Role of Thyroid Transcription Factor-1 in Pulmonary Adenocarcinoma

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9 Role of Thyroid Transcription Factor-1 in Pulmonary Adenocarcinoma Yasushi Yatabe

Introduction Tissue-specific gene expression is mediated largely by transcription factors, and a master regulatory gene is thus a potential marker of cellular lineage. The myoD gene family is an example of such a lineage marker (Weintraub et al., 1991). The expression of these genes is restricted to the skeletal muscle cell lineage, and ectopic expression switches the cell type to a myogenic phenotype. Thus, the myoD family fulfills most criteria for a lineage marker due to its restricted expression and its function as a master regulatory molecule (Weintraub et al., 1991). It is noted that the gene is not expressed after cell differentiation is complete. Using this property, the expression of myoD in tumor cells indicates both neoplastic nature and differentiation into skeletal muscle cells, implying the diagnosis of rhabdomyosarcoma. In this manner, the lineage markers decide the fate of the cells, and the cancer is also likely to be characterized by the features of the originating cells, acquired through the lineage marker. Thyroid transcription factor-1 (TTF-1) also known as Nkx2.1 or thyroid-specific enhancer-binding protein, (Korfhagen et al., 1997; Mendelson, 2000) is a Handbook of Immunohistochemistry and in situ Hybridization of Human Carcinomas, Volume 1: Molecular Genetics; Lung and Breast Carcinomas

homeodomain-containing transcription factor that regulates tissue-specific expression of the surfactant apoprotein A (SPA) (Bruno et al., 1995), surfactant apoprotein B (Yan et al., 1995), surfactant apoprotein C (Kelly et al., 1996), Clara cell antigen (Toonen et al., 1996), and T1α (Ramirez et al., 1997) by directly binding to these promoters. The expression of TTF-1 is initiated at a very early stage of lung morphogenesis. In the developing mouse lung, TTF-1 expression first appears at emergence of the laryngotracheal diverticulum and is localized primarily in the branching bronchial epithelium for the next 6 days (Kimura et al., 1996; Lazzaro et al., 1991; Zhou et al., 1996). Once peripheral airway tubes develop, the expression shifts to the peripheral airway epithelium, and this pattern is retained until death (Kimura et al., 1996; Lazzaro et al., 1991; Zhou et al., 1996). TTF-1−/− knockout mice show a tracheoesophageal fistula and severe pulmonary hypoplasia, suggesting TTF-1 also plays a crucial role in lung morphogenesis (Kimura et al., 1996; Minoo et al., 1999). TTF-1 is indispensable for lung function. Several reports have already described the differences in TTF-1 expression between histologic types.

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170 The expression of TTF-1 appears in 71–76% (mean, 72%) of adenocarcinomas and 81–92% (mean, 89%) of small cell carcinomas (SCLC), whereas it was not found, or only found at very low frequency, in other types of non–small cell carcinoma (NSCLC) and carcinoma of the other organs, except thyroid cancers (Di Loreto et al., 1997; Fabbro et al., 1996; Kaufmann et al., 2000; Ordonez, 2000b; Puglisi et al., 1999; Yatabe et al., 2002). Therefore, most of the reports emphasized the practical usefulness of TTF-1 in the differential diagnosis of lung cancer from nonpulmonary cancers, as a result of the tissue specific expression (Di Loreto et al., 1997; Fabbro et al., 1996; Katoh et al., 2000). In contrast, few studies referred to the possible significance of TTF-1 expression in lung carcinogenesis, despite its crucial roles in lung development and maintenance of pulmonary function by means of SPA and Clara cell-specific 10-KD protein (CC10) induction. Based on the recent development of moleculetargeted drugs, tumor classification schemes are shifting to that based on the molecular mechanisms of carcinogenesis. Imatinib mesylate (Demetri, 2001; Joensuu et al., 2001) elucidated the molecular basis of relatively rare gastrointestinal tumors and proposed new subclassification within the ordinary classification schema (Berman et al., 2001). Because of its multistep derivation, the tumors should be quite heterogeneous. To understand the underlying mechanism, it is important to recognize what categorization is biologically significant and what discriminator is used for the categorization. In this chapter, we attempt to describe the biological significance of TTF-1 expression in lung adenocarcinomas and show that TTF-1 sheds light on a subtype of lung adenocarcinomas, in terms of cellular lineage and molecular carcinogenesis (Yatabe et al., 2002).

MATERIALS 1. Phosphate-buffered saline (PBS) (for 20 L). a. 9 g of NaH2PO42H2O, 64.5g of NaH2PO412H2O and 160 g of NaCl. b. Place in the carboy/plastic container, pour 40 L of distilled water, and mix thoroughly. c. Adjust pH to 7.4, if necessary (unnecessary most times). 2. McIIvaine citrate buffer for antigen retrieval (for 500 mL). a. 3.76g of citrate acid monohydrate and 23g of NaH2PO412H2O. b. Dissolve well with 500 mL of distilled water, and adjust pH to 6.4. 3. Antibody dilution buffer. a. Filtered PBS with 0.001% thimerosal (for an antiseptic) and 0.01% bovine serum albumin.

4. Aminobenzidine tetrachloride (DAB) solution: DAB tablets are commercially available (Dako Copenhagen, Denmark, or Novocastra, Newcastle upon Tyne, U.K.). 5. Mayer’s hematoxylin. 6. Vextastain elite ABC kit (Vector Laboratories, Inc., Burlingame, CA); follow the manufacturer’s instructions.

METHODS The staining procedure is the same as that used for common immunohistochemical analysis, except for the use of freshly prepared sections. This is critical for TTF-1 staining, and the staining must be performed within a week of preparation. Non-neoplastic type II pneumocytes served as internal controls for antigen preservation. 1. Deparaffinize with four changes of xylene for 5 min each, followed by four changes of 100% ethanol for 4 min each. 2. Blocking of endogenous peroxidase in methanol with 0.1% H2O2 for 20 min. 3. Rinse sections in three changes of prechilled PBS. 4. Antigen retrieval: a. Place sections in a heat-stable plastic Coplin jar. b. Fill with an excess amount of citrate buffer (pH 6.4). c. Autoclave for 10 min. d. Keep the Coplin jar in the processor until the solution is cooled down to room temperature (∼ 60 min). 5. Rinse sections in three changes of prechilled PBS. 6. Remove the excess PBS, place slides horizontally in a humidity incubation chamber, and cover specimens with 3–4 drops of 10% normal serum for 30 min (swine serum is commonly used) to avoid nonspecific antibody binding. 7. Remove the excess normal serum, place slides horizontally in a humidity incubation chamber, and cover specimens with 3–4 drops of TTF-1 antibody (8G7G3, Dako, Copenhagen, Denmark; 1:150 diluted with the antibody dilution buffer) for 1 hr at room temperature or overnight at 4°C. 8. Wash sections in three changes of prechilled PBS. 9. Remove the excess PBS, place slides horizontally in a humidity incubation chamber, and cover specimens with 3–4 drops of anti-mouse antibody for 30 min. 10. Wash sections in three changes of prechilled PBS. 11. Remove the excess PBS, place slides horizontally in a humidity incubation chamber, and cover specimens with 3–4 drops of ABC complex for 30 min.

9 Role of Thyroid Transcription Factor-1 in Pulmonary Adenocarcinoma 12. Wash sections in three changes of prechilled PBS. 13. Develop the color with DAB solution (0.6 mg/mL PBS + 0.1% H2O2) for 5–10 min. 14. Wash sections with tap water. 15. Counterstain with hematoxylin. 16. Dehydrate using four changes of ethanol, and then clear with three changes of xylene, 3 min each. 17. Coverslip sections with Permount (Fisher Scientific, Pittsburgh, PA). The positive signals are seen as a brown color that is localized in the nuclei.

RESULTS AND DISCUSSION T TF-1 Expression as a Marker of Terminal Respiratory Unit in the Normal Lung It has been reported that TTF-1 expression during rat development is restricted to the thyroid, lung, and forebrain. In the former two organs, the expression is observed at the earliest stage of development and is preserved throughout lifetime (Kimura et al., 1996; Minoo et al., 1999; Stahlman et al., 1996). Expression of TFF-1 in the human lung is also restricted to the peripheral portion in both fetal and adult lungs (Stahlman et al., 1996, Yatabe, 2002), and expressing cells include pneumocytes (both type I and type II) and a part of small-sized bronchioles (Figure 24). These epithelia stained very uniformly and consistently, and TTF-1 appeared to label a series of cells that represented a certain functional unit or common lineage. Taken together with transcriptional activity of TTF-1 for functional molecules, such as surfactant apoprotein and Clara cell antigen, we therefore considered these epithelia as a functional or lineage unit, termed the terminal respiratory unit (TRU). It is of note that the morphological difference between TTF-1 positive and negative bronchioles was obscured, and that not all, but only a part of the bronchioles, (more peripheral portion of bronchioles) included the TRU.

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(Fabbro et al., 1996; Sturm et al., 2002), the expression is not related to neuroendocrine differentiation. Recent evidences in stem cell research indicate that stem cells expressed a broad range of genes (Akashi et al., 2003; Shamblott et al., 2001). Morphologically, SCLCs appear as very primitive or undifferentiated cells, and TTF-1 expression in SCLCs is suggested to have some association with the multilineage gene expression of the stem cells. Indeed, ectopic expression of c-Kit, stem cell factor (Hibi et al., 1991; Sekido et al., 1991) and some cancer testis antigens (Sugita et al., 2002) are more common in SCLC than in NSCLC. Although expression of TTF-1 in nonpulmonary small cell carcinomas is controversial (Agoff et al., 2000; Kaufmann et al., 2000; Oliveira et al., 2001; Ordonez, 2000b), the hypothesis is supported by our data showing frequent positivity in nonpulmonary small cell carcinoma. Of the nine cases, six of the nonpulmonary small cell carcinoma expressed TTF-1, and in the combined small cell and ordinary carcinomas, only portions showing the small cell carcinoma morphology were positive for TTF-1. As for the tumors with small cell carcinoma morphology, the TTF-1 expression may represent undifferentiated features of the cancer cells. In thyroid carcinomas, TTF-1 is invariably expressed in follicular neoplasm and papillary carcinoma (Bejarano et al., 2000; Katoh et al., 2000), reflecting the normal expression pattern. However, negative expression of TTF-1 in anaplastic carcinoma is rather common. This was explained by “dedifferentiation” of the follicle-derived tumors (Bejarano et al., 2000; Heldin et al., 1991). It is of note that one case of breast cancer was shown to express TTF-1. The case is a 43-year-old female without prior history of any cancers. Two cancer nodules (25 mm and 5 mm in the largest dimensions) in the right breast were surgically resected. Although histology showed an ordinary infiltrating lobular carcinoma (Figure 24). TFF-1 was expressed in both tumors. The presence of in situ lesions and no other cancers during 8 years of follow-up indicates the cancers are actually of breast origin.

T TF-1 Expression in the Neoplasia To examine whether the restricted expression in normal tissue is reflected in the tumors, we applied a tissue microarray method in addition to regular examination with whole sections. The results are summarized in Table 10. The distribution of the TTF-1 expressing tumors is quite similar to that of the pattern in normal tissues, and the expression is restricted to lung adenocarcinoma and thyroid tumors, with a few exceptions. One of the exceptions is SCLC (Figure 24). Because none of the carcinoid tumors expressed TTF-1

T TF-1 Expression in the Lung Adenocarcinoma The expression of TTF-1 was observed in 75.7% of lung adenocarcinomas in our series, and about 70–75% of the adenocarcinomas have been reported to be positive in the literature (Kaufmann et al., 2000; Ordonez, 2000a; Pelosi et al., 2001; Puglisi et al., 1999). The expression pattern is characterized by quite uniform staining; when the tumor cells are positive, almost all cancer cells express TTF-1, regardless of malignancy potential, differentiation, and cytologic atypia. For example,

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Figure 24 TTF-1 expression in normal peripheral lung (upper left), lung adenocarcinoma (TRU-type, upper right), small cell lung cancer (lower left), and an unusual case of breast cancer (lower right).

adenocarcinoma frequently presents as a low-grade lesion in the periphery and high-grade in the center; TTF1 is expressed in both the low-grade peripheral lesion and central high-grade lesion in the individual TTF-1 positive tumors. This feature is maintained even in the metastatic sites. This is contrasted to the expression pattern of surfactant apoprotein A, a functional marker of the lung, which was frequently lost in the metastatic site. In this sense, it is quite reasonable to use TTF-1 to detect micrometastasis in this lymph nodes and malignant effusion of lung adenocarcinoma. Conversely, ∼ 75% of TTF-1 positivity indicated that lung adenocarcinomas could be subdivided by their TTF-1 expression status, which is a functionally important transcription factor in the lung and a marker of

TRU in the normal lung. This raised two questions: 1) whether TTF-1 is useful as a marker of adenocarcinoma derived from the TRU, and 2) what is the significance of subdividing by TTF-1 status.

Morphologic Characteristics of TTF-1 Expressing Adenocarcinoma To obtain the morphologic characteristics of TTF-1 expressing tumors, we focused on the similarity to normal counterparts of the airway epithelium. Based on the morphological resemblance to type II pneumocytes, Clara cells, and small-sized bronchioles, we simply categorized the tumor as TRU or non-TRU type (Figure 25). This morphological classification is very

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Table 10 T TF-1 Expression in Various Tumors Organ

Subtype

n=

Positive

Nonneuroendocrine Tumors Breast Cervix uteri Colon Endometrium Esophagus Head and neck Lung

Ovary Pancreas Parotid glands Stomach Thyroid

Lung Esophagus Stomach Colon Bladder Uterine cervix Thymus aIncluding b64

AD 14 AD 5 SQ 10 AD 16 AD 10 AD 1 SQ 7 SQa 20 AD 296 b SQ 74 LA 25 AS 11 Adenoma 4 AD 10 AD 3 Benign tumors 38 AD 60 AD 19 Adenomatous goiter 6 Follicular adenoma 18 Follicular carcinoma 13 Papillary carcinoma 20 Anaplastic carcinoma 2 Neuroendocrine Tumors

1(7.1%) 0(0%) 0(0%) 0(0%) 0(0%) 0(0%) 0(0%) 0(0%) 224(75%) 2(2.7%) 7(28.0%) 2(18.2%) 0(0%) 0(0%) 0(0%) 0(0%) 0(0%) 0(0%) 6(100.0%) 18(100.0%) 12(92.3%) 19(95.0%) 0(0%)

Typical carcinoid LCNEC SCLC Small cell carcinoma Carcinoid Carcinoid Small cell carcinoma Small cell carcinoma Carcinoid

0(0%) 13(86.7%) 25(96.2%) 4(66.7%) 0(0%) 0(0%) 1(50%) 1(100%) 0(0%)

2 15 26 6 3 4 2 1 1

three head and neck cancer and one esophageal cancer cases that were weakly positive.

were examined with regular whole sections; the other results were from tissue microarrays.

AD, adenocarcinoma; SQ, squamous carcinoma; LA, large cell carcinoma; LCNEC, large cell neuroendocrine tumor; SCLC, small cell lung cancer.

close to Shimosato’s cytological classification of lung adenocarcinoma (Shimosato, 1989), except dealing with adenocarcinoma resembling bronchioles. We include the adenocarcinoma resembling bronchioles into TRU type. This results from the unexpected findings that TTF-1 labeled small-sized bronchioles in addition to pneumocytes, like a unique functional or lineage unit. Because bronchial-surface type and bronchiole-like adenocarcinoma mimic each other, we therefore paid most attention to distinguishing between them. Major differential characteristics of bronchiole-like adenocarcinoma includes low columnar cells with a dome-shaped

protrusion of each luminal cellular border, but not showing a smooth line at luminal border (Figure 25). With this simple classification of lung adenocarcinomas, 48 of 64 (75%) lung adenocarcinomas in our series were categorized as the TRU subtype and 16 cases (25%) as the non-TRU subtype (Table 11). This classification is justified by the frequent expression of the surfactant apoprotein A (SPA) in the TRU subtype, and also by the fact that SPA is expressed in half of the TRU adenocarcinomas, in contrast to none of the non-TRU adenocarcinomas (Table 11). The majority of TTF-1 positive cases showed TRC morphology (42/46, 91%),

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Figure 25 Histologic features of TRU (upper right and left) and non-TRU (lower left and right) types of adenocarcinomas. Upper left shows bronchiole-like adenocarcinoma (TTF-1 positive), which mimics bronchial surface epithelium-like adenocarcinoma (TTF-1 negative, lower left).

whereas a major proportion of TTF-1 negative adenocarcinomas (12/18, 66.7%) belong to the non-TRU subtype. Conversely, 88% (42/48) of adenocarcinomas with the TRU morphology were TTF-1 positive, whereas only 25% (4/16) of non-TRU tumors expressed TTF-1 (Table 12). These results implied that TTF-1 expression is largely maintained even after malignant transformation of TRU cells. Therefore, TTF-1 is capable of being used as a lineage marker for TRU cells.

Difference in Carcinogenetic Mechanism by T TF-1 Expression Status Susceptibility to a particular carcinogen is likely to differ among cell types due to intrinsic cellular features or anatomic features, such as carcinogen accessibility and clearance rates. For example, an erythroid precursor

cell is particularly susceptible to benzene toxicity (Corti et al., 1998), whereas the size of the particles inhaled as carcinogen determines the anatomic sites to which it will be deposited. Therefore, it is quite reasonable to suggest that cellular lineage affects the carcinogenetic mechanism significantly. Accordingly, clinicopathologic features and cancer-associated genes were compared between TTF-1 positive and negative lung adenocarcinomas. The results are summarized in Table 12. Tumors with TTF-1 expression were significantly more prevalent in females and nonsmokers. Because gender and smoking status were tightly correlated with each other (i.e., 4 of 31 females were smokers and only 4 of 33 men were nonsmokers) it was unclear which of them affected TTF-1 status more significantly in the cohort. The close relationship reflected cultural background, and recently this has been

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Table 11 Clinicopathologic Features by T TF-1 Expression Status TTF-1 (+) 46(72%)

TTF-1(-) 18(28%)

p-valuea

Morphological Characteristics SPA (<1+/2≥) TRC/non-TRC morphology

22/24 42/4

2/16 6/12

<0.01 <0.01

Clinicopathologic Characteristics Female/male ratio Smoker/nonsmoker pStage (I/II/IIIA) pN (0/1/2)

27/19 26/20 31/2/13 31/2/13

4/14 13/5 11/2/5 12/2/4

<0.01 0.04 0.60 0.57

Cancer-Associated Genes p53 accumulation (+/−) p53 mutation/wildtypeb G:T > C:A transversionb K-Ras mutation/wildtypeb CCND1 (+/−) Rb (+/−) p27 (1+/2+/3+/4+) COX2 (+/−)

12/34 21/51 3/14(18%) 4/32 29/17 41/5 0/10/14/22 35/10

12/6 17/16 6/9(40%) 3/19 7/11 11/7 3/7/5/3 15/3

<0.01 0.03 0.24 1.00 0.100 0.02 <0.01 0.74

n=

aEach

value represents the results from Fisher’s exact test or chi-square test.

bAnalysis

using a different cohort from that of the other analysis.

Note: Underlined values indicate statistically significant differences. SPA, surfactant apoprotein A; TRC, terminal respiratory unit.

changing. Using another recent cohort, the logistic regression analysis suggested that smoking status was more highly associated with TTF-1 expression than gender. In contrast to this relationship, pathologic stage as well as local tumor-factor (pT) and nodal status are not associated with TTF-1 expression status, supporting the idea that TTF-1 is not a marker of malignant potential or advanced disease, but a lineage marker of the originating cellular feature. The prognostic imact of TTF-1 status further confirmed the idea, and the Cox-proportional hazard model resulted in no prognostic significance of TTF-1 expression in lung adenocarcinoma. Cancer-associated molecules, p53 accumulation, p53 mutation, K-Ras mutation, and expression of cyclin D1 (CCND1), Rb, p27KIP1, and COX2 were compared between TTF-1 positive and negative lung adenocarcinomas. The well-known tumor suppressor gene p53 is frequently involved in a variety of human cancers. Approximately 50% of NSCLC and 90% of SCLCs exhibit the p53 mutation. Meanwhile K-Ras is also a well-known oncogene, and the mutation of K-Ras is frequently detected in adenocarcinoma but is quite rare in SCLC. In some reports (Kobayashi et al., 1990; Marchetti et al., 1996), the K-Ras is mutated preferentially in mucinous bronchioloalveolar carcinomas, which morphologically resemble pancreatic cancer.

We reported that the lack of the expression in the NSCLCs (61%) was significantly associated with shorter survival by the Cox-proportional hazard model (Nishio et al., 1997). This tendency was particularly remarkable in adenocarcinoma. One of the causative genes for retinoblastoma is Rb, and nearly all SCLCs and ∼ 20% of NSCLCs lack Rb expression (Nishio et al., 1997) due to inactivation of the Rb gene. The CDK inhibitor p27KIP1 is considered responsible for the onset and/or maintenance of the quiescent state (Rivard et al., 1996; Zhang et al., 2000), and only differentiated cells expressed this inhibitor in vivo (Endl et al., 2001). Whereas the majority of SCLCs show increased staining when compared with normal epithelium, p27KIP1 was reduced in 72% of NSCLCs and its reduction was correlated with poor prognosis (Yatabe et al., 1998b). COX2 is an enzyme involved in the conversion of arachidonic acid to prostanoids and plays a well-known role in inflammatory reactions. In additions, recent reports suggest that it has various other functions, including an association with carcinogenesis (Vane, 1994; Williams et al., 1997). The molecule was initially investigated in association with colon carcinogenesis, because COX2 was up-regulated in ∼ 50% of adenoma and 80–85% of adenocarcinoma of the colon (Eberhart et al., 1994; Sano et al., 1995), and mice

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176 without a functional COX2 gene had a significantly reduced incidence of intestinal polyps (Oshima et al., 1996). In the lung, we have reported, increased expression of COX2 was detected in about one-third of precancerous and carcinoma in situ, and in 70% of invasive adenocarcinomas (Yatabe et al., 1998a). Interestingly, we found that the expression is heterogeneous, and the up-regulation was remarkable, especially in the invasive portion of the adenocarcinoma. This was evidenced by homogeneous up-regulation in the metastatic sites and the invasion portion in the corresponding primary tumor. This finding was subsequently confirmed (Niki et al., 2002) and further studies from our group demonstrated that a COX2-specific inhibitor suppressed the invasive phenotype of LNM35, which was established as a cell line with consistent lymphogenous metastasis (Kozaki et al., 2001). These findings suggest that COX2-is associated with invasive growth of lung adenocarcinomas. In comparison with these cancer-associated molecules, TTF-1 status was associated with p53 accumulation, p53 mutation, lack of Rb, and level of p27KIP1 expression. The TTF-1 positive TRU type of adenocarcinoma demonstrated less p53 alteration, infrequent Rb inactivation, and frequent p27KIP1 expression, preserved. As previously mentioned, alteration of p53 and Rb is a hallmark of SCLC and squamous cell carcinoma, both of which are suggested to be associated with cigarette smoking. In terms of the association with p53, Rb, and smoking, TTF-1 negative adenocarcinomas are more closely aligned to SCLCs and squamous cell carcinoma. A difference in the carcinogenetic mechanism between the two subtypes was also supported by frequent G->T transversion in TTF-1 negative adenocarcinoma, although the difference did not reach statistical significance, possibly because of the small number of cases. Taken together with all of these findings,

TTF-1 status divides lung adenocarcinoma into two subtypes from the aspect of molecular carcinogenesis. The characteristics are summarized in Table 13.

Correlation with the Other Subclassifications of Lung Adenocarcinomas Several subclassification schemes for lung adenocarcinomas have been proposed, and thus the question how the distinction by TTF-1 status is related with the previous subclassification may arise. In this chapter, we select three subclassifications of lung adenocarcinomas and compare them with TTF-1 positive and negative adenocarcinoma. First, Dr. Shimosato, of the National Cancer Center in Japan, has proposed a subclassification system based on cytologic features (Shimosato, 1989). As previously mentioned, TTF-1 expressing adenocarcinomas correspond to their classification of type II pneumocyte type, Clara cell type, and their mixed type. Most of the other types, including bronchial surface types and bronchial gland types, do not express TTF-1 in our experience. Second, in comparison with the World Health Organization (WHO) classifications (Travis et al., 1999), bronchioloalveolar, acinar, and papillary (Clara/type II pneumocyte type) subtypes are a major source of TTF-1 positive tumors, whereas most of the TTF-1 negative tumors are composed of acinar (bronchial gland-like or bronchial lining cell-like) and solid tumors with mucin. Classifications by the WHO are mostly based on growth structure but not cellular features, and thus it is difficult to fit the subtypes to TTF-1 expression status. Finally, Noguchi et al. (1995) reported prognostic differences based on their own histologic classification. They focused on the structure and divided adenocarcinoma into six subtypes: localized bronchioloalveolar carcinoma (LBAC), LBAC with foci of

Table 12 Characteristics of TTF-1 Positive and Negative Lung Adenocarcinoma TTF-1 Positive Adenocarcinoma

TTF-1 Negative Adenocarcinoma

Location in the Lung Prevalent Population Carcinogenesis Associated Molecules Precancerous Lesions Characteristic Morphology

Terminal respiratory unit (TRU-deriving carcinoma) Periphery Nonsmoker Probably unique Unknown Atypical adenomatous hyperplasia Lepidic growth, central scar

Frequency in Lung Adenoca

70–75%

Central bronchial epithelium (bronchogenic carcinoma) Hilum, sites associated with larger bronchi Smoker Similar to SCLCs or SQ p53,Rb Not known (de novo?) Solid, homogenous appearance Necrosis, frequent high-grade tumor 25–30%

Putative Originating Cells

TRU, terminal respiratory unit; SCLC, small cell lung cancer; S2, squamous cell carcinoma.

9 Role of Thyroid Transcription Factor-1 in Pulmonary Adenocarcinoma collapse (type B), LBAC with foci of active fibroblastic proliferation (type C), poorly differentiated adenocarcinoma (type D), tubular adenocarcinoma (type E), and papillary adenocarcinoma with destructive growth (type F). The first three were grouped as “replacement type” and are characterized by lepidic growth. In addition, the study emphasized that patients with the LBAC (type A) and LBAC with foci of collapse (type B) showed an excellent prognosis, and thus suggested that these subtypes represent a preinvasive lesion. Adenocarcinoma with TTF-1 expression is equivalent to the replacement type and a part of papillary adenocarcinoma with destructive growth. They further subdivided the replacement type/TTF-1 positive subgroup into an invasive and noninvasive group, by means of the presence or absence of active fibroblastic foci, representing a stromal reaction because of invasion. It is of note that TTF-1 is not a marker of aggressiveness. Indeed, micropapillary adenocarcinoma, a recently suggested aggressive form of adenocarcinoma, frequently expressed TTF-1 (Amin et al., 2002). Almost all atypical adenomatous hyperplasia, a putative precancerous lesion of adenocarcinoma. Expression profiling analysis gave insight into classification based on molecular signatures of tumors. One of the earliest reports on breast cancers categorized three major subtypes of cancers (i.e., luminal, basal, and HER-2/neu amplification subtypes). Interestingly, the expression profiles of each subtype were characterized by cellular lineage and a feature represented by a peculiar gene alteration. In regard to lung cancers, the expression profiling analysis addressed the issue on diversity of lung adenocarcinoma. Two excellent reports had been published in tandem. Using a nonsupervised clustering method, both results identified particular subtypes of adenocarcinoma, some of which were equivalent to the TTF-1 positive TRU type of adenocarcinoma. In the article by Garber et al., (2001), lung cancers were divided into six types according to the molecular signature-adenocarcinoma type 1 to 3, squamous cell carcinoma, small cell carcinoma, and large cell carcinoma. Hierarchical clustering of the relationship among them largely grouped them into two arms; one was composed of the latter four groups (adenocarcinoma type 3, squamous cell carcinoma, small cell carcinoma, and large cell carcinoma), and the other included adenocarcinoma types 1 and 2, of which the profile was close to that of normal lung. Conversely, there were two distinct groups in lung adenocarcinoma (i.e., adenocarcinoma type 1 and 2 versus adenocarcinoma type 3). To distinguish the two groups, molecular hallmarks were extracted among thousands of genes examined, and TTF-1 was listed as one of the hallmarks for a highly expressed gene in adenocarcinoma type 1 and 2.

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On the other hand, Bhattacharjee et al., (2001) described four distinct subtypes (types 1–4) of adenocarcinoma, in addition to squamous cell carcinoma, small cell carcinoma, and metastatic carcinoma, based on their expression profiles. Type 1 was poorly differentiated adenocarcinoma, and this type showed high levels of cell division genes, similar to squaous and small cell carcinomas. Type 2 is characterized by the expression of neuroendocrine molecules and demonstrated poor prognosis, confirming the previous report (Carnaghi et al., 2001; Graziano et al., 1994). Type 3 and 4 shared high expression of the surfactant apoproteins, and morphologically, type 4 are largely bronchioloalveolar carcinoma. Therefore, types 3 and 4 appeared to be consistent with TTF-1 positive adenocarcinoma. Both reports were summarized to suggest the existence of distinct subtypes of adenocarcinoma, correspond to TTF-1 positive adenocarcinoma, and that again, TTF-1 positive adenocarcinoma could be further subdivided, probably reflecting invasive growth. In conclusion, we indicated that TTF-1 is expressed consistently throughout the life stages and uniformly in the TRU. The TTF-1 positive adenocarcinomas, which are suggested to derive from the TRU, share distinct clinicopathologic and molecular characteristics, suggesting different mechanisms of molecular carcinogenesis from the TTF-1 negative adenocarcinomas. We suspect that TTF-1 is a marker that discriminates the molecular mechanism of carcinogensis, based on the originating cellular lineage.

Acknowledgments The author thanks Yoshitsugu Horio for critical review of the manuscript, Takashi Takahashi and Tetsuya Mitsudomi for insightful discourse, and Kaori Hayashi for technical assistance.

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