High Neuroendocrine Research Articles

Abstract 3042: Unraveling the mechanism of YAP toxicity in neuroendocrine solid tumors: Insights from Merkel cell carcinoma

Abstract The Hippo pathway, a pivotal regulator orchestrating organ size and tissue growth, has emerged as a central player in cancer biology. The downstream transcription factor YAP within this pathway has been implicated in diverse oncogenic processes. YAP activation contributes to drug resistance, metastasis, and poor clinical outcomes in many solid tumors. Conversely, YAP exhibits growth-suppressive effects in neuroendocrine (NE) solid cancers, a paradox that remains poorly understood. Analysis of RNAseq data from the Cancer Cell Line Encyclopedia (CCLE) revealed a distinct classification of tumors into YAP1-expressing (YAPon) and non-expressing (YAPoff) subgroups, with hematopoietic malignancies and NE solid tumors featured prominently in the latter. Merkel cell carcinoma (MCC) is a rare, high-grade, NE carcinoma of the skin with two distinct etiologies. Virus-positive MCC contains integrated Merkel cell polyomavirus that expresses the viral T antigens and virus-negative MCC caused by sunlight exposure and extensive UV damage. Bulk RNAseq performed with more than 60 MCC tumors revealed that YAP expression displayed an inverse correlation with an NE gene expression signature. To gain insight into why YAP expression is so low in MCC, we generated MCC cell lines with inducible expression of YAP. Expression of YAP led to growth arrest and cell death. We performed a genome-wide CRISPR-Cas9 screen to identify genes that when lost enable MCC cells to tolerate YAP expression. Functional enrichment analysis of the positively selected genes uncovered a variety of signaling pathways. Intriguingly, the CRISPR screen revealed a positive enrichment of non-canonical BAF complex members, including BRD9 and SMARCD1. To validate this finding, we treated MKL-1 cells with a BRD9 inhibitor, observing a substantial reduction in YAP toxicity. These findings open avenues for the development of targeted therapeutic strategies for high grade NE cancers, addressing a critical gap in our understanding of YAP biology in the context of MCC and beyond. Citation Format: Furkan Bahar, Thomas C. Frost, Varsha Ananthapadmanabhan, Julia L. Schnabel, Manisha Thakuria, James A. DeCaprio. Unraveling the mechanism of YAP toxicity in neuroendocrine solid tumors: Insights from Merkel cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3042.

Application of Small Cell Lung Cancer Molecular Subtyping Markers to Small Cell Neuroendocrine Carcinoma of the Cervix: NEUROD1 as a Poor Prognostic Factor.

Cervical small cell neuroendocrine carcinoma (CSCNEC) is a rare, aggressive type of cervical cancer. The treatment for CSCNEC follows the chemotherapeutic regimens used for small cell lung cancer (SCLC), with which it shares similar clinical and histologic features. For the first time, we applied neuroendocrine (NE) and SCLC molecular subtyping immunohistochemical markers [achaete-scute homolog 1 (ASCL1), neurogenic differentiation factor 1 (NEUROD1), POU class 2 homeobox 3 (POU2F3), and yes-associated protein 1] in 45 patients with CSCNEC. For the combined NE score, 51.1% of NE-high and 48.9% of NE-low subtypes were identified. The NE-high subtype tended to show worse progression-free survival and overall survival (OS) than the NE-low subtype ( P =0.059 and P =0.07, respectively). Applying the SCLC molecular subtyping, 53.3% of cases were identified as NEUROD1-dominant, 17.8% as ASCL1-dominant, 13.3% as YAP-dominant, and 4.4% as POU2F3-dominant, while 11.1% of cases showed negative expression for all markers; the distribution was different from that of SCLC. The NEUROD1-dominant subtype exhibited the worst OS, while the POU2F3 subtype exhibited the best OS ( P =0.003), similar to SCLC. In addition, the ASCL1-dominant and NEUROD1-dominant subtypes showed high NE scores, while yes-associated protein 1-dominant and POU2F3-dominant subtypes showed low NE scores ( P =0.008). In multivariate analysis, the NEUROD1 expression was further identified as the independent prognostic factor for worse OS, together with the high FIGO stage. CSCNEC was revealed to be a heterogeneous disease with different biological phenotypes and to share some similarities and differences with SCLC. Regarding the ongoing development of tailored treatments based on biomarkers in SCLC, the application of biomarker-driven individualized therapy would improve clinical outcomes in patients with CSCNEC.

Identification of Lineage-specific Transcriptional Factor–defined Molecular Subtypes in Small Cell Bladder Cancer

Small cell/neuroendocrine bladder cancers (SCBCs) are rare and highly aggressive tumors that are associated with poor clinical outcomes. We discovered that lineage-specific transcription factors (ASCL1, NEUROD1, and POU2F3) defined three SCBC molecular subtypes that resemble well-characterized subtypes in small cell lung cancer. The subtypes expressed various levels of neuroendocrine (NE) markers and distinct downstream transcriptional targets. Specifically, the ASCL1 and NEUROD1 subtypes had high NE marker expression and were enriched with different downstream regulators of the NE phenotype (FOXA2 and HES6, respectively). ASCL1 was also associated with the expression of delta‐like ligands that control oncogenic Notch signaling. POU2F3, a master regulator of the NE low subtype, targeted TRPM5, SOX9, and CHAT. We also observed an inverse association between NE marker expression and immune signatures associated with sensitivity to immune checkpoint blockade, and the ASCL1 subtype had distinct targets for clinically available antibody-drug conjugates. These findings provide new insight into molecular heterogeneity in SCBCs with implications for the development of new treatment regimens. Patient summaryWe investigated the levels of different proteins in a specific type of bladder cancer (small cell/neuroendocrine; SCBC). We could identify three distinct subtypes of SCBC with similarity to small cell/neuroendocrine cancers in other tissues. The results may help in identifying new treatment approaches for this type of bladder cancer.

Abstract 2153: ATR inhibition augments the efficacy of the lurbinectedin in small cell lung cancer

Abstract Small cell lung cancer (SCLC) is a recalcitrant disease with only a 9% five-year survival rate. SCLCs display intertumoral heterogeneity; high neuroendocrine (NE) tumors evolve with treatment and time in a largely MYC driven manner to a chemotherapy resistant non-NE state. Lurbinectedin, an RNA Pol-II inhibitor which causes DNA damage through binding ERCC5 was approved as second line therapy in 2020, with an overall response rate of 35% in patients with relapsed SCLC. However, these responses are short-lived and the majority of patient still succumb to their disease. A trial testing the combination of lurbinectedin and doxorubicin failed to improve survival compared with standard-of-care. We assessed lurbinectedin in combination with 43 other agents in SCLC cells. The top synergistic hit was the clinically relevant ATR inhibitor berzosertib which was 4-fold more synergistic than doxorubicin. Compared with lurbinectedin alone, the combination with berzosertib caused a 3.5-fold increase in DNA damage while significantly reducing the ability of cells to sense this damage by inhibiting γH2AX induction. This led to continued DNA replication even in the presence of lurbinectedin induced DNA damage, causing mitotic catastrophe and cell death. The combination was highly synergistic in 6 of 9 SCLC cell lines tested. Synergy was inversely correlated with p21 RNA and protein expression. The canonical role of p21 is to inhibit G1/S transition, and we found that thymidine induced G1 arrest inhibited synergy, while p21 knock-down increased the synergy. Interestingly, we observed synergy was inversely correlated with NE differentiation. In a high NE high p21 mouse model of SCLC, lurbinectedin alone was highly effective, with the combination showing little improvement. Conversely in a non-NE low p21 mouse model, lurbinectedin treatment alone was less effective, however the addition of berzosertib was synergistic and significantly improved efficacy. Overall, this work demonstrated berzosertib augments lurbinectedin efficacy in the recalcitrant non-NE SCLC subsets, and supported the development of a clinical trial testing lurbinectedin and berzosertib in SCLC patients NCT04802174. Citation Format: Christopher William Schultz, Yang Zhang, Haiqing Fu, Yasuhisa Murai, Darawalee Wangsa, Devon Atkinson, Liton Saha, Brian Elenbaas, Chien-Fei Lee, Astrid Zimmerman, Thomas Ried, Frank Zenke, Zoe Weaver, Rajaa Elmeskini, Yves Pommier, Anish Thomas. ATR inhibition augments the efficacy of the lurbinectedin in small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2153.

Abstract 1906: Relationship between neuroendocrine and immune gene expression in small cell lung cancer

Abstract Small cell lung cancer (SCLC) is classified as a high-grade neuroendocrine (NE) tumor, but a subset of SCLC has been termed “variant” due to the loss of NE characteristics. In this study, we computed NE scores for patient-derived SCLC cell lines and xenografts, as well as human tumors. We aligned NE properties with transcription factor-defined molecular subtypes. Then we investigated the different immune phenotypes associated with high and low NE scores. We found repression of immune response genes as a shared feature between classic SCLC and pulmonary neuroendocrine cells of the healthy lung. With loss of NE fate, variant SCLC tumors regain cell-autonomous immune gene expression and exhibit higher tumor-immune interactions. Pan-cancer analysis revealed this NE lineage-specific immune phenotype in other cancers. Additionally, we observed MHC I re-expression in SCLC upon development of chemoresistance. These findings provide a new framework to guide design of treatment regimens in SCLC. Citation Format: Ling Cai, Hongyu Liu, Fang Huang, Junya Fujimoto, Luc Girard, Yang Xie, John Minna, Adi Gazdar. Relationship between neuroendocrine and immune gene expression in small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1906.

Circulating Tumor Cell Chromosomal Instability and Neuroendocrine Phenotype by Immunomorphology and Poor Outcomes in Men with mCRPC Treated with Abiraterone or Enzalutamide

While the detection of AR-V7 in circulating tumor cells (CTC) is associated with resistance to abiraterone or enzalutamide in men with metastatic castration-resistant prostate cancer (mCRPC), it only accounts for a minority of this resistance. Neuroendocrine (NE) differentiation or chromosomal instability (CIN) may be additional mechanisms that mediate resistance. PROPHECY was a multicenter prospective study of men with high-risk mCRPC starting abiraterone or enzalutamide. A secondary objective was to assess Epic CTC CIN and NE phenotypes before abiraterone or enzalutamide and at progression. The proportional hazards (PH) model was used to investigate the prognostic importance of CIN and NE in predicting progression-free survival and overall survival (OS) adjusting for CTC number (CellSearch), AR-V7, prior therapy, and clinical risk score. The PH model was utilized to validate this association of NE with OS in an external dataset of patients treated similarly at Memorial Sloan Kettering Cancer Center (MSKCC; New York, NY). We enrolled 118 men with mCRPC starting on abiraterone or enzalutamide; 107 were evaluable on the Epic platform. Of these, 36.4% and 8.4% were CIN positive and NE positive, respectively. CIN and NE were independently associated with worse OS [HR, 2.2; 95% confidence interval (CI), 1.2-4.0 and HR 3.8; 95% CI, 1.2-12.3, respectively] when treated with abiraterone/enzalutamide. The prognostic significance of NE positivity for worse OS was confirmed in the MSKCC dataset (n = 173; HR, 5.7; 95% CI, 2.6-12.7). A high CIN and NE CTC phenotype is independently associated with worse survival in men with mCRPC treated with abiraterone/enzalutamide, warranting further prospective controlled predictive studies to inform treatment decisions.

Cell-autonomous immune gene expression is repressed in pulmonary neuroendocrine cells and small cell lung cancer

Small cell lung cancer (SCLC) is classified as a high-grade neuroendocrine (NE) tumor, but a subset of SCLC has been termed “variant” due to the loss of NE characteristics. In this study, we computed NE scores for patient-derived SCLC cell lines and xenografts, as well as human tumors. We aligned NE properties with transcription factor-defined molecular subtypes. Then we investigated the different immune phenotypes associated with high and low NE scores. We found repression of immune response genes as a shared feature between classic SCLC and pulmonary neuroendocrine cells of the healthy lung. With loss of NE fate, variant SCLC tumors regain cell-autonomous immune gene expression and exhibit higher tumor-immune interactions. Pan-cancer analysis revealed this NE lineage-specific immune phenotype in other cancers. Additionally, we observed MHC I re-expression in SCLC upon development of chemoresistance. These findings may help guide the design of treatment regimens in SCLC.

Adaptability to acute stress among women survivors of intimate partner violence: protocol for a mixed-methods cross-sectional study in a laboratory setting (BRAW study)

IntroductionIntimate partner violence (IPV) is the most common and alarming form of violence against women, affecting around 30% of all women around the world. Using an integrative methodology, we approach...

1971P Neuroendocrine (NE) expression profiling in non-castrate tumours is associated with poor therapy benefit and adverse clinical outcome in metastatic castration-resistant prostate cancer (mCRPC) patients

NE dedifferentiation is associated to clinical aggressiveness and resistance to androgen receptor (AR) inhibition in prostate cancer. We analyzed the correlation of a NE expression signature in non-castrate tumors with the clinical outcome of mCRPC patients treated with taxanes or AR signaling inhibitors (ARSI; abiraterone or enzalutamide). Patients with mCRPC were retrospectively tested for a customized signature of 45 NE-related genes in total RNA from formalin-fixed paraffin-embedded non-castrate tumor samples by the nCounter platform (Nanostring Technologies). Patients were grouped as high and low NE profile according to the unsupervised clustering of gene expression data and correlated with treatment response and clinical outcome. Ninety-six patients were included, with a median follow-up of 20 (1.17-81.3) months. Median age was 69.2 (55.8-87.1) years. All patients received taxanes (68 docetaxel, 10 cabazitaxel and 18 both), 29 received ARSI as first line in CRPC and 53 received ARSI after taxanes. Patients classified as high NE expression profile (59 vs. 37 patients) were associated with a shorter time of CRPC development (median 12.6 vs. 30.8 mo, HR 3.3, 95%CI 2-5.5, P<0.001) and shorter OS from CRPC diagnosis (median 24.3 vs. 42.2 mo, HR 2.2, 95%CI 1.4-3.4, P=0.001). Regarding taxanes outcome, high NE profile patients correlated with lower PSA-PFS (HR 1.7, 95%CI 1.1-2.6, P=0.011), radiologic (RX)-PFS (HR 1.8, 95%CI 1.1-2.8, P=0.012) and overall survival (OS) (HR 1.7, 95%CI 1.1-2.6, P=0.009), and they were independently associated with shorter PSA-PFS (HR 1.8, 95%CI 1.1-3, P=0.023). In first-line ARSI-treated patients, high NE profile patients (15 vs. 14 patients) correlated with shorter PSA-PFS (HR 3, 95%CI 1.3-7, P=0.011), RX-PFS (HR 2.5, 95%CI 1-5.9, P=0.042) and OS (HR 5.8, 95%CI 2.3-14.9, P<0.001). NE-related gene expression in non-castrate tumor samples is associated with adverse clinical outcome and both poor taxanes and ARSI benefit in mCRPC patients. Thus, molecular characterization of primary tumors may be useful to guide treatment strategies in mCRPC.

Association of circulating tumor cell chromosomal instability with worse outcomes in men with mCRPC treated with abiraterone or enzalutamide.

183 Background: While AR-V7 is a known driver of hormonal resistance, most men with mCRPC lack AR-V7 detection and commonly experience cross-resistance to abiraterone and enzalutamide (abi/enza). Loss of AR dependence through neuroendocrine (NE) differentiation or chromosomal instability (CIN) may explain AR therapy cross-resistance in additional men. Methods: PROPHECY was a multicenter prospective study of men with poor risk mCRPC starting abi/enza. We assessed Epic CTC AR-V7, CIN and NE phenotypes before abi/enza and at progression. Radiographic/clinical progression free survival (PFS) and overall survival (OS) were associated with CIN (&gt;3 CTCs) and NE (&gt;3 CTCs) CTC phenotypes using the proportional hazards model adjusting for Cellsearch CTC, AR-V7, and clinical risk score. Results: 118 men with mCRPC starting on abi/enza were enrolled; 106 had evaluable CTCs for AR-V7, CIN, and NE on the Epic platform. Of these, 22.6% and 9.4% of men exhibited high CTC CIN and NE scores, respectively. High pre-treatment CIN and NE phenotypic scores were observed in 63 and 27% of AR-V7 (+) and in 17 and 7% of AR-V7 (-) men. CTC CIN phenotype but not NE phenotype was associated with a lower confirmed PSA response rate and OS (TABLE) with abi/enza, adjusting for CTC number, AR-V7 and risk score. Conclusions: A high chromosomal instability CTC phenotype is associated with worse outcomes in men with mCRPC treated with abi/enza and warrants further study as a prognostic or predictive biomarker. Clinical trial information: NCT02269982. [Table: see text]

Association of neuroendocrine (NE) mRNA expression profiling in hormone-sensitive tumors samples with adverse clinical outcome in castration-resistant prostate cancer (CRPC) patients.

165 Background: NE dedifferentiation is associated to clinical aggressiveness and resistance to androgen receptor inhibition in prostate cancer. We investigated impact of a NE expression signature in the clinical outcome of mCRPC patients treated with taxanes. Methods: This is a multicenter retrospective study. A customized panel of 45 NE-related gene signature was tested in total RNA from formalin-fixed paraffin-embedded hormone-sensitive tumor samples, by the nCounter platform (Nanostring Technologies). Patients were grouped according to their molecular profile by unsupervised clustering. Expression levels were correlated with taxanes response and clinical outcome. Independent association with survival was evaluated by multivariate Cox modeling. Results: Eighty seven patients were included in the study, 79 were treated with docetaxel and 8 with cabazitaxel. Median age was 64.8 (44-88.3) years and median follow-up was 20.7 (1.17-74.4) months. High expression of the NE signature was associated with a shorter time of CRPC development (N=60, median 12.8 vs 21.6, HR 2.4, 95%CI 1.3-4.3, P=0.003) and shorter OS from CRPC diagnosis (median 24.1 vs 41.33, HR 2.3, 95%CI 1.4-3.8, P=0.001). Moreover, according to the outcome to taxanes, high NE signature correlated with lower PSA-PFS (median 6.6 vs 10.1 mo P=0.047, HR 1.6, 95%CI 1-2.7, P=0.05) and OS (median 19 vs 22 mo, HR 1.8, 95%CI 1.1-2.8, P=0.014), and it was independently associated to a lower OS (HR 1.9, 95%CI 1.1-3.2, P=0.016). Conclusions: NE-related gene expression in hormone-sensitive tumor samples is associated with adverse clinical outcome and lower taxane benefit in metastatic CRPC patients. Thus, molecular characterization of primary tumors may be useful to guide treatment strategies in metastatic prostate cancer.

ES04.02 Pathology Overview for Carcinoid and NE Spectrum

ES04.02 Pathology Overview for Carcinoid and NE Spectrum

MS32.04 Molecular Phenotypes of SCLC

Heterogeneity of neuroendocrine (NE) differentiation in small cell lung cancer (SCLC) determines very different molecular phenotypes Background and Purpose: SCLC is usually regarded as a homogenous tumor treated in a standard fashion. We have established and validated a 50 gene expression quantitative scoring system for NE lung tumors, consisting of both positive and negative scores [1]. While almost all carcinoid tumors have positive scores, SCLC and large cell neuroendocrine carcinomas have positive and negative subpopulations. Methodology: We utilized the NE gene expression score to demonstrate intertumor heterogeneity of gene expression patterns and their relationship to signaling pathways and to immune and inflammatory response. We used cluster analysis to divide the SCLC samples into high and low NE subtypes. We studied two public SCLC tumor data sets (n = 140) as well as 70 cell lines established by us and a pair of high/low sublines established from a single SCLC cell line. The NE score cluster analysis indicated that ∼10% of cell lines and ∼20 of SCLC tumors were of the low NE subtype. The low NE subtype samples had RB1 mutations or low or absent RB1 expression, confirming their SCLC origin. Differences between the high and low subtypes were divided into those common to tumors and cell lines and those largely or entirely limited to tumors. Differences common to tumors and cell lines: At least three different lineage specific transcription factors were expressed by SCLC tumors and cell lines. Most NE high cells expressed ASCL1 in conjunction with NKX2-1. NEUROD1, occasionally with ASCL1, was expressed by a smaller subset of NE high and occasionally by NE low cells. POU2F3 was expressed by most of the NE low subtypes, and about half of these also expressed ASCL2. However, about 4% of tumors and cell lines (all belonging to the NE low subtype) lacked an identified lineage specific transcription factor. The NE high subtype expressed all of the classic NE markers, as well as numerous neural markers covering neural and glial differentiation. In addition they selectively expressed markers for DNA damage, the FGFR pathway, calcium signaling and Notch inhibitors. The NE low subtype lacked all or most of the NE cell program, NKX2-1 and DLL3 expression, while MYC and REST expression, the Hippo, TGFb and Notch pathways and EMT were greatly upregulated. These and other findings were predicted to impact on the potential responses of the two subtypes to chemotherapy, radiotherapy, and multiple targeted therapies. Differences limited to tumors and absent in cell lines: Despite having a high mutational burden, SCLC is considered to be an “immunological desert”, with few infiltrating immune or inflammatory cells, low or absent PDL-1 expression, and modest responses to PD-1 blockade. While the high NE tumors fitted this description most of the low NE tumors had strands of fibrosis encircling tumor islets, with varying numbers of inflammatory cells in the fibrous strands. Major differences at multiple steps were present in the low NE subtype involving the over 200 genes regulating the Cancer-Immunity Cycle, including the major cytokine families, toll like receptors, the inflammasome pathway, the JAK/STAT pathway, HLA antigens, CD47 antigen, perforin and granzymes. These changes were accompanied by highly significant increases in the relative numbers of multiple types of infiltrating immune and inflammatory cells. In addition, the low NE subtype had increased expression of PDL-1 and the interferon gamma signature, suggesting enhanced responses to immunotherapy. SCLC tumors and cell lines can be divided into high and low NE subtypes and these subtypes have major differences in lineage specific transcription factors, expression of the NE program, as well as some oncogenes and pathways, predicting them to respond very differently to conventional and targeted therapies. In addition, very different infiltrating cellular and gene expression patterns of immune responses were present in the low NE tumors, involving multiple aspects of Innate and Adaptive Immunity, and predicting that the low NE subtype tumors are more likely to respond to immunotherapy. In addition, several possibilities of combining cytotoxic, targeted and immunotherapies are suggested from our findings. I thank my collaborators for their invaluable contributions: Ling Cai, Tao Wang, Guanghua Xiao, Luc Girard and John Minna. Reference: 1. Zhang W, Girard L, Zhang YA, et al. Small cell lung cancer tumors and preclinical models display heterogeneity of neuroendocrine phenotypes. Transl Lung Cancer Res 2018;7(1):32-49.

Small cell lung cancer tumors and preclinical models display heterogeneity of neuroendocrine phenotypes.

Small cell lung cancer (SCLC) is a deadly, high grade neuroendocrine (NE) tumor without recognized morphologic heterogeneity. However, over 30 years ago we described a SCLC subtype with "variant" morphology which did not express some NE markers and exhibited more aggressive growth. To quantitate NE properties of SCLCs, we developed a 50-gene expression-based NE score that could be applied to human SCLC tumors and cell lines, and genetically engineered mouse (GEM) models. We identified high and low NE subtypes of SCLC in all of our sample types, and characterized their properties. We found that 16% of human SCLC tumors and 10% of SCLC cell lines were of the low NE subtype, as well as cell lines from the GEM model. High NE SCLC lines grew as non-adherent floating aggregates or spheroids while Low NE lines had morphologic features of the variant subtype and grew as loosely attached cells. While the high NE subtype expressed one of the NE lineage master transcription factors ASCL1 or NEUROD1, together with NKX2-1, the entire range of NE markers, and lacked expression of the neuronal and NE repressor REST, the low NE subtype had lost expression of most NE markers, ASCL1, NEUROD1 and NKX2-1 and expressed REST. The low NE subtype had undergone epithelial mesenchymal transition (EMT) and had activated the Notch, Hippo and TGFβ pathways and MYC oncogene . Importantly, the high and low NE group of SCLC lines had similar gene expression profiles as their SCLC tumor counterparts. SCLC tumors and cell lines can exhibit distinct inter-tumor heterogeneity with respect to expression of NE features. Loss of NE expression results in major alterations in morphology, growth characteristics, and molecular properties. These findings have major clinical implications as the two subtypes are predicted to have very different responses to targeted therapies.

High neuroendocrine component is a factor for poor prognosis in gastrointestinal high-grade malignant mixed adenoneuroendocrine neoplasms

BackgroundHigh-grade malignant mixed adenoneuroendocrine carcinoma (MANEC) is a highly malignant combined neoplasm formed by an adenocarcinomatous component and a poorly differentiated (Grade 3) neuroendocrine (NE) carcinoma. MethodsTumors from 21 patients with gastrointestinal high-grade malignant MANECs or tumors with varying percentages of Grade 3 NE component were examined, and the NE component was confirmed by morphological analysis and immunohistochemical staining. Patients were divided into high NE (NE component > 50% in the primary tumor) and low NE (NE component ≤ 50% in the primary tumor) component groups. ResultsHigh NE component was a poor prognostic factor for patients with high grade MANEC (p = 0.021). Out of 13 patients with high-grade malignant MANEC, eight had a pure NE component, one had a pure adenocarcinomatous component, and four had mixed-type cancer in the metastatic lymph nodes. We further enrolled eight patients who had a Grade 3 NE component in the primary tumor and found that the pure NE component in tumor emboli and distant liver metastases were more frequent in the high NE than in the low NE component group (p = 0.012 and p = 0.046, respectively). ConclusionThe predominant tumor component in primary tumors was a prognostic factor and could predict tumor emboli and liver metastases pathology in high-grade malignant MANECs.

Human achaete-scute homolog-1 expression in neuroendocrine breast carcinoma

Neuroendocrine (NE) breast carcinoma is defined by morphological features similar to those of NE tumors of other organs and NE marker expression in at least 50% of neoplastic cells. However, a NE morphology may be observed even in breast carcinomas lacking NE markers. Human achaete-scute homolog-1 (hASH-1) is a transcription factor that plays a key role in the regulation of mammalian neural and NE cell development and has been identified in several human NE tumors. The aim of this study was to investigate hASH-1 expression in human breast cancers. hASH-1 expression was evaluated in 482 consecutive non-NE invasive breast carcinomas, in a series of 84 breast cancers with >50% NE marker expression (high NE differentiation) and 21 carcinomas with NE histology but negative or focally (<50%) positive for NE markers (low NE differentiation). hASH-1 protein was evaluated by a specific monoclonal antibody using immunohistochemistry and gene expression by real-time polymerase chain reaction. None of the non-NE invasive breast carcinomas expressed hASH-1 at any levels. hASH-1 was expressed in tumor cell nuclei of 63 and 38% of cases with high and low NE differentiation, respectively. Strong correlation with protein and gene expression levels was observed (p < 0.0001). hASH-1 expression was correlated to a low mitotic count (p = 0.02) and a low Ki67 proliferative index (p = 0.0062). hASH-1 expression occurs in breast cancers with NE differentiation regardless of the extent of the NE cell population, and it is restricted to a subset of tumor cells having a low proliferative potential.

Lung tumours with neuroendocrine differentiation

Neuroendocrine (NE) tumours of the lung include the low grade typical carcinoid (TC), intermediate grade atypical carcinoid (AC) and the high grade large cell NE carcinoma (LCNEC) and small cell carcinoma (SCLC) (Table 1). NE tumours of the lung comprise approximately 20–25% of all invasive lung malignancies. The most common NE lung tumour is SCLC, which accounts for 15–20% [ 1 Travis WD Travis LB Devesa SS Lung cancer. Cancer. 1995; 75 (Erratum:): 191-202Cancer. 1995; 75 (Erratum:): 2979 Crossref PubMed Google Scholar ], followed by LCNEC, which is about 3% in surgical series, and carcinoid, which accounts for 1–2% of lung cancers. The rarest NE lung tumour is atypical carcinoid which comprises approximately 10% of all lung carcinoids, accounting for approximately 0.1–0.2% of invasive lung cancers [ 2 Chen LC Travis WD Krug LM Pulmonary neuroendocrine tumors: What (little) do we know?. J Natl Compr Cancer Netw. 2006; 4: 623-630 Crossref PubMed Google Scholar ]. These tumours have certain morphologic, ultrastructural, immunohistochemical and molecular characteristics in common [ 3 Gosney JR Pulmonary endocrine pathology. Butterworth-Heinemann, Oxford; Boston1992 Google Scholar , 4 Thunnissen FB Van Eijk J Baak JP et al. Bronchopulmonary carcinoids and regional lymph node metastases. A quantitative pathologic investigation. Am J Pathol. 1988; 132: 119-122 PubMed Google Scholar , 5 Jones DJ Hasleton PS Moore M DNA ploidy in bronchopulmonary carcinoid tumours. Thorax. 1988; 43: 195-199 Crossref PubMed Google Scholar , 6 Rusch VW Klimstra DS Venkatraman ES Molecular markers help characterize neuroendocrine lung tumors. Ann Thorac Surg. 1996; 62: 798-809 Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar , 7 Brambilla E Negoescu A Gazzeri S et al. Apoptosis-related factors p53, Bcl2, and Bax in neuroendocrine lung tumors. Am J Pathol. 1996; 149: 1941-1952 PubMed Google Scholar ], but there are important differences in incidence, clinical, epidemiologic, histological, survival, and molecular characteristics [ 8 Travis WD Brambilla E Müller-Hermelink HK Harris CC Pathology and Genetics: Tumours of the Lung, Pleura, Thymus and Heart. IARC, Lyon2004 Google Scholar ]. Diffuse idiopathic NE cell hyperplasia (DIPNECH) is a pre-invasive lesion for carcinoid tumors, but it is very rare. Table 1The spectrum of neuroendocrine (NE) proliferations and neoplasms † Modified from reference [8]. (I)NE cell hyperplasia and tumourlets (A)NE cell hyperplasia 1.NE cell hyperplasia associated with fibrosis and/or inflammation 2.NE cell hyperplasia adjacent to carcinoid tumours 3.Diffuse idiopathic NE cell hyperplasia with or without airway fibrosis/obstruction (B)Tumourlets (less than 0.5 cm) (II)Tumours with NE Morphology (A)Typical carcinoid (0.5 cm or larger) (B)Atypical carcinoid (C)Large cell NE carcinoma Combined large cell NE carcinoma ‡ The histological type of the other component of non-small cell carcinoma should be specified. (D)Small cell carcinoma Combined small cell carcinoma ‡ The histological type of the other component of non-small cell carcinoma should be specified. (II)Non-small cell carcinomas with NE differentiation (NED) (IV)Other tumours with NE properties (A)Pulmonary blastoma (B)Primitive neuroectodermal tumour (C)Desmoplastic round cell tumour (D)Carcinomas with rhabdoid phenotype (E)Paraganglioma † Modified from reference [ 8 Travis WD Brambilla E Müller-Hermelink HK Harris CC Pathology and Genetics: Tumours of the Lung, Pleura, Thymus and Heart. IARC, Lyon2004 Google Scholar ]. ‡ The histological type of the other component of non-small cell carcinoma should be specified. Open table in a new tab

Influence of neuroendocrine tumor cells on proliferation in prostatic carcinoma

Neuroendocrine (NE) tumor cells in prostatic carcinoma (PCa) may influence tumor proliferation by a paracrine stimulus. The role of NE tumor cells is discussed controversially. This study investigates the influence of NE tumor differentiation on proliferation in PCa. Neuroendocrine differentiation, Ki-67, and Polo-like kinase 1 were studied immunohistochemically in 73 consecutive prostatectomies. Polo-like kinase 1 (PLK1) expression was also studied by Western and Northern blot analysis. Tumors were classified as high NE (HNE) and low NE differentiated (LNE), and depending on the growth pattern, with solitary and clusters of NE tumor cells. Low NE differentiated tumors were defined as less than 30 and HNE as 30 or more NE tumor cells per hot spot. Patients were followed by serum prostate-specific antigen (PSA) analysis. Neuroendocrine differentiation was present at least focally in 70% of tumors; 57% were HNE and 43% LNE. Solitary NE tumor cells were more often found in low-grade PCa, whereas clusters of NE tumor cells were more frequent in high-grade PCa. PLK1 messenger RNA and protein as well as Ki-67 were overexpressed in tumor tissue compared with tumor-free tissue. A stronger proliferation as determined by Ki-67 and PLK1 expression was present in HNE tumors compared with LNE tumors and in tumors with clusters in contrast to tumors with solitary NE tumor cells. Analysis for PSA relapse-free survival showed an earlier progression in HNE than in LNE tumors and in PCa with clusters of NE tumor cells. A significant and clustered NE differentiation in PCa may lead to an increased proliferation and earlier tumor progression, whereas few and solitary NE tumor cells have no prognostic impact.

Expression of p15 and p15.5 products in neuroendocrine lung tumours: relationship with p15(INK4b) methylation status.

The cell cycle inhibitor p15(INK4B) is frequently inactivated by homozygous deletions together with p16(INK4a)/p14(ARF) in many tumour types. Although it is now well established that p16(INK4a) and p14(ARF) act as tumour suppressor genes, the role of p15(INK4b) remains to be well defined. In order to explore the possibility of a selective deregulation of p15(INK4b) in human lung carcinogenesis, we studied p15(INK4b) status in neuroendocrine (NE) lung tumours where homozygous deletions of the p16(INK4a)/p14(ARF) locus are rarely observed. Expressions of p15 and p15.5 protein isoforms were analysed in a series of eight control normal lung, 12 tumour-associated normal lung, five low grade and 15 high grade neuroendocrine (NE) lung tumours and relationship with a specific p15(INK4b) methylation status was studied. Using Western blot analysis, we showed that p15 and p15.5 isoforms displayed a high heterogeneous pattern of expression in both normal and tumour tissues. P15 and p15.5 expressions were correlated in control normal lung (P<0.04) whereas they were not in tumours and associated normal lung. The level of p15.5 was significantly higher in associated normal lung and in tumours (P<0.02 respectively), specially in low grade tumours (P<0.01), than in control normal lung. Furthermore, p15.5 expression was more variable in tumours than in normal lung (P<0.01) and in low grade than in high grade NE lung tumours (P<0.02). Levels of p15 and p15.5 were distinct (up- or downregulated) from those observed in paired normal lung in 4/12 (33%) and 10/12 (83%) tumours respectively. Aberrant methylation at the 5' end of p15(INK4b) gene was observed in 15% of NE lung tumours using PCR-based assay, in a region proximal to the translation start where methylation did not occur in control and associated normal lung. However, no correlation could be assessed with protein status. MSP analysis of CpG islands proximal to the transcription start revealed methylation in all normal and tumour samples. No correlation was found between p15(INK4b) and p16(INK4a) or p14(ARF) status. These data suggest that complex deregulation of p15.5 is implicated in the carcinogenesis of human NE lung tumours independently of p16(INK4a) and p14(ARF) status.

Correlation between neovascularisation and neuroendocrine differentiation in prostatic carcinoma

Neuroendocrine (NE) differentiated tumor cells are found in almost all prostatic carcinomas. Prostatic carcinomas with a high NE differentiation have a poor prognosis and increased metastatic potential. A relationship between the neovascularisation density in the tumor and the metastatic potential in prostatic carcinoma is well known. NE cells and microvessels were demonstrated immunohistochemically on 102 radical prostatectomy specimens using antibodies against Chromogranin A and CD34. Standard areas (7.9 mm2) of maximal Chromogranin A expression and highest vascularisation were determined and topographically related by light microscopy. Area density of microvessels was evaluated morphometrically. NE tumor cells were present in all prostatic carcinomas studied. High grade prostatic carcinomas expressed significantly more NE tumor cells and exhibited a higher neovascularisation than low grade carcinomas. There was significantly higher neovascularisation in high grade tumors with many, as compared to high grade tumors with few, NE tumor cells. Poorer pathological staging correlated with increased neovascularisation and stronger NE differentiation. A topographical relationship between the area of maximal NE tumor cells and the area of highest neovascularisation was found in 80.4% of all cases. An analysis of variance revealed a large number of NE tumor cells as the only predictor of an increased neovascularisation (p = 0.0006). These observations support the concept that increased neovascularisation is influenced not only by poor pathological grading but also by a high NE differentiation.