%) LCNEC, 56 (80%) of which showed cytoplasmic/membranous staining. Median H-score was 55 (interquartile range 0-160). DLL3 staining was not different in pRb immunohistochemistry negative and positive patients (DLL3+ in 53/70 (76%) vs. 14/21 (67%), p = 0.409) or RB1 mutated and wildtype patients (DLL3+ in 27/34 (79%) vs. 23/33 (70%), p = 0.361). Nevertheless, 6/6 (100%) STK11 mutated, 10/11 (91%) KEAP1 mutated and 9/9 (100%) TP53 wildtype tumors were DLL3+ . Furthermore, DLL3 expression was associated with expression of ASCL1 and at least 2 out of 3 neuroendocrine markers. CONCLUSION: The high percentage (74%) of DLL3 expression in stage IV LCNEC denotes the potential of DLL3 targeted therapy in this patient group.

Prevalence and prognostic value of PD-L1 expression in molecular subtypes of metastatic large cell neuroendocrine carcinoma (LCNEC).

BACKGROUND: Pulmonary large cell neuroendocrine carcinoma (LCNEC) is a rare tumor with high mutational burden. Two subtypes of LCNEC are recognized, the co-mutated TP53 and RB1 group and the TP53 and STK11/KEAP1 group. We investigated PD-L1 and CD8 expression in a well characterized stage IV LCNEC cohort and compared expression in the two subtypes. METHODS: Immunohistochemical (IHC) analysis for PD-L1 and CD8 was performed on pathological reviewed pretreatment tumor samples for 148 stage IV LCNEC. Data about targeted next generation sequencing (TNGS) (TP53, RB1, STK11, KEAP1) and IHC for RB1 were available for most tumors. IHC staining for PD-L1 (DAKO 28-8) was performed and scored positive if tumors showed ≥1% membranous staining. CD8 was scored for intra-tumor T-cells and stromal cells. RESULTS: PD-L1 IHC expression data could be generated in 98/148 confirmed LCNEC samples along with RB1 IHC (n = 97) of which 77 passed quality control for TNGS. PD-L1 expression was positive in 16/98 cases (16%); 5 (5%) with ≥50%. PD-L1 expression was equal in RB1 mutated and RB1 wildtype tumors. None of STK11 mutated tumors (n = 7) expressed PD-L1. PD-L1 expression was correlated with superior overall survival (OS), hazard ratio 0.55 ((95% Confidence Interval 0.31-0.96), p = 0.038). Intra-tumor CD8 was associated with PD-L1 expression (p = 0.021) and stromal and intra-tumor CD8 were correlated with improved OS (p = 0.037 and p = 0.026 respectively). CONCLUSIONS: PD-L1 expression was positive in 16% of stage IV LCNEC tumors. This was independent of molecular subtype but associated with CD8 expression. In LCNEC patients with PD-L1 and/or CD8 expression superior OS was observed.