The centrioles duplicate once per cell cycle. The formation of the daughter centriole on each mother centriole occurs during the late G1 and S phases of the cell cycle. The daughter and mother centrioles are tightly associated in an orthogonal manner until the end of mitosis, and centriole disengagement occurs during mitotic exit. The initiation of centriole duplication requires the activity of several proteins, such as Cdk2-cyclin E and PLK4 kinases. The procentriole starts to assemble, and elongation depends on several proteins including centrin, CEP135, and -tubulin. During G2 phase, additional PCM proteins are recruited, and centrosome maturation requires the activity of Aurora A and PLK1 kinases. During late G2, the daughter centriole of the parental pair acquires subdistal appendages. Then, the two duplicated centrosomes separate and move to opposite end of the cell (centrosome separation). Finally, the two centrosomes form the poles of the bipolar mitotic

**Centriole elongation**

**M**

**G2**

**Centrosome maturation Centrosome separation**

Fig. 1.  With regard to primary lung cancer, Koutsami et al. (2006) examined 68 primary non-small cell lung carcinomas (NSCLCs) for the presence or absence of centrosome amplification using an immunofluorescence analysis with a monoclonal antibody for -tubulin, a centrosome marker; they showed that 36 (53%) of the 68 NSCLCs exhibited centrosome amplification. Centrosome amplification was not associated with clinicopathological markers such as stage, tumor grade, and histological subtype, but was associated with aneuploidy. Jung et al. (2007) examined 175 NSCLCs for centrosome abnormalities using an immunofluorescence analysis with an anti--tubulin antibody; they showed that 50 (29%) of the 175 NSCLCs exhibited a centrosome abnormality. Aneuploidy, p16 expression, and the loss of pRB expression were significantly associated with centrosome abnormalities. Shinmura et al. (2008) examined 182 primary lung carcinomas for the presence or absence of centrosome amplification using an immunohistochemical analysis with an anti--tubulin antibody and showed that 67 (37%) of the 182 cancers exhibited centrosome amplification. Thus, centrosome amplification is a common abnormality seen in human primary lung cancers.

#### **2.3 Mechanisms inducing centrosome abnormalities**

An immunofluorescence analysis using an antibody for centrosome or centriole markers in cultured cell lines can be used to determine the status of the centrosome number in the cells. The involvement of many kinds of agents and genes in centrosome regulation has been examined using such analyses. Here, these analyses are divided into those using lung cells and those using cells derived from other organs.

#### **2.3.1 Mechanisms identified by using the lung cells**

Holmes et al. (2006) showed that chronic exposure to lead chromate causes centrosome abnormalities and aneuploidy using WTHBF-6 cells, a cell line derived from normal human bronchial fibroblasts. Hexavalent chromium compounds [Cr(VI)] are human lung carcinogens (Le´onard & Lauwerys, 1980), and "particulate" Cr(VI) compounds are one of the most potent forms. They reported centrosome amplification in interphase and mitotic cells in response to treatment with lead chromate as a model particulate Cr(VI) compound. They suggested that one possible mechanism for lead chromate–induced carcinogenesis is through centrosome dysfunction, leading to the induction of aneuploidy. The same group (Holmes et al., 2010) also showed that chronic exposure to zinc chromate, another particulate Cr(VI) compound, induces centrosome amplification and spindle checkpoint bypass using human lung fibroblasts.

Arsenic is another environmental toxicant, and the biological effects of arsenic have been studied. Liao et al. (2007) showed that arsenic promotes centrosome abnormalities and cell colony formation in p53 compromised human lung cells. They used H1355 (a lung adenocarcinoma cell line with a p53 mutation), BEAS-2B (immortalized lung epithelial cells with functional p53) and pifithrin--treated BEAS-2B (p53-inhibited cells) and reported an increase in centrosome abnormalities in both arsenite-treated p53 compromised cell lines, compared with that in arsenite-treated BEAS-2B cells. Their findings provided evidence of the carcinogenic promotional role of arsenic, especially in the presence of p53 abnormalities. The group also showed that arsenite promoted centrosome abnormalities in the presence of a p53-compromised status induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (nicotine-derived nitrosamine ketone, NNK) using BEAS-2B cells (Liao et al., 2010). Their findings provided evidence of an interaction between arsenite and cigarette smoking.

Benzo[*a*]pyrene diol epoxide (B[*a*]PDE), the ultimate carcinogenic metabolite of benzo[*a*]pyrene, has been implicated in the mutagenesis of the *p53* gene, which is involved in smoking-associated lung cancer. Shinmura et al. (2008) showed that the exposure of *p53* deficient H1299 lung cancer cells to B[*a*]PDE resulted in S-phase arrest, leading to abnormal centrosome amplification. They also revealed that the centrosome amplification could be primarily attributed to excessive centrosome duplication, rather than to centriole splitting, and the forced expression of POLK DNA polymerase, which has the ability to bypass B[*a*]PDE–guanine lesions in an error-free manner, suppressing B[*a*]PDE-induced centrosome amplification. The B[*a*]PDE exposure also led to chromosome instability, which was likely to have resulted from centrosome amplification. Thus, they concluded that B[*a*]PDE contributes to neoplasia by inducing centrosome amplification and consequent chromosome destabilization in addition to its mutagenic activity.

Fig. 2. Induction of centrosome amplification in *p53*-deficient H1299 lung cancer cells by exposure to benzo[*a*]pyrene diol epoxide (B[*a*]PDE).

H1299 cells were exposed to 0.6 M B[*a*]PDE for 72 hr and then immunostained with mouse anti--tubulin monoclonal antibody (GTU-88; Sigma-Aldrich, St. Louis, MO, USA). Alexa Fluor 546 (red)-conjugated anti-IgG antibody (Molecular Probes, Eugene, OR, USA) was used to detect the antibody–antigen complexes. The nuclei were stained with 4',6 diamidino-2-phenylindol (DAPI, blue). An increase in the number of centrosomes, i.e., centrosome amplification, was observed in both interphase cells and mitotic phase cells. The arrows indicate the positions of centrosomes.

the carcinogenic promotional role of arsenic, especially in the presence of p53 abnormalities. The group also showed that arsenite promoted centrosome abnormalities in the presence of a p53-compromised status induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (nicotine-derived nitrosamine ketone, NNK) using BEAS-2B cells (Liao et al., 2010). Their findings provided evidence of an interaction between arsenite and cigarette smoking. Benzo[*a*]pyrene diol epoxide (B[*a*]PDE), the ultimate carcinogenic metabolite of benzo[*a*]pyrene, has been implicated in the mutagenesis of the *p53* gene, which is involved in smoking-associated lung cancer. Shinmura et al. (2008) showed that the exposure of *p53* deficient H1299 lung cancer cells to B[*a*]PDE resulted in S-phase arrest, leading to abnormal centrosome amplification. They also revealed that the centrosome amplification could be primarily attributed to excessive centrosome duplication, rather than to centriole splitting, and the forced expression of POLK DNA polymerase, which has the ability to bypass B[*a*]PDE–guanine lesions in an error-free manner, suppressing B[*a*]PDE-induced centrosome amplification. The B[*a*]PDE exposure also led to chromosome instability, which was likely to have resulted from centrosome amplification. Thus, they concluded that B[*a*]PDE contributes to neoplasia by inducing centrosome amplification and consequent chromosome

**Interphase Mitotic phase**

Fig. 2. Induction of centrosome amplification in *p53*-deficient H1299 lung cancer cells by

H1299 cells were exposed to 0.6 M B[*a*]PDE for 72 hr and then immunostained with mouse anti--tubulin monoclonal antibody (GTU-88; Sigma-Aldrich, St. Louis, MO, USA). Alexa Fluor 546 (red)-conjugated anti-IgG antibody (Molecular Probes, Eugene, OR, USA) was used to detect the antibody–antigen complexes. The nuclei were stained with 4',6 diamidino-2-phenylindol (DAPI, blue). An increase in the number of centrosomes, i.e., centrosome amplification, was observed in both interphase cells and mitotic phase cells. The

destabilization in addition to its mutagenic activity.

**B[***a***]PDE**

exposure to benzo[*a*]pyrene diol epoxide (B[*a*]PDE).

arrows indicate the positions of centrosomes.

**untreated**

The lung is easily subjected to many kinds of environmental agents, some of which may be derived from cigarette smoking or occupational exposure. As described in the above three paragraphs, some environmental carcinogens induce centrosome amplification. Other environmental carcinogens attacking DNA may also induce centrosome amplification, since cell cycle arrest has been shown to occur during centrosome amplification. Further precise analyses of environmental agent-related centrosome amplification are needed to understand the relationship between environmental carcinogens and lung cancer more clearly.

The S-phase kinase-interacting protein-2 (SKP2) plays a key role in the progression of cells from a quiescent to proliferative state, and the SKP2 protein is overexpressed in lung cancer. Jiang et al. (2005) showed that the RNA silencing of SKP2 inhibits proliferation and centrosome amplification using the lung cancer cell lines A549 and H1792. Their results suggest that SKP2 plays an oncogenic role in lung cancer and has a centrosome regulating function.

NORE1 (RASSF5) is a member of the *RASSF* gene family, and NORE1A is the longest and major splice isoform of the *NORE1* gene (Nakamura et al., 2005). Its product, NORE1A, is a nucleocytoplasmic shuttling protein and has a growth-suppressive function (Moshnikova et al., 2006). Shinmura et al. (2011) showed that NORE1A suppresses the centrosome amplification induced by hydroxyurea using a *p53*-deficient H1299 lung cancer cell line, and NORE1A expression was down-regulated in NSCLC. Both of these findings imply that NORE1A has a key preventative role against the carcinogenesis of NSCLC.

#### **2.3.2 Mechanisms identified using cells derived from other organs**

CDK2–cyclin E, a known inducer of S-phase entry (Heichman, 1994), has an important role in the regulation of centrosome duplication (Hinchcliffe et al., 1999; Matsumoto et al., 1999). The activation of CDK2–cyclin E during late-G1 phase coordinates the initiation of centrosome and DNA duplication. Several CDK2–cyclin E targets, including nucleophosmin (NPM) (Okuda et al., 2000), have been identified. NPM binds and modulates the activities of multiple proteins including tumor suppressor proteins (e.g.*,* p53) and some oncogenic proteins (e.g., ROCK2) (Colombo et al., 2002; Ma et al., 2006b). The reduced as well as increased expression of NPM can lead to the oncogenic transformation of cells. Actually, NPM is frequently mutated, lost or overexpressed in cancers (Grisendi et al., 2006), and both the overexpression and the depletion of NPM in cultured cells can lead to neoplastic transformation (Kondo et al., 1997; Grisendi et al., 2005). NPM localizes between the paired centrioles of the unduplicated centrosome, probably functioning in centriole pairing (Shinmura et al., 2005). When NPM is phosphorylated by CDK2–cyclin E, most of the NPM dissociates from the centrosomes, leading to the centrosome duplication. In this context, NPM negatively controls centrosome duplication; indeed, the depletion of NPM leads to centrosome amplification (Grisendi et al., 2005; Wang et al., 2005). NPM was reported to have the ability to control centrosome duplication in association with ROCK2 (Ma et al., 2006b), a member of the Rho-associated, coiled-coil containing protein kinase family that is frequently overexpressed in cancer (Nishimura et al., 2003). After NPM phosphorylation by CDK2–cyclin E, the binding between NPM and ROCK2 increases and ROCK2 is activated at centrosomes, leading to centrosome duplication (Ma et al., 2006b). In ROCK2 activation, the binding of Rho small GTPase to the auto-inhibitory region is also required (Kanai et al., 2010). Among three isoforms of Rho, both RhoA and RhoC, but not RhoB, promoted centrosome duplication and centrosome amplification.

Another target of CDK2–cyclin E in centrosome regulation is MPS1, a spindle checkpoint kinase that is localized at the centrosome (Fisk et al., 2003). MPS1 is stabilized and activated by CDK2–cyclin E phosphorylation and involved in centrosome duplication. Mortalin, a member of the heat-shock protein 70 molecular chaperone family, is localized at the centrosome and physically interacts with and is phosphorylated by MPS1. The phosphorylation of mortalin activates MPS1 in a positive-feedback manner, and this phenomenon is important for MPS1-related centrosome duplication (Kanai et al., 2007). Mortalin is frequently upregulated in cancers (Wadhwa et al., 2006).

CDK2 forms a complex with cyclin A in addition to cyclin E, and CDK2–cyclin A has been implicated in the regulation of centrosome duplication (Meraldi et al., 1999). CDK2–cyclin A and CDK2–cyclin E share some substrates (Tokuyama et al., 2001). The CDK2–cyclin A complex is active in S and G2 phases during the cell cycle, and CDK2–cyclin A may have a crucial role in centrosome over-duplication and/or amplification (Hanashiro et al., 2008). As another type of CDK-cyclin complex, the overactivation of CDK4/6–cyclin D has been shown to induce centrosome amplification (Nelsen et al., 2005). The major target of CDK4/6–cyclin D is the RB tumor-suppressor protein (Duensing et al., 2000). The conditional loss of *Rb* in mice results in centrosome amplification (Balsitis et al., 2003; Iovino et al., 2006).

CDK2 activity is also negatively controlled by the CDK inhibitor p21, one of the major transactivation targets of the p53 tumor-suppressor protein (Bálint & Vousden, 2001). p53 is involved in the regulation of centrosome duplication, which was first demonstrated in cells and tissues from *p53*-deficient mice (Fukasawa et al., 1996; Fukasawa et al., 1997). When cells are exposed to DNA-synthesis inhibitors such as hydroxyurea, centrosomes undergo reduplication without DNA synthesis, resulting in centrosome amplification (Balczon et al., 1995). Centrosome reduplication occurs efficiently when *p53* is mutated or lost (Tarapore et al., 2001a). In normal cells, p53 is stabilized under cellular stresses by the inhibition of MDM2, leading to the upregulation of p21, which blocks the initiation of centrosome reduplication through the inhibition of cyclin–CDK2 complexes (Bálint & Vousden, 2001). On the other hand, p21 is not upregulated in cells lacking *p53*, allowing the activation of CDK2, which in turn triggers centrosome reduplication.

Besides the p53–p21 pathway, p53 has the ability to control centrosome duplication. p53 is localized at centrosomes (Blair Zajdel & Blair, 1988; Brown et al., 1994; Tarapore et al., 2001b; Tritarelli et al., 2004; Ma et al., 2006a; Shinmura et al., 2007) and appears to control centrosome duplication independently of its transactivation function. Even if p53 is a mutant without transactivation function, p53 retains the ability to localize to centrosomes and partially suppresses centrosome duplication (Shinmura et al., 2007). However, the mechanism underlying this role of p53 is currently unknown.

The proteins that control p53 stability are also involved in the regulation of centrosome duplication. The ectopic expression of human papilloma virus (HPV) E6 protein, which promotes the degradation of p53, induces centrosome amplification (Duensing et al., 2000). MDM2 is an E3 ubiquitin ligase that promotes the degradation of p53 and is often overexpressed in cancers (Manfredi, 2010). The forced expression of MDM2 in cells containing wild-type p53 efficiently leads to centrosome amplification (Carroll et al., 1999). Aurora A kinase (AURKA) phosphorylates p53 at Ser315, resulting in MDM2-mediated p53 destabilization (Katayama et al., 2004), and the forced expression of Aurora A induces centrosome amplification (Zhou et al., 1998).

Another target of CDK2–cyclin E in centrosome regulation is MPS1, a spindle checkpoint kinase that is localized at the centrosome (Fisk et al., 2003). MPS1 is stabilized and activated by CDK2–cyclin E phosphorylation and involved in centrosome duplication. Mortalin, a member of the heat-shock protein 70 molecular chaperone family, is localized at the centrosome and physically interacts with and is phosphorylated by MPS1. The phosphorylation of mortalin activates MPS1 in a positive-feedback manner, and this phenomenon is important for MPS1-related centrosome duplication (Kanai et al., 2007).

CDK2 forms a complex with cyclin A in addition to cyclin E, and CDK2–cyclin A has been implicated in the regulation of centrosome duplication (Meraldi et al., 1999). CDK2–cyclin A and CDK2–cyclin E share some substrates (Tokuyama et al., 2001). The CDK2–cyclin A complex is active in S and G2 phases during the cell cycle, and CDK2–cyclin A may have a crucial role in centrosome over-duplication and/or amplification (Hanashiro et al., 2008). As another type of CDK-cyclin complex, the overactivation of CDK4/6–cyclin D has been shown to induce centrosome amplification (Nelsen et al., 2005). The major target of CDK4/6–cyclin D is the RB tumor-suppressor protein (Duensing et al., 2000). The conditional loss of *Rb* in mice results in centrosome amplification (Balsitis et al., 2003; Iovino

CDK2 activity is also negatively controlled by the CDK inhibitor p21, one of the major transactivation targets of the p53 tumor-suppressor protein (Bálint & Vousden, 2001). p53 is involved in the regulation of centrosome duplication, which was first demonstrated in cells and tissues from *p53*-deficient mice (Fukasawa et al., 1996; Fukasawa et al., 1997). When cells are exposed to DNA-synthesis inhibitors such as hydroxyurea, centrosomes undergo reduplication without DNA synthesis, resulting in centrosome amplification (Balczon et al., 1995). Centrosome reduplication occurs efficiently when *p53* is mutated or lost (Tarapore et al., 2001a). In normal cells, p53 is stabilized under cellular stresses by the inhibition of MDM2, leading to the upregulation of p21, which blocks the initiation of centrosome reduplication through the inhibition of cyclin–CDK2 complexes (Bálint & Vousden, 2001). On the other hand, p21 is not upregulated in cells lacking *p53*, allowing the activation of

Besides the p53–p21 pathway, p53 has the ability to control centrosome duplication. p53 is localized at centrosomes (Blair Zajdel & Blair, 1988; Brown et al., 1994; Tarapore et al., 2001b; Tritarelli et al., 2004; Ma et al., 2006a; Shinmura et al., 2007) and appears to control centrosome duplication independently of its transactivation function. Even if p53 is a mutant without transactivation function, p53 retains the ability to localize to centrosomes and partially suppresses centrosome duplication (Shinmura et al., 2007). However, the

The proteins that control p53 stability are also involved in the regulation of centrosome duplication. The ectopic expression of human papilloma virus (HPV) E6 protein, which promotes the degradation of p53, induces centrosome amplification (Duensing et al., 2000). MDM2 is an E3 ubiquitin ligase that promotes the degradation of p53 and is often overexpressed in cancers (Manfredi, 2010). The forced expression of MDM2 in cells containing wild-type p53 efficiently leads to centrosome amplification (Carroll et al., 1999). Aurora A kinase (AURKA) phosphorylates p53 at Ser315, resulting in MDM2-mediated p53 destabilization (Katayama et al., 2004), and the forced expression of Aurora A induces

Mortalin is frequently upregulated in cancers (Wadhwa et al., 2006).

CDK2, which in turn triggers centrosome reduplication.

mechanism underlying this role of p53 is currently unknown.

centrosome amplification (Zhou et al., 1998).

et al., 2006).

Polo-like kinase 1 (PLK1) is a key regulator of centrosome maturation (Barr et al., 2004; Bettencourt-Dias and Glover, 2007). Its deregulation is linked to centrosome abnormalities and oncogenesis (Zyss and Gergely, 2009). PLK1 belongs to the mammalian PLK family, which is comprised of five members (PLK1 - PLK4 and PLK5P) (Lens et al., 2010). PLK1 is involved in a variety of mitotic events, including centrosome maturation and separation, G2/M transition, mitotic spindle formation, chromosome segregation, and cytokinesis, and several kinds of PLK1 substrates are known (Barr et al., 2004; Petronczki et al., 2008). PLK1 targets multiple centrosomal proteins (e.g., -tubulin) to fulfill the mitotic function of centrosomes. Ninein-like protein (NLP) interacts with TuRC during interphase, and participates in the establishment of the cytoplasmic microtubule network (Casenghi et al., 2003; Rapley et al., 2005). At the onset of mitosis, the cooperation of PLK1 and NLP promotes the centrosomal localization of -tubulin and other mitosis specific PCM components, resulting in a higher microtubule nucleation capacity of the mitotic centrosome (Casenghi et al., 2003; Rapley et al., 2005). The phosphorylation of NEDD1 by PLK1 is required for the targeting of TuRC to the centrosome (Zhang et al., 2009). In mitosis, centrosomes must withstand the pulling forces exerted by chromosome-attached microtubules. To withstand such forces, PLK1 also plays a role in maintaining the structural integrity of the centrosome during mitosis (Oshimori et al., 2006). Kizuna is localized at the centrosomes and is phosphorylated by PLK1 during mitosis. The reduced expression of kizuna results in centrosome fragmentation and the dispersion of PCM, leading to the formation of aberrant mitotic spindles and chromosome segregation errors.

Another PLK, PLK4, is involved in recruiting the structural components required for the formation of procentrioles at the proximal side of the older centriole, in cooperation with CDK2-cyclin E (Habedanck et al., 2004). The upregulation of PLK4 expression is a strong stimulus for centriole multiplication (Kleylein-Sohn et al., 2007). The timely degradation of PLK4 by the SCF slimb ubiquitin ligase is important for the restriction of procentriole formation (Cunha-Ferreira et al., 2009). The SCF component CUL1 also functions as a centrosomal suppressor of centriole multiplication by regulating the PLK4 protein level (Korzeniewski et al., 2009). PLK4 kinase activity also regulates its own stability (Holland et al., 2010; Guderian et al., 2010). CEP152 interacts with PLK4 and CPAP and controls centrosome duplication in human cells (Dzhindzhev et al., 2010). PLK4 is transcriptionally regulated by p53 (Li et al., 2005). Clinically, the expression of PLK4 is upregulated in colon cancer (Macmillan et al., 2001), while the expression of PLK4 is downregulated in hepatocellular carcinoma because of promoter hypermethylation and the loss of heterozygosity (LOH) (Pellegrino et al., 2010; Rosario et al., 2010).

The role of the morgana/chp-1 in centrosome regulation has been reported by Ferretti et al. (2010). Mutations in morgana result in centrosome amplification. Morgana forms a complex with Hsp90, ROCK1 and ROCK2, and directly binds to ROCK2. Morgana downregulation promotes the interaction between ROCK2 and NPM, leading to an increase in ROCK2 activity, which in turn results in centrosome amplification. Morgana is downregulated in a large fraction of lung and breast cancers. They suggested that morgana plays a role in preventing centrosome amplification and tumorigenesis.

NLP, a previously described substrate of PLK1 (Casenghi et al., 2003), is a BRCA1-associated centrosomal protein that is involved in microtubule nucleation and spindle formation (Jin et al., 2009). NLP is overexpressed as a result of *NLP* gene amplification in lung cancer, and NLP overexpression causes centrosome amplification (Shao et al., 2010).

The *BRCA1* gene is responsible for susceptibility to familial breast/ovarian cancer and participates in diverse cellular functions (Venkitaraman, 2002). The BRCA1 is localized at the centrosomes (Hsu & White, 1998; Okada & Ouchi, 2003) and is involved in the regulation of centrosome duplication (Xu et al., 1999). BRCA1 is associated with BARD1, and this association mediates the ubiquitylation of -tubulin, which is important for maintaining the numeral integrity of centrosomes. The *BRCA2* gene is another causative gene of familial breast/ovarian cancer and its protein product functions in homologous recombination (HR) repair (Venkitaraman, 2002). The loss of *BRCA2* results in centrosome amplification (Tutt et al., 1999), implying a relationship between a defect in DNA repair and the abnormal amplification of the centrosomes. HR repair is mediated by several proteins including RAD51, and the downregulation of RAD51 leads to centrosome amplification (Bertrand et al., 2003). The reduced expression or loss of XRCC2, XRCC3, and RAD51B-D, which are other HR components, induces centrosome amplification and chromosome instability (Griffin et al., 2000; Smiraldo et al., 2005; Date et al., 2006; Renglin Lindh et al., 2007; Cappelli et al., 2011).

Centrosome amplification induced by DNA damage occurs during a prolonged G2 phase (Dodson et al., 2004). A centrosome-autonomous signal that involves centriole disengagement causes centrosome amplification in G2 phase after DNA damage (Inanç et al., 2010), suggesting that genotoxic stress can decouple the centrosome cycle and chromosome cycle.

The active nucleocytoplasmic transport of proteins is mediated by the nuclear localization signal (NLS) and nuclear export signal (NES) (Turner & Sullivan, 2008). NLS-containing proteins are transported from the cytoplasm to the nucleus, whereas NES-containing proteins are exported from the nucleus to the cytoplasm by XPO1, the human homolog of yeast Crm1. The inhibition of XPO1 causes centrosome amplification via the disruption of the nucleocytoplasmic transport of NPM (Forgues et al., 2003; Shinmura et al., 2005; Wang et al., 2005). XPO1 is involved in the centrosomal localization of various proteins (Han et al., 2008). Importin and RANBP1 are other proteins involved in nucleocytoplasmic transport, and these proteins also have the ability to regulate centrosomes (Di Fiore et al., 2003; Ciciarello et al., 2004).

SGOL1 interacts with protein phosphatase 2A, is localized in the centromere, and prevents the cohesin complex from precocious cleavage at the centromere via the dephosphorylation of SA2, one of the cohesin subunits (Kitajima et al., 2006; Riedel et al., 2006). Clinically, SGOL1 expression is downregulated in colorectal cancer, and SGOL1-knockdown leads to centrosome amplification and chromosome instability in a colon cancer cell line (Iwaizumi et al., 2009; Dai et al., 2009). A SGOL1-P1 transcript containing an exon-skip of exon 3, resulting in the formation of a premature stop codon, is expressed in colorectal cancer, and the overexpression of SGOL1-P1 in a colon cancer cell line resulted in an increased number of cells with aberrant chromosome alignment, precociously separated chromatids, delayed mitotic progression, and centrosome amplification (Kahyo et al., 2011). Furthermore, the overexpression of SGOL1-P1 inhibited the localization of endogenous SGOL1 and cohesin subunit RAD21/SCC1 to the centromere, suggesting that SGOL1-P1 may function as a negative factor to native SGOL1 (Kahyo et al., 2011).

#### **2.4 Relationship between centrosome abnormalities and chromosome instability**

Chromosome instability is defined as a persistently high rate of the gain and loss of whole chromosomes (Thompson et al., 2010). Chromosome instability is a major source of

The *BRCA1* gene is responsible for susceptibility to familial breast/ovarian cancer and participates in diverse cellular functions (Venkitaraman, 2002). The BRCA1 is localized at the centrosomes (Hsu & White, 1998; Okada & Ouchi, 2003) and is involved in the regulation of centrosome duplication (Xu et al., 1999). BRCA1 is associated with BARD1, and this association mediates the ubiquitylation of -tubulin, which is important for maintaining the numeral integrity of centrosomes. The *BRCA2* gene is another causative gene of familial breast/ovarian cancer and its protein product functions in homologous recombination (HR) repair (Venkitaraman, 2002). The loss of *BRCA2* results in centrosome amplification (Tutt et al., 1999), implying a relationship between a defect in DNA repair and the abnormal amplification of the centrosomes. HR repair is mediated by several proteins including RAD51, and the downregulation of RAD51 leads to centrosome amplification (Bertrand et al., 2003). The reduced expression or loss of XRCC2, XRCC3, and RAD51B-D, which are other HR components, induces centrosome amplification and chromosome instability (Griffin et al., 2000; Smiraldo et al., 2005; Date et al., 2006; Renglin Lindh et al.,

Centrosome amplification induced by DNA damage occurs during a prolonged G2 phase (Dodson et al., 2004). A centrosome-autonomous signal that involves centriole disengagement causes centrosome amplification in G2 phase after DNA damage (Inanç et al., 2010), suggesting that genotoxic stress can decouple the centrosome cycle and chromosome cycle. The active nucleocytoplasmic transport of proteins is mediated by the nuclear localization signal (NLS) and nuclear export signal (NES) (Turner & Sullivan, 2008). NLS-containing proteins are transported from the cytoplasm to the nucleus, whereas NES-containing proteins are exported from the nucleus to the cytoplasm by XPO1, the human homolog of yeast Crm1. The inhibition of XPO1 causes centrosome amplification via the disruption of the nucleocytoplasmic transport of NPM (Forgues et al., 2003; Shinmura et al., 2005; Wang et al., 2005). XPO1 is involved in the centrosomal localization of various proteins (Han et al., 2008). Importin and RANBP1 are other proteins involved in nucleocytoplasmic transport, and these proteins also have the ability to regulate centrosomes (Di Fiore et al., 2003;

SGOL1 interacts with protein phosphatase 2A, is localized in the centromere, and prevents the cohesin complex from precocious cleavage at the centromere via the dephosphorylation of SA2, one of the cohesin subunits (Kitajima et al., 2006; Riedel et al., 2006). Clinically, SGOL1 expression is downregulated in colorectal cancer, and SGOL1-knockdown leads to centrosome amplification and chromosome instability in a colon cancer cell line (Iwaizumi et al., 2009; Dai et al., 2009). A SGOL1-P1 transcript containing an exon-skip of exon 3, resulting in the formation of a premature stop codon, is expressed in colorectal cancer, and the overexpression of SGOL1-P1 in a colon cancer cell line resulted in an increased number of cells with aberrant chromosome alignment, precociously separated chromatids, delayed mitotic progression, and centrosome amplification (Kahyo et al., 2011). Furthermore, the overexpression of SGOL1-P1 inhibited the localization of endogenous SGOL1 and cohesin subunit RAD21/SCC1 to the centromere, suggesting that SGOL1-P1 may function as a

**2.4 Relationship between centrosome abnormalities and chromosome instability**  Chromosome instability is defined as a persistently high rate of the gain and loss of whole chromosomes (Thompson et al., 2010). Chromosome instability is a major source of

2007; Cappelli et al., 2011).

Ciciarello et al., 2004).

negative factor to native SGOL1 (Kahyo et al., 2011).

aneuploidy (Lengauer et al., 1997; Rajagopalan and Lengauer, 2004), and chromosome instability is thought to be involved not only in cancer initiation, where aneuploidy may have a causal role, but also in cancer development, where increased rates of chromosome missegregation may enable the clonal expansion of cells with a greater malignant potential (Rajagopalan & Lengauer, 2004; Weaver et al., 2007; Gao et al., 2007; Ganem et al., 2009). Defects in chromosome cohesion, weakened spindle assembly checkpoint (SAC) signalling, impaired microtubule-kinetochore attachment, defects in cell cycle regulation, and centrosome abnormalities can cause chromosome instability (Lingle et al., 1998; Draviam et al., 2004; Thompson & Compton, 2008; Weaver & Cleveland, 2008; Thompson et al., 2010). Regarding centrosome abnormalities, two mechanisms underlying chromosome instability have been proposed. The first mechanism is that centrosome amplification generates chromosome instability by promoting multipolar anaphase, which is an abnormal division that produces more than three aneuploid daughter cells (Nigg, 2002). The other mechanism is that centrosome amplification generates chromosome instability by promoting merotelic kinetochore–microtubule attachments (Ganem et al., 2009; Silkworth et al., 2009). Merotely is a type of error in which single kinetochores attach to microtubules emanating from different poles (Salmon et al., 2005; Cimini, 2008) and is common in cells showing chromosome instability (Thompson & Compton, 2008). Cells with centrosome amplification often coalesce the extra centrosomes during mitosis to ensure that anaphase occurs with a bipolar spindle (Quintyne et al., 2005). The extra centrosomes induce transient multipolar spindle intermediates prior to the coalescence of the centrosomes into bipolar spindles; this event increases the incidence of merotelic kinetochore–microtubule attachments and elevates the chromosome missegregation rates (Ganem et al., 2009; Silkworth et al., 2009). Ganem et al. (2009) showed that the presence of extra centrosomes is correlated with an increase in lagging chromosomes (Figure 3), promoting chromosome missegregation through excessive merotely induced by transient multipolar spindle intermediates. Since merotelic attachments are poorly sensed by the SAC (Salmon et al., 2005; Cimini, 2008), the merotelic attachments arising from centrosome amplification are not fully repaired and give rise to lagging chromosomes during anaphase, possibly leading to missegregation events.

Fig. 3. Lagging chromosomes in human cancer cells.

(A, B) Lagging chromosome formation detected in a B[*a*]PDE-treated H1299 lung cancer cell line. (A) Normal segregation; (B) an anaphase cell showing lagging chromosome formation. The nuclei were stained with DAPI (blue). (C) Lagging chromosomes are shown in a hematoxylin-and-eosin-stained section of a squamous cell carcinoma of the lung. In (B) and (C), the arrows indicate lagging chromosomes.

## **3. Conclusion**

The progress in our understanding of the relationship between centrosome abnormalities and cancer during the past 15 years has been enormous. We have learned that centrosome abnormalities are common among diverse human cancers including lung cancer. Many molecules are involved in the control of the numeral and/or functional integrity of centrosomes, and the abrogation of these mechanisms results in centrosome abnormalities, which promote chromosome instability. From a therapeutic standpoint, anti-cancer drugs targeting the centrosome have now been developed (Mazzorana et al., 2011). Future studies using a genome-wide approach and new scientific technologies will further increase our knowledge of the role of the centrosome in human cells, and such knowledge will likely help to establish effective cancer therapies.

## **4. Acknowledgment**

This work was supported by grants from the MHLW (21-1), the JSPS (22590356), the MEXT (20014007 and 221S0001), and the Smoking Research Foundation.

#### **5. References**

The progress in our understanding of the relationship between centrosome abnormalities and cancer during the past 15 years has been enormous. We have learned that centrosome abnormalities are common among diverse human cancers including lung cancer. Many molecules are involved in the control of the numeral and/or functional integrity of centrosomes, and the abrogation of these mechanisms results in centrosome abnormalities, which promote chromosome instability. From a therapeutic standpoint, anti-cancer drugs targeting the centrosome have now been developed (Mazzorana et al., 2011). Future studies using a genome-wide approach and new scientific technologies will further increase our knowledge of the role of the centrosome in human cells, and such knowledge will likely

This work was supported by grants from the MHLW (21-1), the JSPS (22590356), the MEXT

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## **Defective Expression and DNA Variants of TGFBR2 in Chinese Small Cell Lung Carcinoma**

ZhenHong Zhao1, Jibin Xu2, Jun Xie2, Yang Bao2,3, Xiaotian Wang1, Lei Wang2, Junjie Wu4, Li Jin1, Zhiyun Xu2\* and Jiucun Wang1,\* *1MOE Key Laboratory of Contemporary Anthropology and State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai, 2Department of Cardiac and Thoracic Surgery, Changhai Hospital, Shanghai, 3Yangzhou No.1 People's Hospital, Jiangsu Province, 4Department of Pneumology, Changhai Hospital, Shanghai, China*

#### **1. Introduction**

188 Lung Diseases – Selected State of the Art Reviews

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Lung cancer is one of the most commonly diagnosed malignant tumors, and has the highest death rate of all cancer types. Both the incidence rate and death rate of lung cancer have increased rapidly worldwide during the last 50 years. Lung cancer has now become the leading cause of cancer death in males, and the second most common cause of cancer death in females, after breast cancer. According to data provided by the International Agency for Research on Cancer, about 1.6 million new lung cancer patients were confirmed in 2008, accounting for 13% of the total cancer cases, while about 1.4 million patients died, amounting to 18% of the total deaths caused by cancer worldwide (Jemal et al., 2011).

Lung cancer can be divided according to histological subtype into non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC), with the latter accounting for about 14% of new lung cancer cases in the USA and Europe in 2004 (Jemal et al., 2004). The clinical and histological features of SCLC were first recognized by Barnard in 1926 as being distinct from those of other types of lung cancer (Barnard, 1926). SCLC cells develop from lung Kulchitsky cells, and SCLC can be further subdivided into three different types: oat-cell type, intermediate-cell type and mixed-cell type (Travis, 1999). Smoking is the key risk factor for SCLC, and more than 95% of patients develop SCLC as a result of tobacco smoking. Smoking more cigarettes and prolonging the duration of smoking can both increase the risk of developing SCLC (Brownson et al., 1992), while stopping smoking reduces its risk, compared to persistent smokers (Khuder and Mutgi, 2001; Jackman and Johnson, 2005). SCLC is very aggressive and the median survival time without treatment is less than 4 months. Chemotherapy and radiotherapy represent the two major treatments for SCLC. According to the standards developed by the Veterans Administration Lung Cancer Study Group, SCLC can be divided into two stages: a limited stage and an extensive stage (Simon, 2003). Cancer cells in limited-stage SCLC are restricted to the ipsilateral hemithorax

<sup>\*</sup> Corresponding Author

and can be treated by both chemotherapy and radiation therapy. About 20% of patients are cured after treatment, and the median survival time is about 18 months. Patients with extensive-stage SCLC have a high response rate to chemotherapy, which is the primary treatment for this disease, but the median survival time is only about 9 months because most patients relapse and the results of salvage therapy are poor (Janne et al., 2002; Demedts et al., 2010).

Transforming growth factor-beta (TGF-β) belongs to a large superfamily of cell cytokines, and is an important component of several cellular metabolic pathways. TGF-β signaling pathways regulate many aspects of cellular function, such as cellular proliferation, differentiation, migration, apoptosis, adhesion, angiogenesis, immune surveillance and survival (Jakowlew, 2006). TGF-β plays a very complex dual role in cancer development, progress and metastasis (Akhurst & Derynck, 2001; Elliott & Blobe, 2005). TGF-β inhibits primary tumor development and growth by inducing cell cycle arrest and apoptosis as a tumor suppressor during the early phase of tumorigenesis (Arteaga, et al. 1993), but also promotes tumor invasion and metastasis by inducing the epithelial-mesenchymal transition in some epithelial cells, indicating that TGF-β can also act as a tumor promoter in the late stage of cancer (Miyazono, 2009).

There are two types of TGF-β signaling pathways; Smad-dependent and Smad-independent pathways. In Smad-dependent TGF-β signaling pathways, autocrine or exogenous TGF-β binds to the TGFBR2 and TGFBR1 membrane receptors. TGFBR2 then phosphorylates TGFBR1, which activates receptor-regulated Smads (also known as R-Smads). The R-Smads usually comprise Smad2 and Smad3. Activated Smad2 and Smad3 form complexes with Smad4, the common-partner Smad (co-Smad) in mammals. The subsequent R-Smad-co-Smad complexes shuttle between the nucleus and cytoplasm, and interact with various transcription factors and transcriptional co-activators such as AP-1, Sp1, p300, and SMIF to regulate the transcription of target genes (Derynck & Zhang, 2003). The phosphorylation of R-Smads can be blocked by inhibitory Smad, which starts the ubiquitination and degradation of the R-Smad-co-Smad complexes, thus inhibiting signal transduction (Itoh & ten Dijke, 2007). This TGF-β signal transduction pathway mainly regulates cell metabolism through this network involving cell cycle capture and apoptosis. In addition to Smadmediated signaling pathways, TGF-β also activates other pathways, including Erk, JNK and p38 MAPK kinase pathways, via Smad-independent mechanisms (Moustakas & Heldin, 2007).

Both Smad-dependent and -independent TGF-β signaling pathways start by binding TGF-β to its transmembrane receptor TGFBR2, which then activates the downstream signal transduction. However, TGFBR2 expression is often reduced or even blocked in tumor cells (Levy & Hill, 2006). In bladder cancer, deficient TGFBR2 expression leads to loss of the growth inhibition function of TGF-β, and loss of expression of TGFBR2 has been shown to correlate with tumor grade (Tokunaga, et al., 1999). Other studies also found that inactivation of TGFBR2 played a central role in the development and progression of human gastric cancer, and TGFBR2 expression has shown a strong association with the degree of malignancy in gastric cancer (Chang, et al., 1997). The expression of TGFBR2 was also reduced in breast cancer (Gobbi, et al. 2000). Although the reasons for defective TGFBR2 expression are still unknown, loss of or reduced expression of TGFBR2 may be caused by histone deacetylation in lung cancer cell lines (Osada et al., 2001).

TGFBR2 mutations have also been observed in tumor cells. A DNA variant with a frameshift mutation in the poly(A)10 repeat, resulting in microsatellite instability (MSI), has been detected in the coding region of the TGFBR2 gene in several types of tumors, including colon cancer, gastric cancer, and gliomas (Markowitz et al. 1995; Pinto et al., 1997; Izumoto et al. 1997). This frameshift could affect gene function and be related to cancer development. This MSI also been detected in both NSCLC and SCLC (Kim et al., 2000; Tani et al., 1997), though the mutation rate seems to be much lower than that of deficient TGFBR2 expression rate in lung cancer. A previous study identified a novel microdeletion (c.492\_507del) in giant cell carcinoma (GCC) and large cell carcinoma (LCC) patients, compared to other NSCLC subtypes. This 16-bp microdeletion introduced a premature stop codon at positions 590–592 of the cDNA, resulting in a truncated TGFBR2 protein with a mutated transmembrane domain and loss of a kinase domain. Although the mutated TGFBR2 played an important role in the abrogation of TGF-β signal transduction in LCC cells (Wang et al., 2007), it was not correlated with the reduced TGFBR2 expression seen in NSCLC (Xu et al., 2007).

However, TGFBR2 has rarely been studied in Chinese SCLC samples and its role in TGF-β insensitivity in this population thus remains unknown. The present study therefore examined the levels of TGFBR2 expression in 27 pairs of formalin-fixed, paraffin-embedded SCLC tumors and compared them with NSCLC samples. The entire cDNA region and promoter of the gene was then sequenced to identify the causal variants in the TGFBR2 gene that accounted for its defective expression.

## **2. Materials and methods**

#### **2.1 Specimens**

190 Lung Diseases – Selected State of the Art Reviews

and can be treated by both chemotherapy and radiation therapy. About 20% of patients are cured after treatment, and the median survival time is about 18 months. Patients with extensive-stage SCLC have a high response rate to chemotherapy, which is the primary treatment for this disease, but the median survival time is only about 9 months because most patients relapse and the results of salvage therapy are poor (Janne et al., 2002; Demedts

Transforming growth factor-beta (TGF-β) belongs to a large superfamily of cell cytokines, and is an important component of several cellular metabolic pathways. TGF-β signaling pathways regulate many aspects of cellular function, such as cellular proliferation, differentiation, migration, apoptosis, adhesion, angiogenesis, immune surveillance and survival (Jakowlew, 2006). TGF-β plays a very complex dual role in cancer development, progress and metastasis (Akhurst & Derynck, 2001; Elliott & Blobe, 2005). TGF-β inhibits primary tumor development and growth by inducing cell cycle arrest and apoptosis as a tumor suppressor during the early phase of tumorigenesis (Arteaga, et al. 1993), but also promotes tumor invasion and metastasis by inducing the epithelial-mesenchymal transition in some epithelial cells, indicating that TGF-β can also act as a tumor promoter in the late

There are two types of TGF-β signaling pathways; Smad-dependent and Smad-independent pathways. In Smad-dependent TGF-β signaling pathways, autocrine or exogenous TGF-β binds to the TGFBR2 and TGFBR1 membrane receptors. TGFBR2 then phosphorylates TGFBR1, which activates receptor-regulated Smads (also known as R-Smads). The R-Smads usually comprise Smad2 and Smad3. Activated Smad2 and Smad3 form complexes with Smad4, the common-partner Smad (co-Smad) in mammals. The subsequent R-Smad-co-Smad complexes shuttle between the nucleus and cytoplasm, and interact with various transcription factors and transcriptional co-activators such as AP-1, Sp1, p300, and SMIF to regulate the transcription of target genes (Derynck & Zhang, 2003). The phosphorylation of R-Smads can be blocked by inhibitory Smad, which starts the ubiquitination and degradation of the R-Smad-co-Smad complexes, thus inhibiting signal transduction (Itoh & ten Dijke, 2007). This TGF-β signal transduction pathway mainly regulates cell metabolism through this network involving cell cycle capture and apoptosis. In addition to Smadmediated signaling pathways, TGF-β also activates other pathways, including Erk, JNK and p38 MAPK kinase pathways, via Smad-independent mechanisms (Moustakas & Heldin,

Both Smad-dependent and -independent TGF-β signaling pathways start by binding TGF-β to its transmembrane receptor TGFBR2, which then activates the downstream signal transduction. However, TGFBR2 expression is often reduced or even blocked in tumor cells (Levy & Hill, 2006). In bladder cancer, deficient TGFBR2 expression leads to loss of the growth inhibition function of TGF-β, and loss of expression of TGFBR2 has been shown to correlate with tumor grade (Tokunaga, et al., 1999). Other studies also found that inactivation of TGFBR2 played a central role in the development and progression of human gastric cancer, and TGFBR2 expression has shown a strong association with the degree of malignancy in gastric cancer (Chang, et al., 1997). The expression of TGFBR2 was also reduced in breast cancer (Gobbi, et al. 2000). Although the reasons for defective TGFBR2 expression are still unknown, loss of or reduced expression of TGFBR2 may be caused by

TGFBR2 mutations have also been observed in tumor cells. A DNA variant with a frameshift mutation in the poly(A)10 repeat, resulting in microsatellite instability (MSI), has been

histone deacetylation in lung cancer cell lines (Osada et al., 2001).

et al., 2010).

2007).

stage of cancer (Miyazono, 2009).

Twenty-seven formalin-fixed, paraffin-embedded SCLC samples and their corresponding normal tissues were collected by the Laboratory of the Department of Thoracic Surgery, Changhai Hospital between 2000 and 2007. All the patients had undergone pulmonary resection for primary SCLC at Changhai Hospital and had provided informed consent, and none had received preoperative radiotherapy or chemotherapy. The demographic and clinical features of these SCLC cases are summarized in Table 1. This research was conducted with the official approval of the academic advisory board of the Institute of Genetics, Fudan University, Shanghai, P. R. China.

An additional 65 formalin-fixed, paraffin-embedded NSCLC samples and their corresponding normal tissues were collected between 2005 and 2007, as a control group to compare with SCLC (Table 2). These tissues were also provided by the Laboratory of the Department of Thoracic Surgery, Changhai Hospital after obtaining the patients' consent. None of these patients had received radiotherapy or chemotherapy prior to surgery.

#### **2.2 Immunohistochemistry**

Expression of TGFBR2 was detected by immunohistochemistry assay using a monoclonal antibody against the extracellular domain of TGFBR2 (R & D Systems, Minneapolis, MN) via two-step immunohistochemical staining using the EnVision system (DAKO Cytomation, Denmark), as described in our previous report (26). In brief, after the paraffin sections were deparaffinized and hydrated, serial 4-μm thick sections were placed into 3% hydrogen peroxide solution for 10 min to block endogenous peroxidase activity. For antigen retrieval, the sections were treated with boiling 0.01 mol/L citrate buffer (pH 6.0) for 25 min and then incubated with 10% fetal calf serum for 20 min at room temperature. After the blocking serum was removed, the sections were incubated with the primary antibody (1:50) at room temperature for 1 h, followed by rinsing three times with phosphate-buffered saline (PBS).

Re: reduced TGFBR2 expression in tumor tissues, Loss: loss of TGFBR2 expression, Pr: preserved TGFBR2 expression.

The staining score of each tissue is the product of the proportion of positive staining cells and intensity scores.

Table 1. Clinical features and TGFBR2 expression of the 27 SCLC patients

Re: reduced TGFBR2 expression in tumor tissues, Pr: preserved TGFBR2 expression.

Table 2. Clinical features and TGFBR2 expression in NSCLC samples

The sections were then incubated with a working solution of horseradish peroxidase-labeled goat anti-mouse immunoglobulin, as provided in the EnVision kit, for 30 min. Finally, the peroxidase activity was developed with 3,3-diaminobenzidine tetrahydrochloride and hydrogen peroxide. Because NSCLC develops from bronchial epithelium precursors, human normal bronchial epithelium was used as a positive control. A negative control for each specimen was provided by treating the sections with PBS instead of the primary antibody.

#### **2.3 Interpretation of the staining and data evaluation**

192 Lung Diseases – Selected State of the Art Reviews

Re: reduced TGFBR2 expression in tumor tissues, Loss: loss of TGFBR2 expression, Pr: preserved

Table 1. Clinical features and TGFBR2 expression of the 27 SCLC patients

The staining score of each tissue is the product of the proportion of positive staining cells and intensity

TGFBR2 expression.

scores.

All sections were examined by standard light microscopy and scored semi-quantitatively on the basis of the percentage of immunoreactive cells and on the intensity of the staining reaction. The samples were initially classified into one of four grades, according to staining intensity: 0 (negative staining, equivalent to the negative control), 1 (weak staining), 2 (medium staining) and 3 (strong staining). The percentages of positively-stained cells were assigned as 0 for 0–25%, 1 for 26–50%, 2 for 51–75% and 3 for 76–100%, respectively. The final score was determined as the product of the proportion and intensity scores, and ranged from 0–9. Samples were considered to be negatively stained if the final score was 0, and positively stained if the final score was 1–9. Moreover, cancer samples were classified as preserved- or reduced-type in terms of TGFBR2 expression, depending on whether the final score was the same as or less than that of its corresponding normal lung tissue.

## **2.4 DNA Extraction and mutation analysis**

Target cells from formalin-fixed, paraffin-embedded tissue sections were microdissected and scraped into microtubes. After deparaffinization with xylene and washing in ethanol, DNA was extracted by standard proteinase K digestion and phenol-chloroform extraction (Sambrook & Maniatis, 1989).

The presence of the 16-bp microdeletion in exon 4, which was previously detected in LCC and GCC, was examined in all SCLC tissues using the following forward and reverse primers to amplify the fragments of 117/101 bp, representing the wild/mutant alleles: 5' caccagcaatcctgacttgttg-3' and 5'-cggttaacgcggtagcagtag-3'. The MSI in exon 3 was detected by the STR method using an ABI 3100 Sequencer and the following forward and reverse primers were used to amplify the exon 3 fragment (normally 242 bp) of the TGFBR2 gene: 5'-tccaatgaatctcttcactc-3' and 5'-cccacacccttaagagaaga-3'. c.1167 C>T in exon 4 of TGFBR2 was detected by direct sequencing using an ABI 3100 Sequencer and the following forward and reverse primers to amplify the exon 4 fragment (242 bp) of the TGFBR2 gene: 5'-

cccaagatgcccatcgtg-3' and 5'-tcccaggctcaaggtaaagg-3'. The other primers used for promoter and exon sequencing are listed in Table 3.

Table 3. Primers used in the study

#### **2.5 Statistical analysis**

Data were analyzed using χ2 tests, corrected χ2 tests, or Fisher's exact tests. A P value of less than 0.05 was considered statistically significant.

## **3. Results**

#### **3.1 TGFBR2 expression was more often reduced in SCLC than in NSCLC**

TGFBR2 expression was assessed using immunohistochemistry. Normal human lung tissues and normal human bronchial epithelium were used as positive controls. Over 75% of cells in these tissues exhibited consistently strong staining, both showing staining scores of 3 × 3 = 9, indicating normal TGFBR2 expression (Figure 1). Immunostaining of TGFBR2 was performed in 27 SCLC tumor tissue samples and their corresponding normal tissues. All the normal tissues showed strong staining in over 75% cells with staining scores of 9. One SCLC sample showed negative TGFBR2 expression (score of 0), while the remaining 26 were TGFBR2-positive. Furthermore, 16 of the total 27 SCLC tumor samples showed reduced TGFBR2 expression (score of 1–6) and 10 showed preserved expression (Table 1).

None of the 65 NSCLC samples showed negative TGFBR2 expression (staining score of 0). In addition, only 35.4% (23/65) of all NSCLC tumor tissues showed reduced (score of 1–6) TGFBR2 expression and 64.6% (42/65) of tumors had preserved expression (score of 9) (Table 2). When adenocarcinoma and squamous cell carcinoma tissues were analyzed separately, the frequencies of preserved type were also higher (63.6% (21/33) and 66.7% (18/27) respectively) than those of reduced type (36.4% (12/33) and 33.4% (9/27) respectively). In contrast, the frequency of preserved type in SCLCs (47%, 10/27) was much lower than that of reduced type (63%, 17/27), indicating that reduced TGFBR2 expression was more frequent in SCLC cells (Table 1 & 2).

Fig. 1. Expression of TGFBR2 in lung cancer by immunohistochemical analysis (×400).

194 Lung Diseases – Selected State of the Art Reviews

cccaagatgcccatcgtg-3' and 5'-tcccaggctcaaggtaaagg-3'. The other primers used for promoter

Data were analyzed using χ2 tests, corrected χ2 tests, or Fisher's exact tests. A P value of less

TGFBR2 expression was assessed using immunohistochemistry. Normal human lung tissues and normal human bronchial epithelium were used as positive controls. Over 75% of cells in these tissues exhibited consistently strong staining, both showing staining scores of 3 × 3 = 9, indicating normal TGFBR2 expression (Figure 1). Immunostaining of TGFBR2 was performed in 27 SCLC tumor tissue samples and their corresponding normal tissues. All the normal tissues showed strong staining in over 75% cells with staining scores of 9. One SCLC

**3.1 TGFBR2 expression was more often reduced in SCLC than in NSCLC**

Fragments Region Direction Sequence (5' - 3')

TGFBR2 promoter Promoter part1 Forward aactacaaaacatgtacaccagg TGFBR2 promoter Reverse ttctttaggtcgaagtctagagg TGFBR2 promoter Promoter part2 Forward atgcagaatctctgcctgcctc TGFBR2 promoter Reverse cgagagctttggccgacttt TGFBR2 promoter Promoter part3 Forward gtaaatacttggagcgaggaactc TGFBR2 promoter Reverse ttctgaacgtgcggtgggat TGFBR2 exon exon 1 Forward tcggtctatgacgagcag TGFBR2 exon Reverse gggaccccaggaagaccc TGFBR2 exon exon 2 Forward gggctggtatcaagttcatttg TGFBR2 exon Reverse ggagacagagatacactgactgtg TGFBR2 exon exon 3 Forward tccaatgaatctcttcactc TGFBR2 exon Reverse cccacacccttaagagaaga TGFBR2 exon exon 4-1 Forward ccaactccttctctccttgttttg TGFBR2 exon Reverse tccaagaggcatactcctcatagg TGFBR2 exon exon 4-2 Forward gtcgctttgctgaggtctataagg TGFBR2 exon Reverse ccaggctcaaggtaaaggggatctagca TGFBR2 exon exon 5 Forward ggcagctggaattaaatgatgggc TGFBR2 exon Reverse tgctcgaagcaacacatg TGFBR2 exon exon 6 Forward tttcctttgggctgcacatg TGFBR2 exon Reverse cctaagaggcaacttggttgaatc TGFBR2 exon exon 7 Forward ccaactcatggtgtccctttg TGFBR2 exon Reverse tctttggacatgcccagcctg TGFBR2 MSI Exon 3 Forward Fam-tccaatgaatctcttcactc TGFBR2 MSI Reverse cccacacccttaagagaaga TGFBR2 LOH Exon 4 Forward cccaagatgcccatcgtg TGFBR2 LOH Reverse tcccaggctcaaggtaaagg

and exon sequencing are listed in Table 3.

Table 3. Primers used in the study

than 0.05 was considered statistically significant.

**2.5 Statistical analysis**

**3. Results**

#### **3.2 No significant relationship was found between TGFBR2 expression and clinical features in SCLC patients**

The associations between TGFBR2 expression and other clinical features were analyzed. No significant associations were found between TGFBR2 expression and gender (P = 1.00), age (P = 0.14), tumor size (P = 1.00), nodal involvement (P = 1.00), metastasis (P = 1.00) or stage (P = 0.12) (Table 4).

Table 4. Association between TGFBR2 expression and clinical features of 27 SCLC patients

#### **3.3 TGFBR2 expression is related to tumor types**

The relationship between TGFBR2 expression and histological type was analyzed. Samples were categorized as SCLC or NSCLC subtypes because they developed from different lung cells. As shown in Table 5, a significant association between TGFBR2 expression and histological type was identified (P = 0.0151), indicating the existence of a significant difference in TGFBR2 expression levels between SCLC and NSCLC subtypes (Table 5).

For further statistical analysis, NSCLC cases were divided into AdC, SqC, Ad-SqC and other subtypes. Because of the sample sizes, comparisons were only made between SCLC and AdC, and between SCLC and SqC. The results demonstrated significant differences in TGFBR2 expression between SCLC and AdC, and between SCLC and SqC (P = 0.0402 and 0.0293, respectively) (Table 5).

#### **3.4 Mutations in exon 4 of TGFBR2**

In a previous study, we identified a microdeletion (c.492\_507del) in patients with GCC and LCC. We therefore investigated the occurrence of this microdeletion in SCLC in the present study. Genomic DNA was extracted from 21 pairs of formalin-fixed, paraffin-embedded SCLC tissues and their corresponding normal tissues. The coding and promoter regions of

Table 5. TGFBR2 expression in different subtypes of tumor

TGFBR2 were sequenced. The DNA from the other six pairs of tissues was degraded and was unsuitable for amplification. No microdeletion was observed in any of the tested SCLC samples.

However, another novel variant in exon 4 of TGFBR2 was identified in 11 of 21 SCLC tumor samples. This variant at c.1167 in the TGFBR2 coding region was T/T homozygous in eight out of 11 cases, and C/T heterozygous in the other three cases, compared with C/C homozygous in normal individuals. The corresponding normal samples for these were C/T heterozygous. In the other 10 pairs of samples, however, the site was C/C homozygous. These results suggest that loss of heterozygosity (LOH) occurred in the eight tumors whose alleles became T/T homozygous from C/T heterozygous (Figure 2 and Table 6).

Interestingly, this change was a synonymous mutation that did not alter the amino acid sequence. We investigated its effect on the expression of TGFBR2, and found that TGFBR2 expression was reduced in nearly all T carriers (81.8%), compared with that in normal tissues, while only 60% of CC carriers had reduced TGFBR2 expression (Table 6).

Fig. 2. LOH in SCLC.

196 Lung Diseases – Selected State of the Art Reviews

Age

Gender

Tumor Size

Nodal involvement

Metastasis

Stage

Cases Gender TGFBR2 expression Age P-value M F Re Pr

≤60 16(59.26%) 11 5 10 6 32-57 P=1.0000

Male 21(77.78%) 21 0 15 6 P=0.1358

T1 4 3 1 3 1 P=1.0000

N0 5 5 0 3 2 P=1.0000

M0 26 20 6 16 10 P=1.0000

Ⅰ 2 2 0 1 1 P=0.1164

Table 4. Association between TGFBR2 expression and clinical features of 27 SCLC patients

The relationship between TGFBR2 expression and histological type was analyzed. Samples were categorized as SCLC or NSCLC subtypes because they developed from different lung cells. As shown in Table 5, a significant association between TGFBR2 expression and histological type was identified (P = 0.0151), indicating the existence of a significant difference in TGFBR2 expression levels between SCLC and NSCLC subtypes (Table 5). For further statistical analysis, NSCLC cases were divided into AdC, SqC, Ad-SqC and other subtypes. Because of the sample sizes, comparisons were only made between SCLC and AdC, and between SCLC and SqC. The results demonstrated significant differences in TGFBR2 expression between SCLC and AdC, and between SCLC and SqC (P = 0.0402 and

In a previous study, we identified a microdeletion (c.492\_507del) in patients with GCC and LCC. We therefore investigated the occurrence of this microdeletion in SCLC in the present study. Genomic DNA was extracted from 21 pairs of formalin-fixed, paraffin-embedded SCLC tissues and their corresponding normal tissues. The coding and promoter regions of

>60 11(40.74%) 10 1 7 4 61-77

T2 16 11 5 14 9 T≥2

N1 10 8 2 14 8 N≥1

Female 6(22.22%) 0 6 2 4

M1 1 1 0 1 0

Ⅱ 11 8 3 5 6 Ⅲ 13 10 3 11 2 Ⅳ 1 1 0 0 1

T3 3 3 0 T4 4 4 0

N2 10 8 2 N3 2 2 0

**3.3 TGFBR2 expression is related to tumor types**

0.0293, respectively) (Table 5).

**3.4 Mutations in exon 4 of TGFBR2**

A. CC sequence in normal lung tissue from SCLC patients;

B. TC sequence in normal lung tissue from SCLC patients;

C. TT sequence in tumor tissue from SCLC patients.

+: tumor tissues; -: normal lung tissue of patients;

Re: reduced TGFBR2 expression in tumor tissues, Pr: preserved TGFBR2 expression.

Table 6. Relationship between LOH in exon 4 and TGFBR2 expression

#### **3.5 MSI in TGFBR2 in SCLC**

Poly(A)10/(A)9 heterozygosity in exon 3 of TGFBR2, representing MSI, was detected in 60% of SCLC samples (9 out of 15), as shown in Table 7. However, no association between MSI and TGFBR2 expression was found (P = 0.264).

+: tumor tissues; -: normal lung tissue of patients;

Re: reduced TGFBR2 expression in tumor tissues, Pr: preserved TGFBR2 expression.

Table 7. MSI detection and relation with TGFBR2 expression

## **4. Discussion**

198 Lung Diseases – Selected State of the Art Reviews

Patient No. Tissue LOH TGFBR2 expression S1 <sup>+</sup> TT Pr - CT

S3 <sup>+</sup> CC Re - CC

S5 <sup>+</sup> CC Re - CC

S6 <sup>+</sup> TT Re - CT

S7 <sup>+</sup> CC Re - CC

S8 <sup>+</sup> TT Re - CT

S9 <sup>+</sup> CC Pr - CC

S11 <sup>+</sup> CC Pr - CC

S12 <sup>+</sup> TT Re - CT

S13 <sup>+</sup> TT Re - CT

S15 <sup>+</sup> CT Re - CT

S16 <sup>+</sup> CC Re - CC

S18 <sup>+</sup> CC Pr - CC

S19 <sup>+</sup> TT Pr - CT

S20 <sup>+</sup> CT Re - CT

S21 <sup>+</sup> CC Pr - CC

S22 <sup>+</sup> CT Re - CT

S23 <sup>+</sup> TT Re - CT

S25 <sup>+</sup> CC Re - CC

S26 <sup>+</sup> TT Re - CT

Poly(A)10/(A)9 heterozygosity in exon 3 of TGFBR2, representing MSI, was detected in 60% of SCLC samples (9 out of 15), as shown in Table 7. However, no association between MSI

Re: reduced TGFBR2 expression in tumor tissues, Pr: preserved TGFBR2 expression. Table 6. Relationship between LOH in exon 4 and TGFBR2 expression

<sup>+</sup> CC Re - CC

S27

**3.5 MSI in TGFBR2 in SCLC**

+: tumor tissues; -: normal lung tissue of patients;

and TGFBR2 expression was found (P = 0.264).

Tumor cells are often able to escape from TGF-β-signaling-induced cell cycle capture and apoptosis. TGF-β has a dual function in tumor development (Akhurst & Derynck, 2001; Elliott & Blobe, 2005); it acts as a tumor suppressor during the initial stages of tumor development (Arteaga, et al. 1993), but promotes tumor progression during the later stages (Miyazono, 2009). High levels of TGF-β expression in tumor cells can induce tumor evolution by stimulating angiogenesis and through other potential immunosuppressive effects, as well as by directly affecting tumor cell invasion and metastasis (Pardali & Moustakas, 2007). These direct effects can be achieved via Smad-dependent pathways, or may be mediated by interference with these pathways (Derynck & Zhang, 2003). Changes in the TGF-β signaling pathway may lead to abnormal signal transduction and cause dysregulated cell growth and differentiation. The first step in any mechanism involves binding of autocrine or paracrine TGF-β to the TGFBR2 receptor on the cell membrane, before activation of various downstream receptors can occur. TGFBR2 thus plays a key role in TGF-β signaling pathways, and its expression is reduced or blocked in many tumors (Chang et al., 1997; Tokunaga et al., 1999; Gobbi et al. 2000;Levy & Hill, 2006), resulting in partial or complete disruption of the TGF-β pathway.

Previous studies demonstrated that TGFBR2 expression in NSCLC differed between LCC and AdC, SqC or non-LCC cases, but the role of defective TGFBR2 expression in the initiation and/or development of SCLC (Xu et al., 2007), and its expression status in SCLC remain largely unknown. Furthermore, SCLC is phenotypically distinct from and much more malignant than NSCLC. We therefore compared TGFBR2 expression between SCLC and NSCLC. Immunohistochemical staining with TGFBR2 antibody revealed significant differences in the incidence of reduced expression in SCLC (63.0% of cases) versus AdC (36.4% of cases, P = 0.0402) and SqC (33.3% of cases, P = 0.0293), or SCLC versus NSCLC (35% of cases, P = 0.0151). These differences in expression levels between SCLC and NSCLC were consistent with the histopathologic classification of these tumors, suggesting that defective TGFBR2 expression might contribute to the initiation and/or development of SCLC.

To determine the reason for the reduced expression of TGFBR2, we examined the mutation status of c.492 507del in exon 4, but found no changes in this sequence in SCLC tumor samples. We subsequently determined the MSI status in exon 3, and identified a DNA variant with a frameshift mutation in the TGFBR2 poly(A)10 repeat (which causes MSI) in the coding region of the TGFBR2 gene. A total of 60.0% of SCLC were poly(A)10/(A)9 heterozygous, but no association was found between the MSI and TGFBR2 expression. However, no MSI was identified in our previous study of NSCLC, suggesting that the MSI in SCLC is at least partly associated with its carcinogenesis. We also sequenced all seven exons and the promoter region of the TGFBR2 gene and identified a novel LOH at c.1167 in 38.1% (8/21) of SCLC tissues. Further analysis showed that most of the mutant T-allele carriers (81.8%) had reduced TGFBR2 expression in tumor tissues, compared with only 60% of C-allele carriers. These results suggest that the change from wild type to mutant type might contribute, at least in part, to the defective expression of TGFBR2 in SCLC patients, though further studies are needed to clarify the mechanisms responsible.

## **5. Conclusion**

The present study identified reduced TGFBR2 gene expression levels in formalin-fixed, paraffin-embedded sections from most SCLC tumors examined, suggesting that this might contribute to the initiation and/or development of SCLC. Sequencing analysis also indicated that change of the wild-type C-allele to the mutant T-allele at c.1167 might contribute to the defective expression of TGFBR2 in SCLC patients. Another DNA variant with a frameshift mutation in the TGFBR2 poly(A)10 repeat, leading to MSI, was found in the coding region of the TGFBR2 gene, but this was not associated with TGFBR2 expression.

These results suggest that defective expression of TGFBR2 might inactivate TGF-β signal transduction, leading to the loss of growth inhibition and acceleration of tumor formation, and that a C>T substitution at c.1167 might be partially responsible for this reduced expression of TGFBR2 in SCLC.

## **6. Acknowledgement**

This study was supported by grants from the National Science Foundation of China, grant number 30971594 to JC Wang and grant number 30890034 to L Jin.

## **7. References**

200 Lung Diseases – Selected State of the Art Reviews

the TGF-β signaling pathway may lead to abnormal signal transduction and cause dysregulated cell growth and differentiation. The first step in any mechanism involves binding of autocrine or paracrine TGF-β to the TGFBR2 receptor on the cell membrane, before activation of various downstream receptors can occur. TGFBR2 thus plays a key role in TGF-β signaling pathways, and its expression is reduced or blocked in many tumors (Chang et al., 1997; Tokunaga et al., 1999; Gobbi et al. 2000;Levy & Hill, 2006), resulting in

Previous studies demonstrated that TGFBR2 expression in NSCLC differed between LCC and AdC, SqC or non-LCC cases, but the role of defective TGFBR2 expression in the initiation and/or development of SCLC (Xu et al., 2007), and its expression status in SCLC remain largely unknown. Furthermore, SCLC is phenotypically distinct from and much more malignant than NSCLC. We therefore compared TGFBR2 expression between SCLC and NSCLC. Immunohistochemical staining with TGFBR2 antibody revealed significant differences in the incidence of reduced expression in SCLC (63.0% of cases) versus AdC (36.4% of cases, P = 0.0402) and SqC (33.3% of cases, P = 0.0293), or SCLC versus NSCLC (35% of cases, P = 0.0151). These differences in expression levels between SCLC and NSCLC were consistent with the histopathologic classification of these tumors, suggesting that defective TGFBR2 expression might contribute to the initiation and/or development of

To determine the reason for the reduced expression of TGFBR2, we examined the mutation status of c.492 507del in exon 4, but found no changes in this sequence in SCLC tumor samples. We subsequently determined the MSI status in exon 3, and identified a DNA variant with a frameshift mutation in the TGFBR2 poly(A)10 repeat (which causes MSI) in the coding region of the TGFBR2 gene. A total of 60.0% of SCLC were poly(A)10/(A)9 heterozygous, but no association was found between the MSI and TGFBR2 expression. However, no MSI was identified in our previous study of NSCLC, suggesting that the MSI in SCLC is at least partly associated with its carcinogenesis. We also sequenced all seven exons and the promoter region of the TGFBR2 gene and identified a novel LOH at c.1167 in 38.1% (8/21) of SCLC tissues. Further analysis showed that most of the mutant T-allele carriers (81.8%) had reduced TGFBR2 expression in tumor tissues, compared with only 60% of C-allele carriers. These results suggest that the change from wild type to mutant type might contribute, at least in part, to the defective expression of TGFBR2 in SCLC patients,

The present study identified reduced TGFBR2 gene expression levels in formalin-fixed, paraffin-embedded sections from most SCLC tumors examined, suggesting that this might contribute to the initiation and/or development of SCLC. Sequencing analysis also indicated that change of the wild-type C-allele to the mutant T-allele at c.1167 might contribute to the defective expression of TGFBR2 in SCLC patients. Another DNA variant with a frameshift mutation in the TGFBR2 poly(A)10 repeat, leading to MSI, was found in the coding region of

These results suggest that defective expression of TGFBR2 might inactivate TGF-β signal transduction, leading to the loss of growth inhibition and acceleration of tumor formation, and that a C>T substitution at c.1167 might be partially responsible for this reduced

though further studies are needed to clarify the mechanisms responsible.

the TGFBR2 gene, but this was not associated with TGFBR2 expression.

partial or complete disruption of the TGF-β pathway.

SCLC.

**5. Conclusion**

expression of TGFBR2 in SCLC.

 Introduction**

202 Lung Diseases – Selected State of the Art Reviews

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Osada, H., et al., Heterogeneous transforming growth factor (TGF)-beta unresponsiveness

Pardali K, Moustakas A: Actions of TGF-beta as tumor suppressor and pro-metastatic factor

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Tani, M., et al., Infrequent mutations of the transforming growth factor beta-type II receptor

Tokunaga, H., et al., Decreased expression of transforming growth factor beta receptor type

Travis WD, C.T., Corrin B, Shimosato Y, Brambilla E. Epithelial tumours. In: Travis WD, CV,

Uchida, K., et al., Somatic in vivo alterations of the JV18-1 gene at 18q21 in human lung

Wang, J.-C., et al., Novel microdeletion in the transforming growth factor beta Type II

Xu, J. B., et al., Defective expression of transforming growth factor P type II receptor

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receptor gene is associated with giant and large cell variants of nonsmall cell lung

(TGFBR2) in the large cell variant of non-small cell lung carcinoma. Lung Cancer,

Lung cancer arises from neoplastic changes of the epithelial cells in the lung. However, it is not known whether all or only a subset of these lung epithelial cells is susceptible to malignant transformation. Specifically, a major question is whether the changes need to take place in lung epithelial cells involving stem-cell–like properties. Lung cancer is a clinically, biologically, histologically, molecularly, and genetically heterogeneous disease. The underlying causes of this heterogeneity are unknown and could reflect changes occurring in cells with various potential for differentiation or represent different molecular changes occurring in the same lung epithelial target cells. Transformation from a normal to malignant lung cancer phenotype is thought to arise in a multistep fashion, through a series of genetic versus epigenetic alterations, ultimately evolving into an invasive cancer by clonal expansion. These progressive pathological changes in the bronchial epithelium occur primarily as one of three distinct morphological forms: squamous dysplasia, atypical adenomatous hyperplasia, and diffuse idiopathic pulmonary neuroendocrine cell hyperplasia. Bronchial squamous dysplasia and carcinoma in situ (CIS) are the recognised preneoplastic lesions for squamous cell carcinoma (SCC); atypical adenomatous hyperplasia (AAH), a putative preneoplastic lesion, for a subset of adenocarcinomas (ADC); and diffuse idiopathic pulmonary neuroendocrine cell hyperplasia (DIPNECH) for neuroendocrine lung carcinomas. Pulmonary neuroendocrine tumours comprise approximately 2% of all lung malignancies. According to the most recent World Health Organization classification, pulmonary neuroendocrine tumours are histologically divided into a three-tier, fourcategory system including low-grade (typical carcinoid), intermediate-grade (atypical carcinoid), and high-grade (small cell carcinoma and large cell neuroendocrine carcinoma) tumours. Nearly all lung cancers exhibit the morphological and molecular features of epithelial cells and are accordingly classified as carcinomas. The cells of origin of virtually all lung cancers reside in the epithelial lining of the airways. As more is learned about the origin of neuroendocrine lung tumours, it is also increasingly clear that the biology of neuroendocrine lung tumours arising in the central airways (i.e., SCLC) is distinct from that of peripheral airway lesions. The purpose of this chapter is not so much to recapitulate the details of the neuroendocrine lung tumour classification but rather to provide an understanding of the main categories of lung carcinoma, to highlight potential pitfalls in the histopathological diagnosis of lung cancer, to summarise current information on molecular properties and cellular origins of individual neuroendocrine lung tumour subtypes, and to relate pathologies to biological behaviours.

#### **2. Neuroendocrine system of the lung**

The endocrine cells within the gut epithelium (foregut, midgut, and hindgut) constitute the largest population of hormone producing cells in the body. So far, approximately 10 different neuroendocrine lineages have been identified, and most of them show a specific rostro-caudal distribution. Pulmonary neuroendocrine cells (PNECs) are part of the diffuse neuroendocrine system (DNES) distributed throughout the body. The PNEC system (solitary PNECs and neuroepithelial bodies, or NEBs) consists of a distinct population of airway epithelial cells displaying endocrine and paracrine secretory mechanisms. Pulmonary neuroendocrine cells were readily demonstrated and uniformly distributed in normal adult human lungs. Overall, as identified by neurone specific enolase immunoreactivity, there were 10.5 neuroendocrine cells per 10 cm of epithelial length and 4 per 10,000 epithelial cells; they extended from the trachea to the alveolar ducts but none was seen in the alveoli (72% were in bronchi, 24% in bronchioles, and 4% in alveolar ducts). Of the cells identified by gastrin-releasing peptide immunoreactivity, there were 6.9 neuroendocrine cells per 10 cm of epithelial length and 2.4 per 10,000 epithelial cells. Of the cells identified by calcitonin immunoreactivity, there were 3.5 neuroendocrine cells per 10 cm of epithelial length and 1.3 per 10,000 epithelial cells. Minor cells contained serotonin (all in the terminal bronchioles), and in a small minority no peptide or amine was detected. It is currently thought that PNECs, like their counterparts in the gastrointestinal tract, are derived from multipotent epithelial progenitors, and that all epithelial cells arise from a single stem cell. All pulmonary epithelial cells including PNECs and non-NE airway epithelial cells are likely to be derived from a single stem cell. Epithelial lung stem cells, as in many organs, are often confined to discretely localised niches that are protected from environmental insults. In the lung, PNECs are associated with the stem cell niches in both the proximal and distal airways. One of the lung stem-cell niches is located in the trachea that reveals two stem-cell niches: gland ducts in the proximal compartment and select foci near the cartilage-intercartilage junction in the distal trachea. Other intrapulmonary stem cell niches include NEBs located at the airway bifurcation. Another stem cell niche is at the bronchoalveolar junctions, although PNECs may play a diminished role at this location. Among many functions assigned to them, there is a possible dual role: the regulation of lung maturation/growth and chemoreception. First, during the early stages of lung organogenesis, PNECs acting via their amine and peptide products may function as local modulators of lung growth and differentiation. Second, later in foetal life and in postnatal stages, PNECs and in particular innervated NEBs could play a role as airway chemoreceptors.  Aggregates of neuroendocrine cells, called neuroepithelial bodies (NEBs), are diffusely spread in the epithelium at all levels of the intrapulmonary airways, preferentially located at the airway bifurcation of the lungs. Neuroendocrine cells are selectively contacted by different nerve fibres. NEBs are contacted by at least three different nerve-fibre

details of the neuroendocrine lung tumour classification but rather to provide an understanding of the main categories of lung carcinoma, to highlight potential pitfalls in the histopathological diagnosis of lung cancer, to summarise current information on molecular properties and cellular origins of individual neuroendocrine lung tumour subtypes, and to

The endocrine cells within the gut epithelium (foregut, midgut, and hindgut) constitute the largest population of hormone producing cells in the body. So far, approximately 10 different neuroendocrine lineages have been identified, and most of them show a specific rostro-caudal distribution. Pulmonary neuroendocrine cells (PNECs) are part of the diffuse neuroendocrine system (DNES) distributed throughout the body. The PNEC system (solitary PNECs and neuroepithelial bodies, or NEBs) consists of a distinct population of airway epithelial cells displaying endocrine and paracrine secretory mechanisms. Pulmonary neuroendocrine cells were readily demonstrated and uniformly distributed in normal adult human lungs. Overall, as identified by neurone specific enolase immunoreactivity, there were 10.5 neuroendocrine cells per 10 cm of epithelial length and 4 per 10,000 epithelial cells; they extended from the trachea to the alveolar ducts but none was seen in the alveoli (72% were in bronchi, 24% in bronchioles, and 4% in alveolar ducts). Of the cells identified by gastrin-releasing peptide immunoreactivity, there were 6.9 neuroendocrine cells per 10 cm of epithelial length and 2.4 per 10,000 epithelial cells. Of the cells identified by calcitonin immunoreactivity, there were 3.5 neuroendocrine cells per 10 cm of epithelial length and 1.3 per 10,000 epithelial cells. Minor cells contained serotonin (all in the terminal bronchioles), and in a small minority no peptide or amine was detected. It is currently thought that PNECs, like their counterparts in the gastrointestinal tract, are derived from multipotent epithelial progenitors, and that all epithelial cells arise from a single stem cell. All pulmonary epithelial cells including PNECs and non-NE airway epithelial cells are likely to be derived from a single stem cell. Epithelial lung stem cells, as in many organs, are often confined to discretely localised niches that are protected from environmental insults. In the lung, PNECs are associated with the stem cell niches in both the proximal and distal airways. One of the lung stem-cell niches is located in the trachea that reveals two stem-cell niches: gland ducts in the proximal compartment and select foci near the cartilage-intercartilage junction in the distal trachea. Other intrapulmonary stem cell niches include NEBs located at the airway bifurcation. Another stem cell niche is at the bronchoalveolar junctions, although PNECs may play a diminished role at this location. Among many functions assigned to them, there is a possible dual role: the regulation of lung maturation/growth and chemoreception. First, during the early stages of lung organogenesis, PNECs acting via their amine and peptide products may function as local modulators of lung growth and differentiation. Second, later in foetal life and in postnatal stages, PNECs and in particular innervated NEBs could play a role as airway chemoreceptors.  Aggregates of neuroendocrine cells, called neuroepithelial bodies (NEBs), are diffusely spread in the epithelium at all levels of the intrapulmonary airways, preferentially located at the airway bifurcation of the lungs. Neuroendocrine cells are selectively contacted by different nerve fibres. NEBs are contacted by at least three different nerve-fibre

relate pathologies to biological behaviours.

**2. Neuroendocrine system of the lung**

populations: vagal sensory calbidin D28k, calcitonin gene related peptide (CGRP)/substance P (SP) innervation, and intrinsic pulmonary nitrergic neurons.

#### **2.1 Neuroendocrine epithelial cells**

Neuroendocrine epithelial cells (NEC) of the respiratory system tend to occur either as single cells that are sparsely distributed throughout the epithelium of the tracheobronchial tract or in small, well-defined clusters that are supported by nonciliated bronchiolar (Clara) cells. The latter are referred to as neuroepithelial bodies (NEBs) and are located only in the epithelium of the intrapulmonary airways, often at or near a bronchiolar bifurcation. The solitary pulmonary NEC cells of most of the investigated species are fusiform or flaskshaped, resting on the basement membrane with an apical process pointing toward the airway lumen. Adult human NEC cells generally lack luminal contact. Although there are some differences between solitary NEC cells and NEBs, a large body of evidence points to their being a member of the amine precursor uptake and decarboxylation (APUD) cell series or of the paraneuron family. The endocrine system of the lung consists of at least two different cell categories. These categories exhibit similar main characteristics. They contain a biogenic amine and neuropeptide mediators, and their cytoplasm harbours neurosecretorylike granules. Regarding morphological features and location, these cells presumably have a receptor secretory function. Consequently, these classes of endocrine cells can be designated as paraneurons. Solitary NEC cells were found to be distributed over almost the entire respiratory system, while NEBs seemed to be restricted to the epithelium of the intrapulmonary airways. Neuroepithelial bodies generally consist of nonciliated, cylindrical cells with a palisade-like arrangement between the airway surface and the underlying connective tissue, although they may also appear as stratified cells. Most of the luminal side of the NEBs is covered by the supporting Clara cells. The NEBs are strategically located on the surface of the airway bifurcations. They, in fact, contain the serotonin-bioactive amine and neuropeptides, leading to the speculation of these cells as a homogeneous or heterogeneous class. They also exert control on pulmonary vessels and airway tone. The NEC cell may function as the transducer of the stimulus or the sensory nerve ending, the activity of the latter being modulated by the release of bioactive substances from NEC cells. Investigations have indicated the influence of NEC cells on epithelial cell differentiation, mucous secretion, and proliferation of local endoderm in developing airways.

#### **2.2 Pulmonary neuroendocrine cells**

Pulmonary neuroendocrine cells (PNECs) are commonly organised into innervated clusters, called NEBs, which have been proposed to serve various functions, including the regulation of embryonic lung growth and maturation through the elaboration of a variety of potent neuropeptides. Several studies have suggested that PNECs are quiescent cells with limited self-renewal capacity. However, it was recently demonstrated that PNECs have a selfrenewal capacity and can be activated to undergo multiple rounds of proliferation after TA (Clara) cell depletion. It has been suggested that PNEC-derived paracrine factors might play a role in the regulation of epithelial cell differentiation and proliferation during foetal lung development and possibly in the normal or injured adult lung. Cell proliferation has also been shown to contribute to the maintenance of PNE cells in the normal lung as well as in hyperplasia of this population in various disease states. PNE cells are known to act as a progenitor cell for the establishment of NEB hyperplasia and represent one of two proliferative populations within hyperplastic NEBs of the naphthalene-injured lung. Participation of non-PNE progenitor cells in this process has also been demonstrated and may contribute to the intermediate phase of NEB hyperplasia. These data suggest that multiple cell types contribute to the maintenance and expansion of the NEB-associated PNE population and that progenitor selection may be a dynamic feature of NEB hyperplasia. Findings from various studies have established PNE cells as a progenitor population that is sufficient for the development of both NEB hypertrophy and hyperplasia. Although NEB dysplasia is correlated with preneoplastic conditions and PNE cells are thought to serve as a precursor for the development of small cell lung carcinoma, mechanisms regulating the expansion of the PNE cell population are not well understood. Based on studies performed in animal models, it has been suggested that NEB-associated progenitor cells that are phenotypically distinct from PNE cells contribute to PNE cell hyperplasia. However, when considering mechanisms that may account for PNE cell hyperplasia, the finding that multiple cell types proliferate in the NEB microenvironment raises the possibility that a non-PNE cell progenitor may yield progeny cells with the capacity to undergo PNE cell differentiation.

#### **2.3 Neuroepithelial bodies**

Neuroendocrine bodies were illustrated in 1949 in the description of neuroendocrine cells in the bronchiolar mucosa. Neuroendocrine bodies consist of a cluster of 4 to 10 neuroendocrine cells. On well-oriented sections, they can extend from the subepithelial basement membrane to the airway lumens. They are found not only in the epithelium of bronchi and bronchioles, but also in alveoli. The neuroepithelial body (NEB) is a highly dynamic structure that responds to chronic airway injury through hyperplasia of the associated PNEC. NEB-associated epithelial cells share many morphological and biochemical characteristics with cells that are distributed throughout the airway. Pulmonary NEBs are prime candidates to serve as sensory end organs in the lung. NEBs consist of highly organised clusters of specialised cells with neuroendocrine characteristics, arranged into organoids that are dispersed throughout the epithelium at all levels of the intrapulmonary airways. Structurally, NEB cells harbour cytoplasmic neurosecretory granules that are known to contain monoamine, peptide, and purine transmitters. Neuroendocrine cells are able to synthesise and release ATP, monoamine, and peptide transmitters, resulting in autocrine, paracrine, or endocrine effects. Morphologically, NEBs resemble other known chemoreceptors, such as taste buds and carotid bodies, and are thought to represent "chemosensors" among other possible functions. Hypoxic conditions appear to depolarise NEB cells via a potassium channel-mediated mechanism. In particular, the extensively innervated aggregates of the neuroendocrine cells, called neuroepithelial bodies (NEBs), are diffusely spread in the epithelium at all levels of the intrapulmonary airways but are preferentially located at the airway bifurcation points in the lungs. Proportionally, most NEBs are found in the bronchioles and in the terminal respiratory bronchioles. The NEB microenvironment may represent an analogous structure within the conducting airway epithelium for maintenance of an airway stem-cell pool. It may influence the phenotype of the CE cells, blocking the differentiation from Clara to ciliated cells and preserving a population of regenerative cells that can contribute to epithelial renewal after exposure to Clara cell toxicants. Regeneration of the chronically injured airway epithelium is associated with alterations in the number and cellularity of the NEBs as well as the enrichment of nascent epithelial cell populations of epithelial cells that are candidate stem cells. The NEB microenvironment is multifunctional, serving to maintain slow-cycling epithelial cells in the steady state epithelium and to stimulate the proliferation of TA cells either after airway injury or during airway development. Many studies have presented extensive evidence that NEBs in the lungs may be selectively contacted by at least 5 distinct nerve-fibre populations that are both sensory and motor in nature. In addition, they have different origins, indicating that NEBs should be regarded as very complex airway receptors that may be capable of accommodating various chemoand mechano-sensory modalities.

#### **2.3.1 Functions**

206 Lung Diseases – Selected State of the Art Reviews

proliferative populations within hyperplastic NEBs of the naphthalene-injured lung. Participation of non-PNE progenitor cells in this process has also been demonstrated and may contribute to the intermediate phase of NEB hyperplasia. These data suggest that multiple cell types contribute to the maintenance and expansion of the NEB-associated PNE population and that progenitor selection may be a dynamic feature of NEB hyperplasia. Findings from various studies have established PNE cells as a progenitor population that is sufficient for the development of both NEB hypertrophy and hyperplasia. Although NEB dysplasia is correlated with preneoplastic conditions and PNE cells are thought to serve as a precursor for the development of small cell lung carcinoma, mechanisms regulating the expansion of the PNE cell population are not well understood. Based on studies performed in animal models, it has been suggested that NEB-associated progenitor cells that are phenotypically distinct from PNE cells contribute to PNE cell hyperplasia. However, when considering mechanisms that may account for PNE cell hyperplasia, the finding that multiple cell types proliferate in the NEB microenvironment raises the possibility that a non-PNE cell progenitor may yield progeny cells with the capacity to undergo PNE cell

Neuroendocrine bodies were illustrated in 1949 in the description of neuroendocrine cells in the bronchiolar mucosa. Neuroendocrine bodies consist of a cluster of 4 to 10 neuroendocrine cells. On well-oriented sections, they can extend from the subepithelial basement membrane to the airway lumens. They are found not only in the epithelium of bronchi and bronchioles, but also in alveoli. The neuroepithelial body (NEB) is a highly dynamic structure that responds to chronic airway injury through hyperplasia of the associated PNEC. NEB-associated epithelial cells share many morphological and biochemical characteristics with cells that are distributed throughout the airway. Pulmonary NEBs are prime candidates to serve as sensory end organs in the lung. NEBs consist of highly organised clusters of specialised cells with neuroendocrine characteristics, arranged into organoids that are dispersed throughout the epithelium at all levels of the intrapulmonary airways. Structurally, NEB cells harbour cytoplasmic neurosecretory granules that are known to contain monoamine, peptide, and purine transmitters. Neuroendocrine cells are able to synthesise and release ATP, monoamine, and peptide transmitters, resulting in autocrine, paracrine, or endocrine effects. Morphologically, NEBs resemble other known chemoreceptors, such as taste buds and carotid bodies, and are thought to represent "chemosensors" among other possible functions. Hypoxic conditions appear to depolarise NEB cells via a potassium channel-mediated mechanism. In particular, the extensively innervated aggregates of the neuroendocrine cells, called neuroepithelial bodies (NEBs), are diffusely spread in the epithelium at all levels of the intrapulmonary airways but are preferentially located at the airway bifurcation points in the lungs. Proportionally, most NEBs are found in the bronchioles and in the terminal respiratory bronchioles. The NEB microenvironment may represent an analogous structure within the conducting airway epithelium for maintenance of an airway stem-cell pool. It may influence the phenotype of the CE cells, blocking the differentiation from Clara to ciliated cells and preserving a population of regenerative cells that can contribute to epithelial renewal after exposure to Clara cell toxicants. Regeneration of the chronically injured airway epithelium is associated with alterations in the number and cellularity of the NEBs

differentiation.

**2.3 Neuroepithelial bodies**

It has been estimated that NEBs represent <1% of the epithelial cells in human lungs. Some of the supposed functions of NEB in mammalian lungs include the following: 1) the ability of NEB to function as transducers (hypoxia); 2) modulation of bronchomotor tone via targeting bronchial smooth muscle and the associated nerves located directly beneath NEB; 3) promotion and regulation of the growth of developing airways by stimulating the proliferation of local endoderm; 4) release of amine and peptide modulators; and 5) neonatal respiratory adaptation. The lung bud epithelium grows into the adjacent mesenchyme and stars branching to form the future bronchial tree. The various stages are divided into the embryonic, pseudoglandular, canalicular, saccullar and alveolar/microvascular periods. Pulmonary neuroendocrine cells are the first specialised epithelial cell type to appear in lung development. In humans, ultrastructurally distinct primitive PNECs (pre-NE cells), which contain serotonin and neuro-specific enolase (NSE), can be detected in the beginning of the pseudoglandular period. Solitary and clustered PNECs contain bombesin, the major neuropeptide in human lungs, which appears in the early weeks of gestation. As the distal segments of the developing airways elongate, a process referred to as the canalicular period, PNECs differentiate first, followed by ciliated and secretory (Clara) cells. Parallel with the increasing number of peripheral airways, the number of PNECs also increases. In the developing bronchioles, small NEBs composed of 3-5 bombesin and serotoninimmunoreactive cells appear at the airway branching points, and rare nerve endings have been demonstrated to be in contact with NEBs already in the human foetal lung. Proposed roles for PNECs in foetal and newborn lung development include the regulation of branching morphogenesis as well as cellular growth and maturation.

#### **2.3.2 Airway oxygen sensors**

Since 1930, evidence has accumulated to suggest that NEBs may function as hypoxiasensitive airway sensors. NEB cells express membrane-bound O2 sensors and are the transducers of the hypoxic stimulus. NEB cells respond to acute hypoxia, but apparently not to hypercapnia with the degranulation of dense core vesicles and release of 5 hydroxytryptamine (5-HT). Morphologic and experimental studies to support NEB functions as hypoxia-sensitive airway chemoreceptors modulated by the central nervous system include the following: a) preferential location of NEB at airway branching points; b) apical microvilli in contact with the airway lumen; c) cytoplasmic neurosecretory granules containing monoamine and neuropeptides; d) afferent sensory innervation derived from the vagus nerve; and e) proximity to blood capillaries. NEBs are predominantly innervated by sensory nerve fibres derived from cell bodies in the nodose ganglion of the vagus nerve. Morphological data support the role of NEBs as hypoxia-sensitive airway sensor systems. Studies on the effects of chronic hypoxia have shown induced cellular hyperplasia and hypertrophy in the peripheral chemoreceptors; chronic normobaric hypoxia showed a significant increase in the number of solitary pulmonary neuroendocrine cells (PNECs) as well as the enlargement of NEBs. NEB cells possess an oxygen-binding protein, cytochrome *b*, an NADPH oxidase located in the cellular membranes that acts as the O2 receptor both during normoxia and hypoxia.

## **3. Pathology of neuroendocrine tumours of the lung**

The neuroendocrine cell system is divided into cell types that form glands and diffusely distributed cells. This second group is collectively known as the diffuse neuroendocrine system (DNES), and its representatives are found in the lung, gastrointestinal tract, or urogenital tract. Neuroendocrine tumours of the lung arise from bronchial mucosal cells known as Kulchitsky cells, which are part of the DNES. The classification of lung neuroendocrine malignancies has been an evolving process (Table 1).

WHO/IASLC histological classification

WHO: World Health Organization; IASLC: International Association for the Study of Lung Cancer \* From Travis WD, Colby TV, Corrin B, et al in collaboration with pathologists from 14 countries. Histological typing of lung and pleural tumors. 3rd ed. Berlin: Springer Verlag, 1999, with permission.

Table 1. Lung tumours with neuroendocrine morphology include the low-grade typical carcinoid (TC), intermediate-grade atypical carcinoid (AC), and the high-grade LCNEC and SCLC.

These classifications date back to 1972, when atypical carcinoids were initially defined according to histological criteria, including the number of mitoses per high-power field (hpf), the presence of necrosis, increased cellularity with disorganisation, nuclear pleomorphism, hyperchromatism, and an abnormal nuclear to cytoplasmic ratio (Table 2).

In 1991, a new classification proposed 4 categories of neuroendocrine lung tumours that included the following: typical carcinoid (TC), which is a low-grade malignancy; atypical carcinoid (AC), which is a medium-grade malignancy; large-cell neuroendocrine carcinoma (LCNEC), which is a high-grade malignancy; and small-cell lung cancer (SCLC), which is also a high-grade malignancy. The 2004 WHO categorisation of tumours with neuroendocrine features included the classic carcinoid low-grade TC and intermediategrade AC, as well as the high-grade malignancies LCNEC and SCLC.

Morphological data support the role of NEBs as hypoxia-sensitive airway sensor systems. Studies on the effects of chronic hypoxia have shown induced cellular hyperplasia and hypertrophy in the peripheral chemoreceptors; chronic normobaric hypoxia showed a significant increase in the number of solitary pulmonary neuroendocrine cells (PNECs) as well as the enlargement of NEBs. NEB cells possess an oxygen-binding protein, cytochrome *b*, an NADPH oxidase located in the cellular membranes that acts as the O2 receptor both

The neuroendocrine cell system is divided into cell types that form glands and diffusely distributed cells. This second group is collectively known as the diffuse neuroendocrine system (DNES), and its representatives are found in the lung, gastrointestinal tract, or urogenital tract. Neuroendocrine tumours of the lung arise from bronchial mucosal cells known as Kulchitsky cells, which are part of the DNES. The classification of lung

during normoxia and hypoxia.

WHO/IASLC histological classification

Large cell neuroendocrine carcinoma (LCNEC)

Large Cells carcinoma (Variants)

Combined small cell carcinoma

Preinvasive Lesions

Carcinoid tumour Typical carcinoid (TC) Atypical carcinoid (AC) Small cell carcinoma

\*

SCLC.

**3. Pathology of neuroendocrine tumours of the lung**

neuroendocrine malignancies has been an evolving process (Table 1).

Combined large cell neuroendocrine carcinoma (C-LCNEC)

grade AC, as well as the high-grade malignancies LCNEC and SCLC.

Diffuse idiopathic pulmonary neuroendocrine cell hyperplasia (DIPNECH)

Non-small Cell Lung Carcinoma with Neuroendocrine Differentiation (NSCLC-NED) WHO: World Health Organization; IASLC: International Association for the Study of Lung Cancer

These classifications date back to 1972, when atypical carcinoids were initially defined according to histological criteria, including the number of mitoses per high-power field (hpf), the presence of necrosis, increased cellularity with disorganisation, nuclear pleomorphism, hyperchromatism, and an abnormal nuclear to cytoplasmic ratio (Table 2). In 1991, a new classification proposed 4 categories of neuroendocrine lung tumours that included the following: typical carcinoid (TC), which is a low-grade malignancy; atypical carcinoid (AC), which is a medium-grade malignancy; large-cell neuroendocrine carcinoma (LCNEC), which is a high-grade malignancy; and small-cell lung cancer (SCLC), which is also a high-grade malignancy. The 2004 WHO categorisation of tumours with neuroendocrine features included the classic carcinoid low-grade TC and intermediate-

From Travis WD, Colby TV, Corrin B, et al in collaboration with pathologists from 14 countries. Histological typing of lung and pleural tumors. 3rd ed. Berlin: Springer Verlag, 1999, with permission. Table 1.  Histopathological Classification of Neuroendocrine Tumours of Lung

#### **3.1 Tumourlets**

Carcinoid tumours that grow in the peripheral lung and are smaller than 5 mm are referred to as tumourlets. By definition, tumourlets are comprised of increased numbers of individual cells, small group cells, or nodular aggregates of cells that are confined to the bronchial/bronchiolar epithelium (with larger lesions bulging into the lumen but not breaking the subepithelial basement membrane).

#### **3.2 Diffuse Idiopathic Pulmonary Neuroendocrine Cell Hyperplasia**

Diffuse idiopathic pulmonary neuroendocrine cell hyperplasia (DIPNECH) is a rare condition in which neuroendocrine cells proliferate throughout the peripheral airways in the form of neuroendocrine cell hyperplasia, tumourlets, and sometimes carcinoid tumours. In DIPNECH, neuroendocrine cell hyperplasia and tumourlets are thought to be a primary proliferation in contrast to the much more common situation where these lesions are seen as a reactive secondary lesion in the setting of airway inflammation and/or fibrosis. This condition is regarded as a precursor to carcinoid tumours because a subset of these patients experience one or more carcinoid tumours. More aggressive forms of lung carcinoma, including SCLC, have not been associated with DIPNECH.

#### **3.3 Carcinoid tumours**

Pulmonary or bronchial carcinoid tumours account for over 25% of all carcinoid tumours and for 1%-2% of all pulmonary neoplasms. Approximately 10%-20% of pulmonary carcinoids are typical carcinoids; the remaining 80%-90% are atypical carcinoids. Most of these tumours occur centrally and involve the main, lobar, or segmental airways. Sometimes they are located distal to the segmental bronchi; such tumours are the so-called peripheral carcinoids. Atypical carcinoids have been reported to be larger than typical carcinoids, with mean diameters of 3.6 cm and 2.3 cm, respectively. Moreover, atypical carcinoids are more likely to occur in the periphery of the lung than are typical carcinoids. It was generally accepted that a carcinoid tumour was a very slow-growing and benign neoplasm with no potential for invasiveness and no tendency to give rise to metastases. Carcinoid tumours have subsequently been reported in a wide range of organs, but they most commonly involve the lungs and the gastrointestinal tract. The histopathologic features that distinguish atypical carcinoids from typical carcinoids are as follows: increased mitotic activity; greater cytological pleomorphism and higher nuclear to cytoplasmic ratios; increased cellularity and architectural irregularities, and more areas of tumour necrosis. In terms of histological features, typical carcinoids show no evidence of necrosis and fewer than 2 mitoses per 10 high-power fields (or 2 mm2) of viable tumour, whereas atypical carcinoids do have areas of necrosis and 2-10 mitoses per 10 high-power fields.

## **3.4 Large-Cell Neuroendocrine Carcinoma**

Large-cell neuroendocrine carcinoma (LCNEC) was proposed as the fourth category of pulmonary neuroendocrine tumours due to its distinct clinical and pathologic findings versus the typical carcinoid, atypical carcinoid, and SCLC. LCNEC is defined as a poorly differentiated and high-grade neuroendocrine tumour that morphologically is between an atypical carcinoid and SCLC. According to the WHO suggestions, the morphologic features of LCNEC represent a spectrum between those of atypical carcinoid and those of SCLC. In 70%-80% of cases, LCNEC appears as a peripheral mass or nodule, whereas 25% manifests as a central mass. Histopathologic diagnosis criteria for LCNEC are as follows: neuroendocrine morphologic features; a high mitotic rate (>10 per 10 high-power fields); necrosis (often large zones); cytologic features different from those of SCLC; and positive immunohistochemical staining for one or more neuroendocrine markers including chromogranin A, synaptophysin , and neural cell adhesion molecular (NCAM/CD56).

## **3.5 Small-cell lung carcinoma**