Pulmonale neuroendokrine Tumoren in der neuen WHO-Klassifikation 2015

Classification

In the recently published 4th edition of the World Health Organization (WHO) classification of tumors of the lungs, pleura, thymus and heart, all neuroendocrine tumors of the lungs (pNET) are presented for the first time in one single chapter following adenocarcinoma and squamous cell carcinoma and before large cell carcinoma. In this classification, high grade small cell lung cancer (SCLC) and large cell neuroendocrine carcinoma (LCNEC) are differentiated from intermediate grade atypical carcinoids (AC) and low grade typical carcinoids as well as from preinvasive lesions (DIPNECH). In the 3rd WHO classification from 2004, which dealt with resection specimens, SCLC and carcinoids each had a separate chapter and LCNEC was previously listed in the chapter on large cell carcinoma of the lungs. The new WHO classification is for the first time also applicable to lung biopsies.

Diagnostics

Normally, common features of all pNET are a neuroendocrine morphology (as far as detectable in small biopsies) and expression of the neuroendocrine (NE) markers (chromogranin A, synaptophysin and CD56/NCAM). An immunohistochemical positive staining of at least one NE marker was already recommended in the 3rd edition of the WHO classification (2004) only for LCNEC. Differentiating features are a small or large cell cytomorphology/histomorphology, nuclear criteria and the mitotic rate (for SCLC >10 with a median of 80, for LCNEC >10 median 70, for AC 2 - 10, for TC <?2 each per 2 mm²). Tumor cell necrosis usually occurs in SCLC and LCNEC, partially in AC and not in TC. The guideline Ki67 proliferation rates are given for the first time in the new WHO classification for SCLC as 50–100?%, for LCNEC 40–80?%, for AC up to 20?% and for TC up to 5?%.

Molecular pathology

Molecular alterations occur in SCLC and LCNEC in large numbers and are very variable in quality. In AC and TC they occur much less frequently and are relatively similar.

Conclusion

The direct comparison of all pNET in one chapter facilitates the differential diagnostics of these tumors, provides a better transparency especially of LCNEC and allows a further comprehensive development of the clinical practical and scientific evaluation of pNET. Although a separate terminology of pNET is maintained for the lungs, a careful approach towards the gastroentero-pancreatic NET (gepNET) can be observed.

     

LKB1 protein expression in neuroendocrine tumors of the lung

During a recent investigation of LKB1 gene abnormality in lung lesions, strong expression of LKB1 protein in normal neuroendocrine (NE) cells of the bronchial epithelium was found. Because LKB1 functions as a tumor suppressor gene, the question of whether alteration of LKB1 expression is related to the development of pulmonary NE tumors of various grades was investigated. LKB1 immunohistochemistry was examined in a total of 68 primary pulmonary NE tumors consisting of 30 specimens of small cell lung carcinoma (SCLC), 23 large cell neuroendocrine carcinomas (LCNEC), two atypical carcinoids, and 13 typical carcinoids. Loss or low expression (<20% immunoreactive cells) of LKB1 protein expression was more frequently observed in high-grade NE tumors (SCLC and LCNEC; 45/53, 84.9%) than in typical and atypical carcinoids (3/15; 20%). The difference in LKB1 immunoreactivity between the high-grade NE tumors and the carcinoid group was statistically significant ( P  < 0.0001). In conclusion, marked reduction of LKB1 expression in high-grade NE tumors of the lung suggests a possible role of LKB1 inactivation in its tumorigenesis. Although a few previous studies indicated rare genetic alterations of LKB1 in SCLC, further studies including analysis of other NE tumors and focusing on epigenetic abnormalities of LKB1 gene are warranted.     

Expression of L-type amino acid transporter 1 (LAT1) in neuroendocrine tumors of the lung

Amino acid transport systems play an important role in cellular proliferation. L-type amino acid transporter 1 (LAT1) has been associated with tumor growth, and is highly expressed in the established tumor cell lines and primary human neoplasms. In this study, we investigated the expression of LAT1 to evaluate the malignant potential and prognostic significance in neuroendocrine (NE) tumors of the lung. Twenty-one surgically resected, large cell neuroendocrine carcinomas (LCNEC), 13 small cell lung cancers (SCLC), five atypical carcinoids (AC), and 10 typical carcinoids (TC) were enrolled in the study. LAT1 expression and Ki-67 labeling index of the NE tumors were analyzed by immunohistochemical staining. LAT1 was overexpressed in 52.4% of the LCNEC, in 46.2% of the SCLC, and in 25% of the AC. LAT1 expression in LCNEC was significantly associated with lymph node metastasis and poor outcome. Moreover, a significant correlation was found between LAT1 expression and Ki-67 in both LCNEC and SCLC. Expression of LAT1 tended to increase from low-grade to high-grade NE tumors. The present results suggest that LAT1 may play a significant role in cellular proliferation, lymph node metastasis, and poor outcome in patients with NE tumors of the lung.     

CGH-Befunde bei neuroendokrinen Tumoren der Lunge

According to the 1999 World Health Organisation classification of lung tumors, the classification of neuroendocrine (NE) tumors is solely based on light-microscopic features. Typical and atypical carcinoid tumors are distinguished from large cell (LCNEC) and small cell neuroendocrine carcinomas (SCLC). We used comparative genomic hybridization (CGH) on 50 samples to investigate the cytogenetic relationships between NE tumors. On average, carcinoid tumors showed markedly fewer chromosomal imbalances (1.8/case, 23 cases) than LCNEC (13.3/case, 17 cases) or SCLC (17/case, 10 cases). The frequency of amplicons increased accordingly. Typical carcinoid tumors exhibited significant defects on chromosomes 11 and 13 only. Interestingly, only the frequency of losses on chromosome arm 11q was very similar for all three tumor entities (about 30%). In conclusion, the CGH results support the classification of typical carcinoid tumors as a separate entity in clear distinction from the NE carcinomas.     

Unraveling Tumor Grading and Genomic Landscape in Lung Neuroendocrine Tumors

Currently, grading in lung neuroendocrine tumors (NETs) is inherently defined by the histological classification based on cell features, mitosis count, and necrosis, for which typical carcinoids (TC) are low-grade malignant tumors with long life expectation, atypical carcinoids (AC) intermediate-grade malignant tumors with more aggressive clinical behavior, and large cell NE carcinomas (LCNEC) and small cell lung carcinomas (SCLC) high-grade malignant tumors with dismal prognosis. While Ki-67 antigen labeling index, highlighting the proportion of proliferating tumor cells, has largely been used in digestive NETs for assessing prognosis and assisting therapy decisions, the same marker does not play an established role in the diagnosis, grading, and prognosis of lung NETs. Next generation sequencing techniques (NGS), thanks to their astonishing ability to process in a shorter timeframe up to billions of DNA strands, are radically revolutionizing our approach to diagnosis and therapy of tumors, including lung cancer. When applied to single genes, panels of genes, exome, or the whole genome by using either frozen or paraffin tissues, NGS techniques increase our understanding of cancer, thus realizing the bases of precision medicine. Data are emerging that TC and AC are mainly altered in chromatin remodeling genes, whereas LCNEC and SCLC are also mutated in cell cycle checkpoint and cell differentiation regulators. A common denominator to all lung NETs is a deregulation of cell proliferation, which represents a biological rationale for morphologic (mitoses and necrosis) and molecular (Ki-67 antigen) parameters to successfully serve as predictors of tumor behavior (i.e., identification of pathological entities with clinical correlation). It is envisaged that a novel grading system in lung NETs based on the combined assessment of mitoses, necrosis, and Ki-67 LI may offer a better stratification of prognostic classes, realizing a bridge between molecular alterations, morphological features, and clinical behavior.     

Controversial issues and new discoveries in lung neuroendocrine tumors

Lung neuroendocrine (NE) tumors consist of four histologic subtypes, which are usually classified based on a three-tiered prognostic scheme. They are typical carcinoid (TC) as low-grade malignant tumors, atypical carcinoid (AD) as intermediate-grade malignant tumors, and large cell NE carcinoma (LCNEC) and small cell lung carcinoma (SCLC), both of which are high-grade malignant tumors. This three-tiered classification is based solely on histologic grounds and is a source of controversy especially when dealing with borderline or “gray zone” categories (TC vs. AC, AC vs. LCNEC, LCNEC and SCLC). In this review, controversial issues regarding the histologic classification will be discussed, and an innovative grading system that incorporates Ki-67 labeling index will be described. In addition, the recently discovered molecular alterations involved in TC/AC, as well as pathways involved in high-grade NE carcinomas, will be discussed in order to elucidate the differences in pathogenesis and biology between carcinoid tumors and high-grade NE carcinomas.     

Expression of amino acid sequences of the chromogranin A molecule and synaptic vesicle protein 2 in neuroendocrine tumors of the lung

Chromogranin A (CgA) and its valuable complement synaptic vesicle protein 2 (SV2) are neuroendocrine (NE) markers. Post-translational processing of CgA has been reported to vary in different NE cell types and tumors, but little is known regarding the expression of various CgA epitopes and SV2 in NE pulmonary tumors. We studied the immunoreactivity to six CgA epitopes and SV2 in ten typical (TC) and ten atypical (ACT) carcinoids, five large-cell NE carcinomas (LCNEC) and five small-cell carcinomas (SCLC), also comparing the results with clinicopathological characteristics of tumors. The sequences CgA 17–38 (vasostatin), 176–195 (chromacin), 375–384 (parastatin) and 411–424 (C-terminal parastatin) and SV2 were relevant markers for the CT/ATC group, whereas the antibody to CgA 176–195 was a better marker for the LCNEC/SCLC group. An inverse correlation was found between proliferative activity and granule-related markers in the CT/ACT group, and a direct correlation in poorly differentiated tumors. The expression of granule-related markers did not correlate with hormone content or clinical characteristics of NE tumors. The expression of CgA epitopes and SV2 occurs in all NE tumors, differing between better differentiated and poorly differentiated tumors but not within the respective groups.     

Molecular alterations of neuroendocrine tumours of the lung

Neuroendocrine tumours of the lung comprise low [typical carcinoid (TC)], intermediate [atypical carcinoid (AC)] and high‐grade [small‐cell lung cancer (SCLC) and large‐cell neuroendocrine carcinoma (LCNEC)] malignancies, while a pre‐invasive lesion [diffuse idiopathic pulmonary neuroendocrine cell hyperplasia (DIPNECH)] may generate a subset of peripheral carcinoid tumours. These neoplasms are differentiated conventionally based on mitotic rate, presence of necrosis and cytological details, according to the 2015 World Health Organisation (WHO) classification. Clinical data and molecular alterations distinguish carcinoids and high‐grade carcinomas into two separate categories. Previous studies have demonstrated a significantly higher rate of chromosomal aberrations in carcinomas (e.g. 3p and 17p deletions), but restriction of multiple endocrine neoplasia type 1 (MEN1) mutations to carcinoids. High‐grade carcinomas are also characterised by TP53 and RB1 gene inactivation. In this review, a critical analysis of the diagnostic and prognostic role of Ki67 labelling index and a concise discussion of the most relevant findings regarding molecular characterisation of lung neuroendocrine neoplasms are reported. In addition, we illustrate how the development of promising therapeutic strategies based on the identification of molecular targets (mTOR inhibitors in carcinoids and targeting of the Notch ligand DLL3 in SCLC) may require the assessment of predictive biomarkers, even in the group of neuroendocrine tumours of the lung.     

Neuroendokrine Tumoren der Lunge

The new World Health Organization (WHO) classification announced for 2015 will for the first time present all neuroendocrine tumors (NET) of the lungs in one single section. In this classification high grade small cell lung carcinoma (SCLC) and large cell neuroendocrine carcinoma (LCNEC) will be discriminated from intermediate grade atypical carcinoid (AC) and low grade typical carcinoid as well as from the preinvasive lesion diffuse idiopathic pulmonary neuroendocrine cell hyperplasia (DIPNECH). The LCNEC was previously listed under the section of large cell carcinomas. The LCNEC could previously be diagnosed according to the current WHO classification from 2004 which is designed for resection specimens. According to this the main diagnostic criteria are a neuroendocrine growth pattern which can be difficult or impossible to detect in biopsy material, non-small cell cytological features, more than 10 mitoses per 2 mm² (mean 70–80 per 2 mm²), tumor cell necrosis, and an immunohistochemical positivity for at least one neuroendocrine marker other than neuron-specific enolase (NSE). The presentation of all neuroendocrine tumors of the lungs in one section allows a more direct comparison and a better differential diagnostic discrimination of the different entities.     

Neuroendocrine differentiation in endocrine and nonendocrine lung carcinomas

Bronchial carcinoids and small cell lung cancer (SCLC) are currently recognized as neuroendocrine (NE) neoplasms. However, non-SCLC (NSCLC) may also express NE properties. Paraffin-embedded sections of a comprehensive panel of 113 lung carcinomas were analyzed for the expression of three general markers common to all NE cells, namely, chromogranin A, Leu-7 and neuron-specific enolase (NSE), five specific NE secretory products, and four other tumor markers by immunohistochemistry using the sensitive avidin-biotinylated peroxidase technique. The authors were able to demonstrate the following: (1) most, but not all carcinoids and SCLCs expressed multiple NE markers in a high percentage of tumor cells; (2) up to a half of NSCLC cases contained small subpopulations of cells expressing NE in a high percentage of tumor cells; (2) up to half of NSCLC cases contained small subpopulations of cells expressing NE markers; and (3) occasional NSCLCs showed staining patterns indistinguishable from SCLC. Specifically, 7 of 77 NSCLCs expressed four or more NE markers. NE markers in NSCLCs were more commonly expressed in adenocarcinomas and large cell carcinomas and rarely in squamous cell carcinomas. For comparison, the mean number of NE markers expressed by all cases of NSCLC was 1.5, carcinoids 6.0, and SCLCs 3.8. Individual "marker counts" were not useful in categorizing lung tumors as carcinoids and SCLC versus NSCLC. Instead, 95% of the tumors were correctly classified, applying a statistical model created from staining indices of the three general NE markers (chromogranin A, Leu-7, NSE) and three other tumor markers (carcinoembryonic antigen, keratin, vimentin). Because NSCLCs with NE features might have different clinical characteristics than other NSCLCs, immunohistochemistry provides an effective manner to identify this biologically interesting subset of NSCLCs in routine paraffin sections.     

Coexpression of c-kit and bcl-2 in small cell carcinoma and large cell neuroendocrine carcinoma of the lung.

It has been shown that tyrosine kinase oncoprotein c-kit and antiapoptotic molecule bcl-2 are overexpressed in several types of malignancy, including small cell carcinoma (SCLC) and large cell neuroendocrine carcinoma (LCNEC) of the lung. Whether these 2 molecules are coexpressed in lung neuroendocrine tumors has not been investigated. Here, we analyzed immunohistochemical results to determine expression and coexpression patterns of c-kit and bcl-2 in the spectrum of lung neuroendocrine tumors. Using a polyclonal antibody against c-kit and a monoclonal antibody against bcl-2, our data demonstrated that all 7 cases (100%) of SCLC included in this study were positive for both c-kit and bcl-2. Among 14 LCNECs, 7 (50%) stained positive for c-kit and 9 (64%) for bcl-2. All cases of high grade neuroendocrine carcinomas (SCLCs and LCNECs) that showed positive staining for c-kit coexpressed bcl-2. In contrast, all typical and atypical carcinoids (TC and AC) were negative for c-kit, and only 1 of 16 (6.3%) TCs and 1 of 6 (16.7%) ACs stained positive for bcl-2. These results indicate a progressive increase in the frequency of c-kit and bcl-2 expression and coexpression, from carcinoid tumors (TC and AC) to LCNEC and to SCLC. High grade neuroendocrine carcinomas are more likely to coexpress c-kit and bcl-2 when compared with carcinoid tumors. The high frequency of coexpression of these 2 molecules in high grade neuroendocrine carcinomas of the lung suggests that they may be involved in the carcinogenic pathway, given their important roles in carcinogenesis. Therapeutic targeting on both c-kit and bcl-2 molecules might be beneficial in the management of patients with high grade neuroendocrine carcinomas of the lung in the future.     

Tumour suppressor gene products, proliferation, and differentiation markers in lung neuroendocrine neoplasms.

Typical carcinoid, atypical carcinoid, and small cell lung cancer (SCLC) fall within the spectrum of neuroendocrine lung neoplasms. This paper investigates the immunohistochemical expression of the products of tumour suppressor genes p53 and retinoblastoma (RB), together with proliferation (PCNA and Ki67) and neuroendocrine differentiation markers, in 14 typical carcinoids, ten atypical carcinoids, four borderline atypical carcinoid/SCLC, and 11 SCLC. We demonstrated that the phosphoprotein p53 and RB product can be immunolocalized on routine histological material. p53 protein was absent in all typical and atypical carcinoids, while it was abnormally expressed in eight SCLC and one borderline case. RB product was detected in all typical carcinoids and in two atypical carcinoids, while it was consistently absent in the other cases. PCNA-labelled cells were less than 4 per cent in typical carcinoids, about 40 per cent in atypical carcinoids, and over 70 per cent in SCLC. PCNA labelling index discriminates between typical and atypical carcinoids. Neuroendocrine differentiation was evaluated by a semi-quantitative method: a mean score value was obtained, which was high in typical carcinoids, intermediate in atypical carcinoids, and low in SCLC. Our data was obtained, which was high in typical carcinoids, intermediate in atypical carcinoids, and low in SCLC. Our data show that the decrease in neuroendocrine features from typical carcinoid to SCLC is paralleled by an increase in proliferative activity and by an altered expression of tumour suppressor gene products. The above findings have diagnostic relevance.     

Recent advances and current controversies in lung neuroendocrine neoplasms✰

In the lung, neuroendocrine tumors (NETs), namely typical and atypical carcinoids, and neuroendocrine carcinomas (NECs), grouping small cell carcinoma (SCLC) and large cell neuroendocrine carcinoma (LCNEC), make up for distinct tumor entities according to epidemiological, genetic, pathologic and clinical data. The proper classification is essential in clinical practice for diagnosis, prognosis and therapy purposes.Through an extensive literature survey, three perspectives on lung NENs have been revised: i) criteria and terminology on biopsy or cytology samples of primaries or metastases; ii) carcinoids with elevated mitotic counts and/or Ki-67 proliferation rates; iii) relevance of molecular landscape to identify new tumor entities and therapeutic targets. Furthermore, a dispute about lung NEN development has been raised according to emerging molecular models.We herein provide a pathology update on practical topics in the setting of lung NENs according to the current classification (recent advances). We have also reappraised the development of these tumors by modeling risk factors and natural history of disease (recent controversies).Combining recent advances and controversies may help clarify our biological understanding of lung NENs and give practical information for the clinical decision-making process.     

High-grade neuroendocrine carcinomas of the lung express K homology domain containing protein overexpressed in cancer but carcinoid tumors do not

K homology domain containing protein overexpressed in cancer (KOC) is a member of the insulin-like growth factor (IGF) messenger RNA-binding protein family and is expressed during embryogenesis and in certain malignancies. KOC, known as L523S and IGF messenger RNA-binding protein 3, was shown to be frequently expressed in high-grade neuroendocrine carcinomas of the lung in our immunohistochemical studies using a monoclonal antibody against human KOC. Specifically, all 10 small cell lung carcinomas (SCLCs) exhibited strong cytoplasmic staining, 9 with diffuse positivity and 1 with focal positivity. Among 14 large cell neuroendocrine carcinomas (LCNECs), 9 exhibited strong and diffuse cytoplasmic staining, and 5 cases showed focal immunoreactivity. In contrast, no KOC was detected in 21 typical and atypical carcinoids, except for one atypical carcinoid with oncocytic cells showing weak cytoplasmic staining. Although SCLCs exhibited a strong and diffuse staining pattern more frequently (90%) than LCNECs (64%), the difference did not reach statistical significance (P = .3408). Interestingly, our immunohistochemical studies demonstrated that IGF-II, reportedly regulated by KOC, was comparably expressed in SCLC, LCNEC, and typical and atypical carcinoids, irrespective of KOC expression status of the tumors. These results support the formulation that KOC may play an important role in the regulation of biologic behavior of high-grade neuroendocrine carcinomas. In addition, detection of KOC expression may be diagnostically useful in distinguishing high-grade neuroendocrine carcinomas from carcinoid tumors. Our findings of equivalent IGF-II expression in KOC-positive SCLC and LCNEC and KOC-negative carcinoid tumors suggest different regulatory mechanisms involved in the control of IGF-II expression in these tumors.     

Combined adenocarcinoma–atypical carcinoid of the lung. Targeted Next-Generation Sequencing (NGS) suggests a monoclonal origin of the two components

Combined neoplasms of the lung are defined by the WHO classification as an admixture of a small or large cell neuroendocrine carcinoma and a non-neuroendocrine component. However, rare cases of carcinoids combined with a non-neuroendocrine component have been described. These tumor types are not included as a specific entity in the WHO classification of pulmonary neoplasms and their pathogenesis is still unknown. We describe the clinico-pathologic and molecular features of a mixed (combined) lung neoplasm composed of adenocarcinoma and atypical carcinoid. The NGS analysis of the two different tumor components shows a similar molecular profile suggesting their monoclonal origin from a transformed stem/progenitor tumor cell which acquires a divergent differentiation during its development and progression. We also suggest the use of the recently proposed term of mixed neuroendocrine/non-neuroendocrine neoplasm (MiNEN) to define mixed (combined) neoplasms of the lung, because it seems to better cover the wide spectrum of different possible combinations of neuroendocrine and non-neuroendocrine components.     

Apoptosis-related factors p53, Bcl2, and Bax in neuroendocrine lung tumors.

Neuroendocrine (NE) lung tumors comprise four classes of progressive aggressiveness for which proliferation and apoptosis rates could both contribute to their distinctive behavior. As p53 mutations may favor escape from apoptosis through changes in Bcl2-Bax expression balance, which are survival and apoptotic genes, respectively, we studied 121 NE lung tumors (16 typical carcinoids (TC), 5 atypical carcinoids (AC), 29 large-cell NE carcinomas (LCNECs), and 71 small-cell lung carcinomas (SCLCs) using immunohistochemistry. We quantified apoptosis by terminal-deoxynucleotidyl-transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) in 31 of these cases. There was a significant increase of p53 mutant immunophenotype (defined as immunoreactivity with at least two antibodies for at least 20% of tumor cells) between atypical/typical carcinoids group and the LCNEC/SCLC group (P = 0.0003). There was an inverse correlation (P < 0.0001) between the scores of Bax and Bcl2 expression in individual tumors and a significant inversion of the Bcl2. Bax ratio between low-grade (typical and atypical carcinoids) and high-grade (LCNECs and SCLCs) tumors with a predominant Bax expression in the first group and predominant Bcl2 expression in the second. Whereas carcinoids had variable apoptotic indexes, LCNECs had high indexes (1.3 to 6.8%), Bcl2 overexpression, Bax down-regulation, and Bcl2.Bax ratio > 1 correlated with lower apoptotic index in both LCNEC and the pool of LCNECs and SCLCs (P < 0.05) and a lower survival rate in the group of atypical and typical carcinoids and LCNECs (P < 0.002). The highest levels of Bcl2 expression and Bcl2.Bax ratios were associated with p53 mutant immunophenotype (P = 0.02). Our results suggest that aggressiveness in NE lung tumors could be linked, in addition to proliferation, to apoptosis-related factors.     

hASH1基因与肺神经内分泌肿瘤的关系

肺神经内分泌肿瘤(neuroendocrine tumor，NET)是由具有多向分化潜能的肿瘤细胞组成，这些肿瘤细胞具有分泌多种活性激素的功能。肺NET的发生、发展与hASH1(human achaete-scute homologue 1), MEN1(multiple endocrine neoplasia type 1), pRB和E2F1等基因相关，其中hASH1基因成为近年来研究的热点，该基因能促进肺NET的内分泌分化，降低肿瘤的分化程度，并且有临床资料显示hASH1基因的表达还与小细胞肺癌的低生存率有关，可以作为临床预后的一个标准。