Molecular and cellular biology of neuroendocrine lung tumors: Evidence for separate biological entities

Biochimica et Biophysica Acta 1826 (2012) 255–271

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Review

Molecular and cellular biology of neuroendocrine lung tumors: Evidence for separate biological entities Dorian R.A. Swarts a,⁎, Frans C.S. Ramaekers a, Ernst-Jan M. Speel a, b a b

Department of Molecular Cell Biology, GROW — School for Oncology and Developmental Biology, Maastricht University Medical Center, Maastricht, The Netherlands Department of Pathology, GROW — School for Oncology and Developmental Biology, Maastricht University Medical Center, Maastricht, The Netherlands

a r t i c l e

i n f o

Article history: Received 13 March 2012 Accepted 4 May 2012 Available online 10 May 2012 Keywords: Neuroendocrine lung tumors Carcinoids Small cell lung cancer Large cell neuroendocrine carcinoma Carcinogenesis Tumor biology

a b s t r a c t Pulmonary neuroendocrine tumors (NETs) are traditionally described as comprising a spectrum of neoplasms, ranging from low grade typical carcinoids (TCs) via the intermediate grade atypical carcinoids (ACs) to the highly malignant small cell lung cancers (SCLCs) and large cell neuroendocrine carcinomas (LCNECs). Recent data, however, suggests that two categories can be distinguished on basis of molecular and clinical data, i.e. the high grade neuroendocrine (NE) carcinomas and the carcinoid tumors. Bronchial carcinoids and SCLCs may originate from the same pulmonary NE precursor cells, but a precursor lesion has only been observed in association with carcinoids, termed diffuse idiopathic pulmonary neuroendocrine cell hyperplasia. The occurrence of mixed tumors exclusively comprising high grade NE carcinomas also supports a different carcinogenesis for these two groups. Histopathologically, high grade NE lung tumors are characterized by high mitotic and proliferative indices, while carcinoids are defined by maximally 10 mitoses per 2 mm2 (10 high-power fields) and rarely have Ki67-proliferative indices over 10%. High grade NE carcinomas are chemosensitive tumors, although they usually relapse. Surgery is often not an option due to extensive disease at presentation and early metastasis, especially in SCLC. Conversely, carcinoids are often insensitive to chemoand radiation therapy, but cure can usually be achieved by surgery. A meta-analysis of comparative genomic hybridization studies performed for this review, as well as gene expression profiling data indicates separate clustering of carcinoids and carcinomas. Chromosomal aberrations are much more frequent in carcinomas, except for deletion of 11q, which is involved in the whole spectrum of NE lung tumors. Deletions of chromosome 3p are rare in carcinoids but are a hallmark of the high grade pulmonary NE carcinomas. On the contrary, mutations of the multiple endocrine neoplasia type 1 (MEN1) gene are restricted to carcinoid tumors. Many of the differences between carcinoids and high grade lung NETs can be ascribed to tobacco consumption, which is strongly linked to the occurrence of high grade NE carcinomas. Smoking causes p53 mutations, very frequently present in SCLCs and LCNECs, but rarely in carcinoids. It further results in other early genetic events in SCLCs and LCNECs, such as 3p and 17p deletions. Smoking induces downregulation of E-cadherin and associated epithelial to mesenchymal transition. Also, high grade lung NETs display higher frequencies of aberrations of the Rb pathway, and of the intrinsic and extrinsic apoptotic routes. Carcinoid biology on the other hand is not depending on cigarette smoke intake but rather characterized by aberrations of other specific genetic events, probably including Menin or its targets and interaction partners. This results in a gradual evolution, most likely from proliferating pulmonary NE cells via hyperplasia and tumorlets towards classical carcinoid tumors. We conclude that carcinoids and high grade NE lung carcinomas are separate biological entities and do not comprise one spectrum of pulmonary NETs. This implies the need to reconsider both diagnostic as well as therapeutic approaches for these different groups of malignancies. © 2012 Elsevier B.V. All rights reserved.

Contents 1. 2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Clinicopathological characteristics of lung NETs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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⁎ Corresponding author at: Department of Molecular Cell Biology, UNS50-17, GROW — School for Oncology & Developmental Biology, Maastricht University Medical Center, P.O. Box 616, 6200 MD Maastricht, The Netherlands. Tel.: + 31 43 3882998; fax: + 31 43 3884151. E-mail address: [email protected] (D.RA. Swarts). 0304-419X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.bbcan.2012.05.001

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2.1.

Epidemiology . . . . . . . . . . . . . . . . . . . . 2.1.1. Carcinoids . . . . . . . . . . . . . . . . . 2.1.2. High grade lung NETs . . . . . . . . . . . . 2.2. Histopathological classification . . . . . . . . . . . . 2.2.1. Combined tumors of the lung . . . . . . . . 2.3. Therapy of lung NETs . . . . . . . . . . . . . . . . 2.3.1. Carcinoid tumors . . . . . . . . . . . . . . 2.3.2. High grade lung NETs . . . . . . . . . . . . 2.4. Conclusion . . . . . . . . . . . . . . . . . . . . . 3. Early tumorigenesis of pulmonary NETs . . . . . . . . . . . 3.1. Cell of origin . . . . . . . . . . . . . . . . . . . . 3.2. Precursor lesions . . . . . . . . . . . . . . . . . . 3.3. Conclusion . . . . . . . . . . . . . . . . . . . . . 4. Genomic DNA alterations in lung NETs . . . . . . . . . . . . 4.1. Meta-analysis of genomic DNA studies in lung NETs . . 4.2. Deletion of 3p . . . . . . . . . . . . . . . . . . . . 4.3. Deletion of 11q . . . . . . . . . . . . . . . . . . . 4.3.1. MEN1 (11q13) . . . . . . . . . . . . . . . 4.4. Deletion of 13q . . . . . . . . . . . . . . . . . . . 4.5. Conclusion . . . . . . . . . . . . . . . . . . . . . 5. Gene expression profiling in lung NETs. . . . . . . . . . . . 5.1. Other genome-wide approaches in lung NETs . . . . . 5.2. Conclusion . . . . . . . . . . . . . . . . . . . . . 6. Tumor suppressor genes and oncogenes involved in pulmonary 6.1. p53 alterations . . . . . . . . . . . . . . . . . . . 6.2. E-cadherin and β-catenin alterations, and EMT . . . . 6.3. Deregulation of the pRb pathway . . . . . . . . . . . 6.4. Apoptosis-related genes . . . . . . . . . . . . . . . 6.5. Conclusion . . . . . . . . . . . . . . . . . . . . . 7. Models of lung NET tumorigenesis . . . . . . . . . . . . . . 7.1. Pulmonary carcinoids . . . . . . . . . . . . . . . . 7.2. Small cell lung cancer . . . . . . . . . . . . . . . . 7.3. Conclusion . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction Neuroendocrine tumors (NETs) of the lung comprise a heterogenous population of tumors, ranging from well-differentiated bronchial carcinoids to highly malignant and poorly differentiated small cell lung cancer (SCLC) and large cell neuroendocrine carcinoma (LCNEC) (Table 1). In classification systems lung NETs are often represented as a spectrum, and a number of 10 mitoses per 2 mm 2 (this usually equals 10 high power fields) is the criterium defined by the WHO to separate pulmonary carcinoids from neuroendocrine (NE) carcinomas [1]. However, in a clinicopathological sense they behave very differently. Virtually all SCLCs and LCNECs display much higher mitotic indices (average between 60 and 75 mitoses per 2 mm 2) and tumors with intermediate mitotic indices (10–20),

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most often classified as LCNECs, are rare [1]. They grow very rapidly and occur almost exclusively in patients with a history of smoking [2]. Lung carcinoids occur frequently in never-smokers and are subdivided into typical (TC) and atypical (AC) carcinoids [1,3]. TCs and SCLCs are more frequently found to be centrally located in the lung, while ACs and LCNECs more often show a peripheral localization [1]. This review will focus on the similarities and differences between carcinoids and high grade NE lung carcinomas, emphasizing on the molecular pathogenesis, and the present theories concerning the cell(s) of origin of these neoplasms. We will argue that pulmonary carcinoids represent a separate entity of lung NETs rather than being part of a continuum of NE neoplasms. This implies the need to reconsider lung NET diagnosis, where tumors with intermediate mitotic activity should be considered either low grade carcinoids or

Table 1 Clinicopathological and epidemiological characteristics of pulmonary neuroendocrine tumors and precursor lesions.

Mitoses per 2 mm2 (10 HPF) Necrosis Most frequent location Mean age at diagnosis Sex ratio (male:female) % of total number of lung cancers % smokers 5-Year overall survival % lymph node metastasis % distant metastasis

DIPNECH

Tumorlet

TC

AC

LCNEC

SCLC

References

Absent Absent Peripherally 50–60 1:4 NR 37% NR NR NR

Absent Absent Peripherally 60–70 1:>4 NR Unknown NR Low Very low

b2 Absent Centrally 40–50 1:1 1–2% 33%a 92–100% 4–14% 1.5%

2–10 Focal Peripherally 50–60 1:1/2:1 0.1–0.2% 64% 61–88% 35–64% 10%

>10 (median 70) Extensive Peripherally 68 4:1/8:1 1.6–3% 98% 13–57% 40% 65%

>10 (median 80) Extensive Centrally 50–70 1:1 15–20% 97% 5% 90% 60–70%

[1,2,79] [5] [1,86,87] [11,221,222] [10,11,221,223,224] [2,12] [8,11,80,222] [5,12] [5,84,225] [1,8,12,18,81]

Abbreviations used: AC, atypical carcinoid; DIPNECH, diffuse idiopathic pulmonary neuroendocrine cell hyperplasia; HPF, high power fields; LCNEC, large cell neuroendocrine carcinoma; NR, not relevant; SCLC, small cell lung cancer; and TC, typical carcinoid. a Equal to the general population.

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high grade carcinomas, as well as therapeutic approaches for these two groups.

LCNECs and SCLCs are almost exclusively seen in patients with a smoking history [1].

2. Clinicopathological characteristics of lung NETs

2.2. Histopathological classification

2.1. Epidemiology

In 1998, Travis et al. proposed new criteria for the classification of NE lung tumors, which were implemented in the WHO classification of 1999 and renewed in 2004 [3], and which is the current standard for lung NETs. It defines TCs as tumors lacking necrosis and having less than 2 mitoses present per 2 mm 2, and ACs as tumors displaying 2–10 mitoses per 2 mm 2 and/or necrosis (Table 1). Furthermore, this classification system recognizes tumorlets as small carcinoid tumors with a diameter b0.5 cm [1]. High grade lung NETs are defined as tumors displaying more than 10 mitoses per 2 mm 2 and are often characterized by extensive necrosis. LCNECs are not listed as a separate entity, but are still grouped together with other large cell carcinomas [1]. Similar to mitotic indices, carcinoid tumors are characterized by lower Ki67 proliferative indices compared to high grade lung NETs (Table 2) [14,15]. The most important differences between SCLCs and LCNECs are cell size (usually smaller than 3 small resting lymphocytes for SCLCs while larger for LCNECs) and the presence of nucleoli, which are absent or inconspicuous in SCLCs and prominently present in LCNECs [1,16]. The differential diagnosis between high grade lung NETs (especially LCNEC) and non-SCLC (NSCLC) is most commonly based on immunostaining of NE markers including neural cell adhesion molecule (NCAM, also known as CD56), synaptophysin and chromogranin A. More than 90% of all SCLCs are positive for at least one of these NE markers [1].

2.1.1. Carcinoids The incidence of pulmonary carcinoids is low, although reported to have increased over the past 30 years. This is mainly due to improved detection methods and diagnostic protocols [4]. TCs comprise approximately 1–2% and ACs only 0.1–0.2% of pulmonary neoplasms (Table 1). In 2003, their combined incidence was 1.57 per 100,000 inhabitants in the USA. In contrast to high grade lung NETs, carcinoids have a relatively favorable prognosis with a 5-year overall survival of 92–100% for TCs and 61–88% for ACs [5]. However, the only curative treatment for these tumors is surgical resection, and inoperable tumors are difficult to treat because of the insensitivity for both radiation and chemotherapy [6]. Furthermore, recurrence or metastasis can occur decades after resection of the primary tumor. Importantly, the occurrence of carcinoids is generally not related to smoking history [7], although Fink et al. described that for their series of ACs the number of smokers is twice as high as that of the general population (Table 1) [8]. 2.1.2. High grade lung NETs SCLC is the most common lung NET, previously reported to account for 15–20% of invasive lung cancers [9] (Table 1). However, the incidence of SCLC has been declining the last 35 years and was around 13% in 2002 in the US [10]. LCNECs comprise 1.6–3% of resectable lung cancers [11,12]. Both LCNECs and SCLCs are more common in males [2], although the sex-ratio for SCLC has become almost equal to 1 due to the increased proportion of smoking women [10]. SCLC has a very poor prognosis, the median survival upon treatment being 15–20 months for patients with limited stage disease and 8– 13 months for patients with extensive disease [13]. The 5-year overall survival is approximately 5% for SCLCs and varying between 13 and 57% for LCNECs [5,12]. In contrast to pulmonary carcinoids, both

2.2.1. Combined tumors of the lung It was previously thought that combined tumors of the lung were rare and incidences were reported to be ≤5% [17]. It has been realized, however, that the incidence of mixed tumors of the lung may be much higher when surgical specimens instead of biopsies are considered [18]. These tumors are included in the WHO classification and are more frequently found in the peripheral areas of the lung [1,19]. Included in the subgroup of mixed tumors of the lung are not only combinations of SCLC–LCNEC, but also mixtures of either SCLCs or

Table 2 Molecular characteristics that distinguish lung carcinoids from SCLC. Molecular characteristic Number of chromosomal alterations Chromosomal instability Telomerase activity 3p lossa FHIT protein expression 4q lossa 5q lossa 13q lossa pRb downregulation 17p lossa p53 mutation p53 upregulation Bcl-2:Bax ratio > 1 CASP8 methylation β-Catenin dislocation E-Cadherin dislocation CDH1 methylation Fascin protein expression Snail protein expression Ki67 proliferative index Cyclin B1 protein overexpression c-Kit protein expression MEN1 mutation NCAM-PSA protein expression

a

Carcinoids

SCLC

Relation to smoking

References

3.8 Rare 7% (TCs) 10% 100% 5% 4% 14% 9–21% (TC–AC) 7% 6% b 10% 5% 18% 11–41% 17–46% ~ 25% b 35% Weak b 5% 4% 11% 18–36% 53%

14.4 Frequent 93% 92% 0% 79% 79% 83% 90% 73% 90% >50% 90% 41% 90–93% 67–90% ~ 60% 100% Strong >25% 84% 56% 0% 95%

Yes Yes Unknown Yes Yes Unknown Yes Yes Unknown Yes Yes Yes Unknown Unknown Unknown Yes Unknown Unknown Probably Unknown Unknown Unknown Unknown Unknown

[106] [94,96,99] [226] [107] [115]

Abbreviations used: AC, atypical carcinoid; SCLC, small cell lung cancer; and TC, typical carcinoid. a Derived from meta-analysis as described in text and in Supplementary Table 1.

[227] [111] [89,96,197–203] [113] [91,173,174] [175,181] [179] [210,211] [189,190] [189,190] [116] [228] [194] [14,15] [202] [32,37–52] [143,144,146,147] [159–163]

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LCNECs with adenocarcinomas and/or squamous cell carcinomas [1]. It has been shown that the LCNEC part of a combined SCLC–LCNEC can be genetically more closely related to the SCLC counterpart than to other LCNECs [20]. Both tumor types in a mixed tumor have been reported to be of monoclonal origin, indicating that they may arise from the same precursor cell [20–24]. In contrast, combinations of carcinoid tumors with either high grade NE carcinomas or NSCLCs are very rare [17,25,26] and are suggested to be collision tumors rather than having a monoclonal origin, although this remains to be proven by molecular studies [21]. 2.3. Therapy of lung NETs 2.3.1. Carcinoid tumors The only curative treatment for pulmonary carcinoids is radical surgery [27]. The preferred treatment for centrally localized TCs is conservative resection (i.e. sleeve resection, segmentectomy or wedge resection), while for ACs, especially in a peripheral location, often anatomic resection (i.e. [bi]lobectomy or pneumonectomy) is required [4,28]. For aggressive peripherally located ACs, but putatively also for early stage ACs, the use of preoperative chemo- and/or radiation therapy have been suggested to be beneficial [28,29]. However, in the study of Kaplan et al. [29] prognosis of patients with ACs was not improved when pretreated, and in a study by Srirajaskanthan et al. [30] patients had a better disease outcome when surgery was their first treatment option as compared to patients which obtained chemotherapy as a first-line treatment. The use of conventional chemotherapeutics for curative purposes is therefore limited [4]. When applied as a palliative treatment option for patients with unresectable disease, the use of chemotherapeutics as well as somatostatin analogs and peptide receptor radionuclide therapy has proven helpful [30]. Unresectable or metastatic carcinoid tumors are most commonly treated with regimens that are also used for SCLC [31]. However, the response rate of carcinoids for chemotherapy (approximately 22%) is much lower as compared to that of SCLCs, which show response rates up to 90%. Because radiotherapy has so far only been used in combination with chemotherapy, its potential as an independent treatment option in pulmonary carcinoids has still to be determined [28]. It has been reported that tyrosine kinase inhibitors are possible candidates for targeted treatment of metastatic pulmonary carcinoid tumors, because subsets of TCs express c-Kit, PDGFRα, PDGFRβ and EGFR [32]. Furthermore, the mTor pathway has been demonstrated to be functionally activated in a subset of carcinoid tumors, which contained higher levels of phospho-mTor and phospho-S6K proteins as compared to high grade lung NETs [33], making lung carcinoids candidates for mTor inhibition. In 15 out of 24 primary cultures from carcinoid tumors, the mTor inhibitor Everolimus reduced cell viability, as well as chromogranin A and VEGF expression levels [34]. In a prospective study with 60 low-grade NET patients, including four with lung carcinoids, a response rate of 22% to Everolimus was found [35]. Ultimate proof for the optimal treatment of pulmonary carcinoid tumors and the possible use of novel cell signaling pathway inhibitors should come from randomized trials and detailed molecular analyses [36], but such studies have not been published so far. 2.3.2. High grade lung NETs In contrast to pulmonary carcinoids, SCLCs are rarely treated by surgical resection because patients most often present with advanced disease, and tumors are very chemosensitive. The therapeutic options for SCLC are reviewed extensively elsewhere [13]. The first-line treatment of choice is 4–6 cycles of etoposide combined with either cisplatin or carboplatin. Despite very high initial response rates to chemotherapy almost all patients with SCLC relapse, probably as a result of resistance of a small number of insensitive cells, which may include tumor stem

cells. Because of their poor condition second line chemotherapy is only an option in a small number of patients [13]. Targeted therapy of tyrosine kinase receptors also holds promise in SCLC. Especially expression of the c-Kit receptor has been widely studied within SCLCs [37–52]. When combining immunohistochemistry studies, 651 out of 1155 cases (56%) were c-Kit positive. Some of these studies, however, used relatively stringent cut-off points for positivity. The actual frequency of positivity may therefore well exceed 70%. LCNECs displayed similar percentages (147/232; 63%) [12,37,42,48,51,53,54], while carcinoid tumors were only rarely c-Kit positive (19/176; 11%) [32,37,42,43,48,51,52] (Table 2). Also PDGFRβ is frequently expressed in SCLCs [55], while the expression of PDGFRα in SCLC has not been extensively studied [56]. LCNECs express both types of PDGF receptors [12]. A growth inhibitory effect of the tyrosine kinase receptor inhibitor imatinib mesylate (Gleevec, formerly known as STI571), which targets c-Kit as well as PDGFR and the Bcr-Abl tyrosine kinase, was observed in SCLC cell lines [57]. However, within clinical trials, there was a lack of anti-tumor activity, possibly caused by the fact that Gleevec mainly targets tumors with c-Kit mutations, which is a rare event in SCLC [40,58]. Hedgehog-signaling may be an additional target for inhibition, because this pathway is implicated in the development and maintenance of SCLC, and a subset of tumors express sonic hedgehog [59,60]. The optimal treatment for LCNECs has not yet been established because of the limited size of clinical studies conducted so far, but for early stage disease surgery is preferred, while for patients with advanced disease a similar treatment as applied for SCLC, i.e. platinum-etoposide based, is strongly recommended [12]. This improves patient-outcome significantly when compared to regimens used for treatment of NSCLC. 2.4. Conclusion Pulmonary carcinoid tumors are characterized by a low mitotic activity and a considerably better prognosis than high grade NE carcinomas. The latter often exist in combined tumors with a SCLC and LCNEC component, or form combined tumors consisting of a NE and NSCLC component. Molecular studies suggest that these mixed tumors are of monoclonal origin. Carcinoids only rarely combine with other lung tumor types, suggesting different mechanisms of tumorigenesis between carcinoids and high grade lung NETs. With respect to lung NET therapy, there are major differences between the different NET subtypes. While carcinoid tumors are mostly chemoresistant and preferably treated by surgery, high grade lung NETs are initially very chemosensitive, although they usually relapse. These differences further stress the likelihood of a different tumor biology. 3. Early tumorigenesis of pulmonary NETs 3.1. Cell of origin Both pulmonary carcinoids and SCLCs were previously reported to arise from serotonin producing Kulchitsky-type cells (also called Feyrter cells, APUD cells or enterochromaffin cells) by Bensch et al. [61]. These authors termed these bronchial NE cells, which were earlier postulated by Feyrter, ‘bronchial Kulchitsky cells’, based upon similarities with the intestinal Kulchitsky cells observed using electron microscopy [62]. The bronchial Kulchitsky-type cells, currently known as pulmonary NE cells (PNECs), are the first cells to form and differentiate in the epithelium during the earliest stages of lung development [63]. Although they resemble neural cells morphologically, they are from endodermal origin because they can be found in immature fetal epithelium in vitro, and are part of the diffuse NE system [1,64,65]. They usually exist as solitary cells, but sometimes aggregate to form small nodules termed neuroepithelial bodies (NEBs), which are located

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within the ciliated epithelium [4]. PNECs comprise approximately 0.4% of bronchial epithelial cells and play an important dual role, firstly as local modulators of lung growth and pulmonary differentiation during prenatal development, and secondly as airway chemoreceptors during adult life [66,67]. In development, PNECs express serotonin and neuron-specific enolase (at 8 weeks of gestation) and gastrinreleasing peptide (GRP, also known as bombesin) (at 10 weeks of gestation) [66]. In adults, NEBs have been described to respond to hypoxia by secretion of serotonin, thereby inducing local vasoconstriction to decrease the bloodstream in poorly ventilated areas of the lung and thereby directing the blood towards better ventilated areas [68]. The descent of lung NETs from these cells has been under debate and alternatives for their origin have been proposed. It has been suggested that SCLCs could arise from normal bronchial epithelial cells acquiring NE characteristics [1], which is supported by gene expression profiling [69]. Alternatively, a common pulmonary stem cell may exist giving rise to both pulmonary NE stem cells and SCLCs, but also to non-SCLC (NSCLC) tumors, including adenocarcinoma and squamous cell carcinoma (see also Fig. 1) [70]. This latter hypothesis is supported by the existence of combined NE and NSCLC tumors of monoclonal origin. Only recently, in transgenic mice experiments performed by Sutherland et al. [71], different pulmonary cell types were targeted with viral vectors introducing selective p53 and Rb mutations in either (1) Clara cells and bronchioalveolar stem cells (BASCs), (2) PNECs or (3) alveolar type II cells. Although PNECs comprised the dominant cell of origin for SCLCs, these tumors also arose from alveolar type II cells, but not from Clara cells or BASCs. Interestingly, peripheral SCLCs arose only from alveolar type II cells, pointing to these cells as possible cells of origin for

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combined SCLCs–NSCLCs. On the other hand, PNECs only gave rise to centrally located SCLCs. The possibility of the descent of SCLCs from other cell types or from a pluripotent precursor stem cell cannot be excluded and is strengthened by recent evidence that PNECs, Clara cells, ciliated cells and alveolar type II cells are all derived from a common ASCL1 (achaete scute complex homolog 1, also known as ASH1) expressing cell [72]. Further evidence that the different types of lung cancer do not comprise strict entities comes from recent experiments with mouse cell lines, in which adenocarcinomas could arise from SCLCs after expression of oncogenic Ras [73] (Fig. 1). The authors furthermore demonstrated cellular heterogeneity within mouse SCLC tumors, consisting of both small NE cells and larger mesenchymal non-NE cells. They suggested that this tumor heterogeneity improves the adaptivity of the tumor to the environment, thereby providing an advantage to the tumor as a whole. The presence of non-NE cells in mouse SCLCs moreover enhanced the metastatic capacity of the NE cells [73]. Because gene expression profiling showed that SCLC clusters together with LCNEC, and because combined SCLC–LCNEC tumors occur frequently [1,74], we suggest that LCNEC originates from similar precursors as SCLC. However, Nasgashio et al. [75] suggested that LCNEC is more closely related to the non-NETs because they observed lower ASH1 staining intensities and higher intensities of the non-NET specific marker HES1 (hairy/enhancer of split). It remains unclear how carcinoids fit into this scheme (Fig. 1) and which cell type next to PNECs – if any – represents the common ancestor of pulmonary carcinoids and NE lung carcinomas. A case report of carcinoid tumor with features of alveolar type II cells has been

Fig. 1. Current model for the cells of origin of the main types of lung cancer.An ASH1 positive precursor cell is considered a stem cell giving rise to both pulmonary neuroendocrine cells (PNECs), bronchioalveolar stem cells (BASCs) and basal cell progenitors. PNECs give rise to all types of neuroendocrine tumors and BASCs are supposed to be stem cells for both types of alveolar cells and Clara cells. Basal cell progenitors give rise to squamous cell carcinomas. Alveolar type II cells, located in the lung periphery (P) are thought to be the cell of origin for adenocarcinomas, although recent evidence showed the possible descent of a subset of small cell lung cancers (SCLCs) from these cells. They may also explain the existence of combined tumors, at least of the SCLC-adenocarcinoma subtype. Diffuse idiopathic pulmonary neuroendocrine cell hyperplasia (DIPNECH) and tumorlets comprise precursor lesions for (peripherally located) pulmonary carcinoids, while for SCLC and large cell neuroendocrine carcinoma (LCNEC) no precursor lesions have been identified. Atypical adenomatous hyperplasia (AAH) and bronchioalveolar carcinoma in situ (BAC) comprise precursor lesions for adenocarcinoma. Bronchial squamous dysplasia precedes squamous cell carcinoma. Solid lines indicate established relationships between precursor cells and the different normal and abnormal cell types in the lung. Dotted lines indicate hypothetical connections. Additional abbreviations used: Ash1, achaete-scute homolog 1; C, centrally located; NCAM, neural cell adhesion molecule; and NE, neuroendocrine.

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published however, indicating that also these tumors might originate from this cell type, in addition to their origin from PNECs [76]. These important issues should be further addressed because they will provide important insights into the degree of similarity between the different subtypes of lung NETs. 3.2. Precursor lesions Precursor lesions for pulmonary carcinoid tumors comprise diffuse idiopathic pulmonary cell hyperplasia (DIPNECH), a linear proliferation of PNECs or NEBs [1], and tumorlets, extraluminal lesions 2–5 mm in diameter which share histopathological features with carcinoids [77,78] (Table 1). Both types of lesions are reported to originate from the same precursor cell, i.e. the bronchial Kulchitsky-type cells (PNECs) [79]. DIPNECH is considered a precursor for carcinoids and tumorlets as concluded from the fact that these lesions are almost always present in patients with this hyperplastic condition [80]. Although only TCs were associated with DIPNECH in early studies, it was recently also described in the context of ACs and in a patient with the MEN1 syndrome (discussed below) [80]. When proliferating PNECs break through the basal membrane to invade locally, they form small aggregates which are known as tumorlets. By definition, a tumorlet >5 mm in diameter is termed carcinoid [77]. Although they were originally described as being of benign nature, tumorlets behave like carcinoids, are reported to cause Cushing's syndrome [81–83], and may metastasize to lymph nodes [82,84] or even distantly [81]. Reactive PNEC hyperplasias resulting from chronic pulmonary disease can also progress to a tumorlet phase. However, these do not progress towards carcinoid tumors in contrast to tumorlets related to DIPNECH, and were recently reported to be also negative for Ki67 and p53 immunostaining, while DIPNECH associated tumorlets and carcinoid tumors were positive [85]. The same authors also showed protein expression of the peptide inactivator MME (membrane metallo-endopeptidase, also known as CALLA and CD10), in DIPNECH but not in PNECs [85]. Importantly, both DIPNECH [86] and tumorlets [87] are associated with a peripheral localization of carcinoids, indicating that they are not necessarily precursor lesions for centrally located carcinoid tumors. No precursor lesions for high grade lung NETs have been described, but genomic changes typical for these high grade lung NETs have been observed in phenotypically normal cells [78,88] (Fig. 1). 3.3. Conclusion Both carcinoids and SCLCs can originate from PNECs or Kulchitskytype cells, while in addition experiments with transgenic mice showed that SCLCs may also arise from alveolar type II cells or a multipotent ASH1 positive precursor cell. The possibility that carcinoid tumors may also arise from these latter cell types remains to be further investigated. DIPNECH is considered a precursor lesion for (peripherally located typical) carcinoids, while precursor lesions for SCLC or LCNEC have so far not been identified. Therefore we conclude that pathways of tumorigenesis for lung carcinoids on the one hand and NE carcinomas on the other are largely unrelated (Fig. 1). 4. Genomic DNA alterations in lung NETs 4.1. Meta-analysis of genomic DNA studies in lung NETs Differences in the extent of genetic alterations between lung carcinoids and NE carcinomas may underline the differences in the aggressiveness of these lesions. Several genome-wide (array) comparative genomic hybridization (CGH) studies as well as other genetic approaches have been conducted to identify chromosomal alterations in pulmonary carcinoids [89–96], high grade lung NETs [97–102], or both [98,103–105]. For the underlying study we performed a meta-analysis

of the specific genomic alterations in lung NETs using those studies in which information for individual tumors was available. As a result, we included 87 TCs, 38 ACs, 33 LCNECs, 48 SCLCs and 11 unclassified high grade lung NETs. The major results are depicted in Fig. 2 and an overview of the frequencies of chromosomal alterations is provided as Supplementary Table 1 [89–91,93,94,96,97,99–101,103,104]. We have chosen to define chromosomal alterations in array CGH studies as copy number alterations >10 Mb, to be able to compare these studies with conventional CGH studies. Chromosomal alterations >10 Mb are much more frequent in SCLCs (on average 18.8 aberrations per tumor) and LCNECs (13.7) as compared to ACs (6.1) and TCs (2.8). Smoking habits may to a large extent explain these differences. Exposure to benzo[a]pyrene, an important carcinogen in tobacco smoke, and its metabolic activation into benzo[a]pyrene diol epoxide (B[a]PDE), which covalently binds DNA, can induce a DNA synthesis block leading to aberrant centrosome amplification [106]. This leads to chromosomal instability in lung cancer cells with abrogated p53 function. In other words, smokers have a higher risk for developing chromosome instable tumors when their p53 function is already disturbed. This is reflected by much higher numbers of chromosomal aberrations in the smoking-associated high grade lung NETs and very low frequencies in TCs. Fig. 2A shows the ten most frequently occurring chromosomal alterations for the high grade NE carcinomas (LCNEC and SCLC) in comparison to the combined group of carcinoids. In general, SCLCs display higher numbers of chromosomal aberrations compared to LCNECs. Although most alterations occur frequently in both high grade tumor types, it has to be noted that loss of chromosome 17p (harboring the p53 gene) and gain of 3q are twice as frequent in SCLCs as compared to LCNECs. The most frequently occurring alterations in high grade lung NETs are only infrequently found in the carcinoids. In Fig. 2B the most prevalent aberrations for pulmonary carcinoid tumors (TC and AC) are depicted in comparison to the combined group of high grade lung NETs. With the exception of 3p (discussed below) and 13q losses, different chromosomal alterations than for the high grade NE carcinomas are most abundant in pulmonary carcinoid tumors. However, even for these alterations, frequencies are usually higher for high grade lung NETs (Supplementary Table 1), with the exception of 11q deletions (discussed below). 4.2. Deletion of 3p The most important chromosomal alteration characteristic of NE lung carcinomas, but infrequently present in lung carcinoids, is a deletion of chromosome 3p (Fig. 2A, Table 2), which is often followed by overrepresentation of chromosome 3q, frequently arranged in an isochromosome formation [1]. The fact that 3p allelic loss is almost universal in the pathogenesis of both SCLC and NSCLC [107] underlines the unique position of carcinoids within the spectrum of lung tumors. Chromosome 3p deletion has been shown to be one of the earliest events in lung cancer and is highly related to smoking. This is similar to the situation in head and neck squamous cell carcinoma [108]. Deletions of 3p precede other chromosomal alterations, such as deletions of 5q, 9p and 17p [109]. Loss of heterozygosity (LOH) at several 3p regions has been found in normal bronchial epithelium of smokers, but not in never-smokers [107]. Gorgoulis et al. [110] showed that hyperplasias preceding NSCLCs obtain allelic imbalances at the common fragile site FRA3B (harboring the FHIT gene, 3p14.2) very early during tumorigenesis. Although they concluded that these imbalances are preceding other losses at this chromosome arm, Wistuba et al. [107] did not identify LOH at the FHIT region in non-dysplastic bronchial epithelium from smokers without lung cancer, while loss of 3p21.3 was present in 59% of smokers without lung cancer [109,111]. Putative tumor suppressor genes located at chromosome 3p are identified in four widely separated areas and include DUTT1 at 3p12, ROBO1 at 3p12–13, FHIT at 3p14.2, BAP1, CTNNB1, FUS1, HYAL2, MLH1, RASSF1A, SEMA3B and SEMA3F at 3p21.3 and VHL and RARB at 3p24-6 [1,112,113]. FHIT is a target of carcinogens

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Fig. 2. Meta-analysis of the major chromosomal alterations in pulmonary neuroendocrine tumors.(A) The ten most frequently occuring chromosomal abnormalities in high grade lung neuroendocrine (NE) tumors are shown, and depicted separately for large cell neuroendocrine carcinoma (LCNEC, n = 33) and small cell lung cancer (SCLC, n = 48). Data are taken from Supplementary Table 1 and the aberrations are sorted left to right according to their frequency in the total group of high grade NETs (n = 92). For comparison, the frequency of these chromosomal alterations in the combined group of carcinoid tumors (n = 125) is included.(B) The ten most frequently occuring chromosomal aberrations in typical carcinoids (TC, n = 87) and atypical carcinoids (AC, n = 38) are shown. Data are arranged from left to right according to the frequency of genomic aberrations in the total group of carcinoid tumors (n = 125). For comparison, the frequency of these chromosomal alterations in the combined group of high grade lung NE tumors (n = 92) is depicted.

present in tobacco smoke and LOH at its region is only very rarely observed in pure non-smokers [114]. Although deletions at this locus have also been observed in carcinoid tumors, FHIT protein expression in this subgroup seems to be retained (Table 2) [115]. Also deletion of hMLH1 (3p21) is strongly associated with smoking [113]. RARB (~50%) and RASSF1A (~85%) are frequently methylated in SCLCs but less often in carcinoids [116]. It has been shown that methylation of these genes in immortalized human bronchial epithelial cells (HBECs) is caused by exposure to cigarette smoke condensate, and that for RASSF1A this was associated with a decrease in gene expression [117]. Also inactivation of genes on other frequently deleted chromosomal regions may reflect the effect of smoking on these tumors. For example, deletion of hMSH3 located at 5q11–13 is reported to be strongly associated with exposure to tobacco carcinogens [113]. 4.3. Deletion of 11q Deletion of 11q is the most frequently observed alteration in pulmonary carcinoids (Fig. 2B and Supplementary Table 1), and deletion of its telomeric part is associated with ACs and an important prognostic factor for these tumors [96]. Since 11q is less frequently lost in TCs

as compared to ACs, it could represent a marker of tumor progression rather than tumor initiation [92,96]. Similar results were reported for neuroblastomas [118] and insulinomas [119], the latter being grouped together as foregut NETs with other pancreatic NETs, thymic carcinoids and bronchial carcinoids. In contrast, chromosome 11q deletions are less common and usually smaller in gastrointestinal carcinoids, which display allelic losses at chromosome 18q more frequently [92,95,120]. Deletion of 11q is the only chromosomal aberration for which the frequency of loss is comparable in carcinoids (28%) and NE carcinomas (32%). It has been previously suggested that 11q deletions reflect that these tumors arise from a pulmonary tissue, because they have also been reported in lung adenocarcinomas [121,122]. However, 11q deletions probably represent a more general hallmark of tumor biology, because they are also involved in many other solid tumors, including cancers from bladder, breast, cervix, colorectum, stomach, nasopharynx, ovary and prostate, as well as intracerebral neoplasms and malignant melanomas [123]. Similar to what we observed for lung carcinoid tumors [96], in these cancers the deletions in 11q are mostly centered around the regions with the most dense concentration of genes, i.e. 11q13, 11q22– q23 and 11q24, and only rarely a deletion of the whole chromosome arm is seen [123]. Next to the MEN1 gene, which is discussed below,

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several (candidate) tumor suppressor genes have been identified at these regions, including ADAMTS8 (11q24.3), which is inactivated by hypermethylation in NSCLC [124], ATM (11q22.3) [125], CADM1 (11q22.3, also known as TSLC1), methylated in NSCLC and downregulated in neuroblastomas [126,127], and SDHD (11q23.1), frequently mutated in paragangliomas and phaeochromocytomas [128]. We did not observe absence of SDH immunohistochemical staining, indicative of mutation [128], in lung carcinoids (our unpublished results). Amplification of 11q13 and associated gain of the gene encoding cyclin D1, a very frequent event in non-HPV related head and neck squamous cell carcinoma [129] and also commonly involved in NSCLCs [130,131], has only been reported in a single SCLC case report, and never in carcinoid tumors [132]. 4.3.1. MEN1 (11q13) Since its discovery in 1997 as the gene responsible for the familial hereditary syndrome multiple endocrine neoplasia type 1 (MEN1), MEN1, located at 11q13, has been widely studied within the NET spectrum. Menin is a complex tumor suppressor protein with many different functions in multiple pathways. The protein plays important roles in cell cycle control, apoptosis and maintenance of genomic stability, but functions also as a transcription factor [133]. Menin inhibits for example the S-phase activator ASK and can convert the growth promoting JunD protein to a growth suppressor by binding directly to it [133]. It can also activate CASP8 and repress IGFBP2 expression [134]. Moreover, Menin has been reported to be a member of multiple histone remodeling complexes [135,136]. By binding to the Polycomb group protein EZH2 (Enhancer of Zeste homolog 2) it enhances histone H3K27 methylation, an epigenetic repressive mark. In this way Menin represses the PTN gene (an activator of the ALK signaling pathway) and as a result inhibits proliferation [135]. More profoundly studied is the role of Menin in the histone H3K4 trimethylation complex. Menin interacts with a member of this complex, i.e. MLL (mixed lineage of leukemia), which is often translocated in the context of human leukemia [136], but not in carcinoid tumors (Swarts, unpublished observations). The complex consists furthermore of Ash2, Rbbp5 and WDR5. The H3K4 trimethylation mark is an activating epigenetic mark and this complex stimulates gene transcription of the tumor suppressor genes p18, p21 and p27 [137,138]. Patients with the MEN1-syndrome have an inherited mutation of the MEN1 gene, predisposing them to the formation of multiple NETs. MEN1 gene mutations have been reported to occur scattered throughout the gene [139], although often the two nuclear localization signals at the N-terminal part of the Menin protein are affected. This region is crucial for the functioning of Menin and is involved in its binding to DNA [133]. NETs that arise in association with the MEN1-syndrome comprise cancers of the parathyroid and pituitary, foregut NETs, including bronchial and thymic carcinoids, and enteropancreatic tumors such as gastrinomas and insulinomas [140,141]. However, neither SCLCs nor LCNECs have been reported in the context of the MEN1-syndrome, while in ≥5% of patients with the MEN1-syndrome bronchial carcinoids do appear [142]. Because they are commonly observed in relation to the MEN1-syndrome, mutations of the MEN1 gene have been investigated in sporadic pulmonary carcinoids [143,144]. The frequency of sporadic lung carcinoids that actually display MEN1 gene mutations is approximately 18%, but the percentage of LOH of the MEN1 region at chromosome 11q13 is higher (approximately 36%). This suggests that MEN1 can be silenced in other ways, for example by transcriptional regulation or epigenetic mechanisms, although we did not observe promoter hypermethylation in lung carcinoids [145]. Finkelstein et al. [77] reported extremely high frequencies of LOH (73%) at 11q13 in TCs, but also showed almost complete absence of allelic imbalance at this region in tumorlets, indicating that LOH at 11q13, like 11q loss, may represent a relatively late event in the tumorigenesis of carcinoids. Importantly, MEN1 mutations are largely absent in sporadic NE lung carcinomas

(Table 2) [146,147], further underscoring differences in carcinogenesis between pulmonary carcinoids and these high grade lung NETs. 4.4. Deletion of 13q Next to 11q deletions, the other important shared alteration between pulmonary carcinoids and NE carcinomas is deletion of chromosome 13q, although present at much higher frequencies in the latter group (Fig. 2). The pRb (retinoblastoma) gene is located at this chromosome arm at 13q14 (see below at Section 6.3). Other tumor suppressor genes at chromosome 13q include BRCA2 at 13q12.3, for which low expression levels in lung adenocarcinoma have been demonstrated to be associated with promoter hypermethylation [148], and ING1 (also known as p33) at 13q34, which is a modulator of p53 transcriptional activity towards e.g. p21, and has been reported to be downregulated in NSCLC [149,150]. Deletion of 13q might be a relatively late event in tumorigenesis because it is more often involved in metastatic SCLCs than in their primary counterparts [102], and has been suggested to be involved in aggressive carcinoid cases [89,96]. 4.5. Conclusion High grade lung NETs are characterized by a considerable higher number of chromosomal alterations as compared to pulmonary carcinoid tumors. Carcinogens present in tobacco smoke are a major cause of both a generally higher number of chromosome aberrations as seen in the high grade NE carcinomas, and the occurrence of specific alterations, e.g. chromosome 3p deletion. This deletion represents one of the earliest events in tumor biology of high grade lung NETs and is almost exclusively associated with smoking. Many tumor suppressor genes located on this chromosome arm are implicated in the carcinogenesis of SCLCs. Deletion of chromosome 11q (containing the MEN1 tumor suppressor gene) is the only chromosomal alteration present in a considerable frequency in carcinoid tumors. Although the frequency of 11q deletions in high grade lung carcinomas is similar to that in carcinoid tumors, direct involvement of MEN1 seems to be restricted to pulmonary carcinoids. 5. Gene expression profiling in lung NETs A limited number of studies describe genome wide gene expression profiling of NE lung carcinomas [69,74,151–157], and only a few of these reports included pulmonary carcinoids [69,74,151,153,156]. Furthermore, only a few genes have been reported to show an altered expression level in either carcinoids, LCNECs or SCLCs by more than one study. Anbazhagan et al. [69] showed that after hierarchical clustering of gene expression profiles of two carcinoids, two SCLCs and two brain tumors (astrocytoma, oligodendroglioma), the carcinoids clustered together with the brain tumors, while the SCLCs were more closely related to normal bronchial epithelium. Bhattacharjee et al. [151] reported that six SCLCs and 20 carcinoids shared only few markers, while a distinct group of genes was specifically associated with carcinoid tumors. Jones et al. [74] proposed an alternative classification of 25 high grade lung NETs based on expression profiling and showed that these tumors clustered in two prognostically different subgroups independent of classification as LCNEC or SCLC. Carcinoids were shown to cluster separately from high grade NETs in this study. In the study by He et al. [153] three SCLCs and two LCNECs clustered separately, but more closely to each other than to 11 carcinoid tumors. Bhattacharjee et al. [151] showed that SCLCs and carcinoids shared high-level expression of NE genes such as INSM1 (insulinoma-associated 1), ASCL1/ASH1 and CHGA (chromogranin A). The importance of expression of these genes for SCLC was confirmed by Taniwaki et al. [155], who also showed that 15 SCLCs clustered separately from NSCLCs. In contrast to the study of Bhattacharjee et al. [151], Jiang et al. [158] observed low frequencies of ASH1 expression in TCs in comparison to the poorly

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differentiated NETs, and concluded that ASH1 in lung NETs may imitate the role that has been attributed to this protein in fetal development. As such it may only be required to initiate and establish the endocrine character and not to maintain this phenotype in TCs. This also implicates that ASH1 is associated with the less well differentiated tumors such as SCLCs [158]. On the other hand, immunohistochemistry for chromogranin A has been reported to be more often positive for carcinoids than for high grade lung NETs [159]. In the study by Jones et al. [74], TCs and the poor prognosis subgroup of high grade lung NETs shared high expression of NRXN1 (neurexin) and NCAM1, an important diagnostic NET marker. While NCAM1 has been reported to be overexpressed in both SCLCs and carcinoids [74,154], and the NCAM (CD56) protein is an important NE marker which is used as a standard in the classification of lung NETs next to chromogranin A and synaptophysin [1], NCAM can be post-translationally modified in several ways, the most important modification being the addition of α2,8-linked polysialic acid (PSA). This has a negative effect on the cell–cell interactions mediated by the NCAM protein. In several immunohistochemical studies the NCAM-PSA variant was reported to be more often present in SCLCs (159/167 cases positive) than in pulmonary carcinoids (66/125 cases positive) [159–163] (Table 2). While the large majority of SCLCs and LCNECs has been reported to be positive for this variant, TCs are mostly negative, while ACs display variable frequencies of positivity [162,163]. 5.1. Other genome-wide approaches in lung NETs Next to gene expression profiling and (array) CGH, few genome-wide approaches have been applied to lung NETs. Pleasance et al. [164] performed next-generation sequencing on a SCLC cell line and identified smoking-specific events as well as the recurrent rearrangement of the chromatin remodeler CHD7 (located at the frequently amplified 8q chromosome arm; see also Fig. 2) in additional SCLC cell lines. Based on microRNA expression profiling, Du et al. [165] observed more differences between SCLC cell lines and immortalized normal HBECs than between NSCLC cell lines and HBECs. Genome-wide methylome analyses remain to be conducted on NETs of the lung. 5.2. Conclusion In gene expression profiling studies LCNECs and SCLCs have both been reported to cluster either separately or together, but in general more closely towards normal bronchial epithelium, while carcinoids group always together in a different cluster, which is more closely related to brain tumors than to the NE lung carcinomas.

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6.1. p53 alterations Mutations in the p53 gene are more common in lung cancers from smokers as compared to non-smokers, and the type of mutation in smoking patients differs from that in non-smoking patients [168]. Lung cancers from smokers are characterized by G → T transversion mutations (30% in smokers vs. 12% in non-smokers), with hotspots at codons 157, 158, 248 and 273. Mutations in the latter two codons are present in cancers from both smokers and non-smokers, but differ considerably in their frequencies (35–45% G → T transversions in smokers and almost no G → T transversions in non-smokers) [169]. Importantly, it has been shown that exposure to BPD[a]E induces G → T transversions at p53 codons 157, 248 and 249 [170]. Mutations in codons 248 and 273 are crucial because the amino acids that they encode are involved in the interaction of p53 with DNA [171]. Codon 157, 248, 249 and 250 mutations are reported to be present in both histologically normal lung tissues adjacent to cancers of smokers, as well as in lung tissues of smokers without lung cancer [170], indicating that p53 mutations are early events in lung cancer tumorigenesis. Mutant p53 immunophenotype has indeed been identified in precursor lesions of NSCLC [172]. The protein expression levels of p53, the mutation frequencies of the p53 gene and the percentage of LOH at the p53 locus differ greatly between lung carcinoids and high grade lung NETs. Mutations of p53 are reported to be rare or absent in carcinoids [173–175], but are present in up to 90% of high grade lung NETs [91] (Table 2). The type of mutation is indeed often G → T for SCLCs [176,177], while three of the four reported p53 mutations in carcinoids were G → A transversions [173,174]. Also the frequency of LOH is rising considerably from carcinoids towards NE carcinomas [178]. Similarly, there is a significant increase of p53 protein expression indicative of mutation when comparing carcinoids to lung carcinomas [179]. Although carcinoids, and particularly ACs, are sometimes immunopositive for p53, its expression pattern is normally focal and sometimes patchy in aggressive ACs, and rarely exceeds 10% of tumor cells [175,180,181]. High grade NETs show a more profound p53 expression (≥50% of the tumor cells positive) and are only rarely p53 negative [175]. Mutations of p53 can be present in dysplasia related to NSCLC [110], and immunostaining in b10% of the cells has been observed in tumorlets and carcinoid tumors related to DIPNECH, but not in early intraepithelial phases of PNEC proliferation [85]. In addition to p53 abrogation, alterations of its main regulator Mdm2 may be involved in carcinogenesis. Eymin et al. [182] detected overexpression of Mdm2 protein in approximately 30% of cases in carcinoid tumors, high grade lung NETs and NSCLCs. However, there is no evidence for amplification of this locus in lung NETs.

6. Tumor suppressor genes and oncogenes involved in pulmonary NET biology

6.2. E-cadherin and β-catenin alterations, and EMT

Several tumor suppressor genes and oncogenes have been related to pulmonary tumorigenesis, including Bcl-2 family members, CDH1 (E-cadherin), FHIT (as described above) and p53. Disturbances in the proper functioning of these genes and their encoded proteins are often induced by inhaled mutagenic compounds. For example, cigarette smoke consists of more than 4800 different compounds from which at least 60 are carcinogenic [166,167]. Exposure to cigarette smoke provokes a process of ‘field cancerization’ yielding different mutations and genetic alterations, thereby promoting the development of multiple primary (pre)malignant lesions as well as the occurrence of tumors of different subtypes adjacent to each other or existing as combined tumors. The substantial differences between pulmonary carcinoids and high grade lung NETs (reviewed below and summarized in Table 2) with regard to gene and protein expression levels, and mutations of important tumor suppressor genes and oncogenes may thus reflect differences in the exposure to tobacco carcinogens.

Recently, it has been shown that epithelial to mesenchymal transition (EMT), an important step in cancer progression and metastasis, can be induced by carcinogens present in tobacco smoke [183,184]. The process is characterized by a decreased E-cadherin expression and increased expression of tyrosine kinase receptors, including EGFR, FGFR and IGFR [185]. EGFR does not play a major role in the tumor biology of pulmonary NETs, in contrast to its role in NSCLC and in particular adenocarcinoma, where its mutations in exons 18–21 in 20–30% of cases have therapeutic implications [186]. Although a subset of carcinoid tumors (48%) is EGFR positive, they are pEGFR negative and do not have EGFR mutations [32,187]. EGFR mutations have been reported in approximately 3% of SCLC patients, mostly in non-smokers [188]. The involvement of FGFR and/or IGFR signaling in pulmonary NETs remains to be investigated. E-cadherin is a transmembrane glycoprotein that interacts with β-catenin, a main transducer of Wnt-signaling to the nucleus. Loss or dislocation of E-cadherin causes disturbance of cell–cell adhesion leading to

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detachment of tumor cells and the start of migration of these cells through the basal lamina into the stroma [183]. E-cadherin protein is expressed in most, and β-catenin in all lung NETs, although a disruption of normal membrane-associated expression of these proteins is seen (see also Table 2) [189,190]. This dislocation of E-cadherin and β-catenin to the cytoplasm gradually increases both in frequency and in severity from low-grade TCs to high grade SCLCs [189,190]. It has been shown that the Menin protein binds directly to β-catenin and transports it out of the nucleus via nuclear-cytoplasmic shuttling [191]. Thereby it suppresses Wnt-signaling, which is stimulated by nuclear β-catenin. Furthermore, increase of the Menin protein concentration leads to accumulation of both β-catenin and E-cadherin at the surface of pancreatic islet cells, mediated by the scaffold protein IQGAP1, thereby increasing cell–cell adhesion [192]. Because alterations of Menin have been described in pulmonary carcinoids but not in high grade lung NETs (see above), the dislocation and deregulation of both β-catenin and E-cadherin expression in lung carcinoids might be explained by these functions of the Menin protein. In high grade lung NETs, dislocation of E-cadherin and reduction of its expression may be explained by effects of tobacco smoke. Nuclear protein expression of Snail, an inducer of EMT and direct transcriptional repressor of the E-cadherin gene CDH1 [193], was intense in high grade lung NETs, while weak for TCs [194]. These expression levels were inversely correlated with E-cadherin protein levels and were (borderline) significantly related to smoking. Snail can be induced by reactive oxygen species, which have been described to form after exposure of lung epithelial cells to tobacco smoke [185]. It is known that both DNMT1, HDAC1 and the repressive chromatin mark H3K9me2 are recruited to sites of DNA damage. After exposure of immortalized human bronchial epithelial cells to the common tobacco carcinogens methylnitrosourea (MNU) and B[a]PDE, expression levels of DNMT1 are rising leading to methylation of several adhesion molecules, including H-cadherin (CDH13) [195]. Furthermore, both CDH1 and three microRNAs (i.e. miR-200b, miR-200c, and miR205), which are indirect activators of CDH1 expression, are epigenetically silenced. This is accomplished by a decrease of transcription promoting chromatin marks (H3K4 methylation) and recruitment of repressive marks (H3K9me2, H3K27me3), and followed by de novo hypermethylation of these genes by DNMT1 [184,195]. Methylation of CDH1 has also been reported in primary lung tumors, in both NSCLCs and NETs. The frequency of CDH1 methylation is more than two times as high in SCLCs (~60%) as compared to carcinoids (~25%) [116]. Loss of CDH1 expression upregulates NCAM which is involved in the initiation and maintenance of EMT and is highly expressed in SCLCs [196]. On the other hand, abrogation of NCAM can reverse EMT. Thus, downregulation of CDH1 expression in response to tobacco consumption is established in at least three different ways, i.e. 1) epigenetic silencing of the CDH1 promotor region by chromatin remodeling and de novo methylation by DNMT1, 2) repression of CDH1 activating microRNAs by similar mechanisms, and 3) upregulation of Snail. Moreover, ASH1 (described above) represses CDH1, further highlighting the importance of E-cadherin, EMT and Wnt signaling in the tumor biology of lung cancer. The induction of EMT by tobacco carcinogens is important because it may give an explanation for the dedifferentiated phenotype of malignant lung carcinomas and might explain differences with pulmonary carcinoids, which have a more differentiated phenotype and are usually not related to smoking. 6.3. Deregulation of the pRb pathway A number of immunohistochemistry studies have evaluated protein expression of pRb in NE lung tumors [89,96,197–203]. In general, pRb is gradually lost when comparing more differentiated NETs with poorly differentiated ones. Taken together, absence of pRb protein expression has been reported in 15/159 TCs, 16/76 ACs, 63/102 LCNECs and 209/232 SCLCs (Table 2). Onuki et al. [178] showed LOH at 13q14.2 in 20% of TCs, 22% of ACs, 62% of LCNECs and 71% of SCLCs,

in agreement with frequencies of 13q loss concluded from our meta-analysis of (array) CGH studies (Supplementary Table 1). Four carcinoid cases described in one of these studies retained pRb expression despite loss of 13q [89]. In addition to loss of pRb, Eymin et al. [204] reported overexpression of E2F1 in 81% of high grade NE carcinomas, while carcinoids were not analyzed. Nuclear immunostaining for p16 has been detected in the whole spectrum of lung NETs, including DIPNECH and tumorlets, and cyclin D1 overexpression is only rarely detected [85,198]. The frequencies of tumors showing cyclin D1 and/or p16 expression were not significantly different between the lung NET subgroups [198]. 6.4. Apoptosis-related genes Because bronchial carcinoids have a very low proliferation rate, evasion from apoptosis has to be essential for these tumors to expand. However, the intrinsic apoptosis pathway, including the BCL2, BCL2L1 (Bcl-X) and BAX genes, is more often inhibited in the high grade NETs, and in particular SCLCs display very low apoptotic indices [179]. Although Brambilla et al. [179] reported variable apoptotic indices in both TCs and ACs, Laitinen et al. [180] demonstrated higher apoptotic activity in ACs as compared to TCs. LCNECs showed high (1–7%) apoptotic indices, especially when compared with SCLCs (b0.1%) [179]. Several immunohistochemical studies have been performed examining this pathway in lung NETs [96,179–181,203,205–209]. In a study by Brambilla et al. [179] there was an inversion in the Bax/ Bcl-2 ratio towards the antiapoptotic (Bcl-2) side in high grade lung NETs, i.e. 90% in SCLC, compared to 5% in carcinoids (Table 2). Other studies showed comparable results, with an almost intact intrinsic apoptotic pathway in TCs, a moderately affected pathway in ACs and a largely compromised pathway in the high grade lung NETs. Similar to expression of mutant p53, upregulation of Bcl-2 and downregulation of Bax, resulting in an inverted Bax/Bcl-2 ratio, was observed in preneoplastic lesions adjacent to lung cancers [85,172]. Data are less comprehensive with regard to alterations of the extrinsic apoptotic pathway in lung NETs. The Fas, TRAIL-R1 and TRAIL-R4 components have been reported to be absent in SCLC tumors and cell lines [210]. This was demonstrated to be at least partly due to epigenetic inactivation, because FAS and TNFRSF10A (TRAILR1) were methylated in 40% of SCLC samples tested. Moreover, also the downstream target caspase-8 has been reported to be hypermethylated in 35–52% of SCLCs and 79% of SCLC cell lines [210,211]. All cell lines that were methylated for CASP8 lost mRNA expression [211]. A homozygous deletion of the gene locus of the CASP8 gene has previously been shown in a SCLC cell line [212]. Also in carcinoid tumors, CASP8 is methylated in 18% of cases (Table 2), although decrease of gene expression remains to be studied [211]. Interestingly, Menin has been reported to upregulate CASP8 gene expression by binding to its locus, thus inducing apoptosis [213,214]. The other components of the extrinsic apoptotic pathway remain to be examined in lung carcinoids. 6.5. Conclusion With regard to alterations of well-characterized tumor suppressor genes and oncogenes in lung NETs, it can be concluded that p53 aberrations are infrequent in carcinoids but are very common in high grade NETs which is in line with the fact that they are much more common in lung cancers from smokers as compared to nonsmokers, especially with respect to G → T transversion mutations. Dislocation of the EMT-related proteins E-cadherin and β-catenin is observed in both carcinoids and high grade lung NETs, but the frequencies and severity are higher in the latter group. The pRb pathway is significantly more often altered in high grade lung NETs than in carcinoids. Although evasion of apoptosis is likely to play a major role in the tumorigenesis of slowly proliferating carcinoids, at least the

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intrinsic apoptosis pathway is more often abrogated in high grade lung NETs. 7. Models of lung NET tumorigenesis Although many studies have stressed the importance of several individual genes and pathways involved in the tumorigenesis of lung NETs, building a comprehensive model for their successive steps in carcinogenesis remains very challenging. From the foregoing it may be evident that we postulate two separate lines of tumorigenesis for pulmonary NETs, one for the carcinoids, and another for the high grade carcinomas, and that carcinoid tumors are certainly not precursor lesions for the latter. It has become more and more apparent that processes of tumorigenesis in the lung depend on their localization and that therefore tumors arising in different parts of the lung may have different cells of origin and/or mechanisms of carcinogenesis. This may well be true for pulmonary carcinoids, for which peripherally located tumors are associated with DIPNECH and tumorlets, while centrally located tumors often arise in the absence of these precursor lesions. Similarly, for SCLC, transgenic mouse studies have suggested that PNECs give only rise to centrally located tumors, while alveolar type II cells may give rise to both centrally and peripherally located SCLCs [215]. When combining molecular and clinicopathological features described in this review (see also Table 2), a model for pulmonary NET carcinogenesis emerges (Fig. 3). An Ash1-positive pulmonary precursor cell may exist that gives rise to PNECs, BASCs and basal

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cell progenitors and possibly also directly to (precursors of) lung NETs (Fig. 1). However, only PNECs are firmly established as the cell of origin for both carcinoid tumors and SCLCs. In the next two paragraphs these two routes of lung NET carcinogenesis will be elaborated. 7.1. Pulmonary carcinoids As discussed above, pulmonary carcinoids (depicted at the right side of Fig. 3) comprise a group of neoplasms that is characterized by a low proliferative index. We have chosen to group TCs and ACs together because of their supposed common origin and the lack of clear discriminative markers for these two subtypes. It still needs to be clarified how the development of PNECs towards carcinoids is initiated and which genes are disturbed during this process. Most likely, at least peripherally located lung carcinoids arise from a linear expansion of PNECs or NEBs called DIPNECH, which is often found in the presence of ‘mature’ carcinoid tumors. When this proliferating NE hyperplastic lesion gains resistance to apoptotic mechanisms, e.g. reflected by increased Bcl-2 levels in cases of DIPNECH [85], a transition towards a tumorlet stage is initiated. Because TCs do not progress into ACs, also evasion of cell cycle control mechanisms has to be amongst the earliest stages of their biology. Indeed, tumorlets acquire further proliferative potential as indicated by the emergence of Ki67 positive cells [85] and it could be envisaged that the carcinoid subtype is determined at this stage. Epigenetic silencing of tumor suppressor genes is also likely to occur during

Fig. 3. Tumorigenesis model for pulmonary carcinoids and small cell lung cancer. A model for the tumor biology of pulmonary carcinoids is shown on the right and the carcinogenesis of small cell lung cancer is depicted on the left. The importance of smoking in the tumorigenesis of high grade lung neuroendocrine tumors (NETs) is highlighted. We have chosen to group typical and atypical carcinoids together in this figure, because they have probably very similar mechanisms of tumorigenesis. In the gray area the lung NET precursor lesions are indicated. The percentages of occurrence of specific aberrations in the primary tumor (i.e. the small cell lung cancer or carcinoid tumor, respectively) are provided. The dotted lines indicate hypothetical relationships. Additional abbreviations used: ASH1, achaete-scute homolog; BASC, bronchioalveolar stem cell; C, central; DIPNECH, diffuse idiopathic neuroendocrine cell hyperplasia; EMT, epithelial to mesenchymal transition; NEB, neuroendocrine body; LOH, loss of heterozygosity; NCAM-PSA, polysialylated neural cell adhesion molecule; NE, neuroendocrine; P, peripheral; PNEC, pulmonary neuroendocrine cell; and SCLC, small cell lung cancer.

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early stages of tumorigenesis, and may involve RASSF1A methylation [116]. A fraction of the tumorlets slowly expands towards a mature carcinoid tumor, either a TC or an AC. However, most carcinoid tumors arise centrally, and centrally located tumors exist often without concomitant tumorlets or DIPNECH, raising the possibility that these lesions may originate directly from PNECs or NEBs (indicated by the dotted line in Fig. 3). Subsequent steps in carcinoid progression may include abrogations of the Menin-pathway, deletion of the telomeric part of chromosome 11q, and CD44 downregulation [216]. Carcinoid metastasis may result from EMT, which can be induced by abrogation of E-cadherin/β-catenin function as well as an increase of the PSA variant of NCAM (Fig. 3). This model for the tumorigenesis of carcinoids remains preliminary in the sense that the exact sequence of events still needs to be elucidated. Because carcinoids recur relatively frequently in the form of distant metastases more than 10 years after diagnosis, additional molecular markers for prediction of prognosis are highly needed. 7.2. Small cell lung cancer SCLC (depicted at the left side of Fig. 3) is the most malignant type of lung cancer, proliferates very rapidly and exhibits metastasis at an early stage. The majority of the steps in SCLC carcinogenesis described below can be related to exposure to tobacco carcinogens, as described in the foregoing sections. The lack of counterparts of high grade lung NETs at other locations in the body where carcinoids occur may support this relationship. Similar to the carcinoid tumors, understanding the tumor biology of SCLC becomes complicated through the occurrence of both centrally and peripherally located tumors, the latter group displaying more often a combined phenotype. As reported recently, the cell of origin for SCLC is a PNEC in the majority of cases, but may also be an alveolar type II cell. The existence of (an) other precursor cell(s) should, however, not be excluded [217] (Fig. 1). As proposed earlier [112], and irrespective of the cell type of origin, this cell remains phenotypically normal while acquiring genetic damage. Early events in carcinogenesis comprise epigenetic changes and allelic imbalances at fragile chromosomal regions, such as several 3p regions [110], comprising FHIT and RASSF1A, which can both be inactivated by promoter hypermethylation. Furthermore, aberration of the intrinsic apoptosis pathway, reflected by an inversion of the Bcl-2/Bax ratio towards the anti-apoptotic side represents an early event, possibly preceding p53 alterations [172]. P53 mutations have been observed in phenotypically normal lung tissues of smokers, either without tumors or adjacent to cancers [170]. It remains to be answered whether 3p deletions either precede or succeed 17p losses and associated p53 mutations. A strict sequence of events in SCLC and LCNEC tumorigenesis as reported for squamous cell carcinoma [112] is therefore unlikely. It is suggested that p53 mutation paves the way for subsequent chromosomal instability and further chromosomal losses of e.g. 9p, 17p and 5q. When all these barriers are breached, the primary SCLC starts proliferating extremely rapidly with an average doubling time of ~ 80 days [218]. More downstream in the process of tumorigenesis, inactivation of the pRb protein and an increase of the PSA variant of NCAM comprise important events. After formation of a primary SCLC, cigarette smoking may induce EMT by abrogating E-cadherin function through epigenetic inactivation and protein dislocation. EMT may be crucial to yield metastatic SCLC, probably in association with MYC overexpression due to gene amplification (Fig. 3) [219,220]. Because there is a major lack of information regarding the LCNEC subtype, we have not included these tumors in the model. They are characterized by fewer chromosomal alterations than SCLCs, but we suppose the mechanisms of their tumorigenesis to be largely similar to those of SCLCs.

7.3. Conclusion Although traditionally depicted as a spectrum of NETs, the similarity between high grade NE carcinomas on the one hand and pulmonary carcinoids on the other hand is probably restricted to their NE component. While high grade NE carcinomas are generally diagnosed in the absence of preneoplastic lesions, and result from accumulation of smoking-induced (epi)genetic aberrations, pulmonary carcinoids arise gradually and may evolve from NE hyperplasia, via a tumorlet stage towards a mature carcinoid. The tumor biology of lung carcinoids is complex because DIPNECH and tumorlets are generally associated with peripheral carcinoids, but are only seldomly detected with centrally located carcinoid tumors. Although the (epi)genetic abnormalities underlying early carcinoid tumorigenesis and disease progression need to be elucidated in further studies, it is clear that their tumor biology is very different from that of high grade NE carcinomas. We therefore conclude that these different tumor types are not part of one and the same spectrum of lung NETs. The large differences between carcinoid tumors and high grade lung NETs may implicate a reconsideration of the current WHO classification for NE lung tumors [1], in the sense that tumors with relatively low mitotic indices, currently classified as high grade lung NETs, may well comprise aggressively growing carcinoid tumors. For borderline cases, it may be valuable to screen for a number of discriminating molecular markers as provided in Table 2. Novel genome-wide techniques, such as exome sequencing and methylome analyses, should be deployed to further unravel the mechanisms of carcinogenesis and tumor progression in lung NETs. These approaches may also identify several novel candidate genes for diagnosis and therapy selection. These techniques may also help elucidate the mechanisms behind the late metastatic progression in a subset of carcinoid tumors and the relapse of SCLCs after initial response to chemotherapy. Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.bbcan.2012.05.001. References [1] W.D. Travis, E. Brambilla, H.K. Müller-Hermelink, C.C. Harris (Eds.), Tumours of the Lung, Pleura, Thymus and Heart, IARC Press, Lyon, 2004. [2] W.D. Travis, Lung tumours with neuroendocrine differentiation, Eur. J. Cancer 45 (Suppl. 1) (2009) 251–266. [3] W.D. Travis, W. Rush, D.B. Flieder, R. Falk, M.V. Fleming, A.A. Gal, M.N. Koss, Survival analysis of 200 pulmonary neuroendocrine tumors with clarification of criteria for atypical carcinoid and its separation from typical carcinoid, Am. J. Surg. Pathol. 22 (1998) 934–944. [4] B.I. Gustafsson, M. Kidd, A. Chan, M.V. Malfertheiner, I.M. Modlin, Bronchopulmonary neuroendocrine tumors, Cancer 113 (2008) 5–21. [5] W.D. Travis, Advances in neuroendocrine lung tumors, Ann. Oncol. 21 (Suppl. 7) (2010) vii65–vii71. [6] I.M. Modlin, K. Öberg, A Century of Advances in Neuroendocrine Tumor Biology and Treatment, 2007. [7] L. Righi, M. Volante, I. Rapa, G.V. Scagliotti, M. Papotti, Neuro-endocrine tumours of the lung. A review of relevant pathological and molecular data, Virchows Arch. 451 (Suppl. 1) (2007) S51–S59. [8] G. Fink, T. Krelbaum, A. Yellin, D. Bendayan, M. Saute, M. Glazer, M.R. Kramer, Pulmonary carcinoid: presentation, diagnosis, and outcome in 142 cases in Israel and review of 640 cases from the literature, Chest 119 (2001) 1647–1651. [9] E. Lim, P. Goldstraw, A.G. Nicholson, W.D. Travis, J.R. Jett, P. Ferolla, J. Bomanji, V.W. Rusch, H. Asamura, B. Skogseid, E. Baudin, M. Caplin, D. Kwekkeboom, E. Brambilla, J. Crowley, Proceedings of the IASLC International Workshop on Advances in Pulmonary Neuroendocrine Tumors 2007, J. Thorac. Oncol. 3 (2008) 1194–1201. [10] R. Govindan, N. Page, D. Morgensztern, W. Read, R. Tierney, A. Vlahiotis, E.L. Spitznagel, J. Piccirillo, Changing epidemiology of small-cell lung cancer in the United States over the last 30 years: analysis of the surveillance, epidemiologic, and end results database, J. Clin. Oncol. 24 (2006) 4539–4544. [11] H. Takei, H. Asamura, A. Maeshima, K. Suzuki, H. Kondo, T. Niki, T. Yamada, R. Tsuchiya, Y. Matsuno, Large cell neuroendocrine carcinoma of the lung: a clinicopathologic study of eighty-seven cases, J. Thorac. Cardiovasc. Surg. 124 (2002) 285–292. [12] G. Rossi, A. Cavazza, A. Marchioni, L. Longo, M. Migaldi, G. Sartori, N. Bigiani, L. Schirosi, C. Casali, U. Morandi, N. Facciolongo, A. Maiorana, M. Bavieri, L.M. Fabbri, E. Brambilla, Role of chemotherapy and the receptor tyrosine kinases KIT, PDGFRalpha, PDGFRbeta, and Met in large-cell neuroendocrine carcinoma of the lung, J. Clin. Oncol. 23 (2005) 8774–8785.

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