Epidermal growth factor receptor mutation status and clinicopathological features of combined small cell carcinoma with adenocarcinoma of the lung

Abstract

In lung cancer, somatic mutations of epidermal growth factor receptor (EGFR) are concentrated in exons 18–21, especially in adenocarcinoma (Ad), but these mutations have rarely been reported in small cell lung carcinoma (SCLC). Combined SCLC is rare, and the EGFR mutation status and its relationship to the clinicopathological features of this tumor type have not yet been elucidated. We retrospectively studied six patients with combined SCLC with Ad components among 64 consecutive patients who underwent resection of SCLC. The clinicopathological features of each patient were reviewed, especially for the distribution pattern of the Ad component and lymph node metastases. EGFR mutations were screened by high‐resolution melting analysis in each case, and were confirmed by sequencing of each mutation in the microdissected SCLC or Ad components. Regarding EGFR, no specific mutation was detected in five of the six patients, whereas one female patient who had never smoked had a missense mutation. In this case, both the SCLC and Ad components shared the same mutation in exon 21 (L858R). We identified a patient with combined SCLC with Ad sharing an identical EGFR mutation in both the SCLC and Ad components. In addition to the clinicopathological characteristics of this rare histological type of lung cancer, these findings provide useful information for better understanding the biology, natural history and clinical management of SCLC. (Cancer Sci 2007; 98: 1714–1719)

Small cell lung carcinoma (SCLC) accounts for 15–20% of all lung cancers worldwide.^{( 1 )} SCLC is known to be more sensitive than non‐SCLC to chemotherapy, but shows a more aggressive clinical course. The median survival time without treatment is 2–4 months.^{( 2} , ^{3 )} Approximately 20% of patients with limited SCLC achieve a cure, but most patients with SCLC will relapse, and relapsed or refractory SCLC has a uniformly poor prognosis with a 5‐year survival rate of less than 5%.^{( 4 )}

According to the 2004 World Health Organization (WHO)/International Association for the Study of Lung Cancer (IASLC) classification of lung and pleural tumors,^{( 5 )}‘combined SCLC’ is defined as SCLC combined with an additional component that consists of any of the histological types of non‐SCLC, usually adenocarcinoma (Ad), squamous cell carcinoma (Sq) or large cell carcinoma. Combined SCLC is rare, and has been reported to account for less than 1–3.2% of all SCLC.^{( 6} , ^{7 )} However, a high proportion (12–26%) of SCLC patients who undergo surgical resection show combination with non‐SCLC.^{( 8} , ⁹ , ¹⁰ , ¹¹ , ^{12 )}

In a clinical setting, the distinction of SCLC from non‐SCLC is critical because of major differences in patient management and prognosis. Recently, molecular targeted therapy has been developed using agents such as epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, which exerts antitumor activity in patients with advanced non‐SCLC (especially Ad) with EGFR mutations. High expression of EGFR has been reported in various epithelial malignant tumors, including lung cancer,^{( 13} , ^{14 )} and somatic mutations in the kinase domain of EGFR are suggested to be strongly correlated with sensitivity to EGFR tyrosine kinase inhibitor.^{( 15} , ^{16 )} These mutations are concentrated in exons 18–21 of EGFR, and approximately 90% of EGFR‐mutant patients with lung Ad have mutations in two hot spots: in‐frame deletion at codons 747–749 (DEL) in exon 19, and a missense mutation at codon 858 (L858R) in exon 21.^{( 17} , ^{18 )} Although these mutations have rarely been reported in SCLC, two recent studies have demonstrated EGFR mutation in SCLC.^{( 19} , ^{20 )}

In the present study, we retrospectively investigated six resected cases of combined SCLC with an Ad component to elucidate the clinicopathological features of this rare tumor, especially the ratio of each tumor component, the distribution patterns of the Ad component, and the status of lymph node metastasis. The EGFR mutation status in surgically resected specimens was also analyzed for each histological type in the same tumor.

Materials and Methods

Patients and histological diagnosis.  A search of our surgical pathology files covering the period January 1982 to December 2004 yielded 64 consecutive patients with SCLC who had undergone surgical resection at the National Cancer Center Hospital, Tokyo, Japan. For the purposes of the present study, we identified six patients with combined SCLC with an Ad component. The research protocol was approved by the Institutional Review Board.

The surgically resected specimens were fixed in 10% formalin. All sections containing both tumor tissues and surrounding lung tissues were embedded in paraffin. Additional consecutive 5 µm‐thick sections were cut from the tissue block and stained with hematoxylin and eosin. All histological diagnoses were reviewed by certificated pathologists (K. T., A. M. M. and Y. M.) based on the most recent WHO/IASLC classification of lung and pleural tumors.^{( 5 )} Both clinical and pathological staging data for each patient have been reported according to the International Staging System for Lung Cancer.^{( 21 )} Patient survival was calculated as the time between operation and death.

Immunohistochemistry and evaluation.  For phenotypic analysis, paraffin section immunohistochemistry was carried out using the primary antibodies listed in Table 1, followed by subsequent labeling with the Envision+ horseradish peroxidase (HRP) system (DAKO, Carpinteria, CA, USA). For heat‐induced epitope retrieval, sections stained for p63 were treated with 1.0 mmol/L ethylenediaminetetraacetic acid buffer (pH 8.0). Sections stained for chromogranin A (1:500, polyclonal; DAKO), synaptophysin (1:100, polyclonal; DAKO), neural cell adhesion molecule (NCAM) (1:200, Lu243; Nihon Kayaku, Tokyo, Japan), thyroid transcription factor (TTF)‐1 (1:100, 8G7G3/1; DAKO), p63 (1:100, 4A4; DAKO) and D2‐40 (1:50, D2‐40; DAKO) were treated with 0.02 mol/L citrate buffer (pH 6.0). The slides were incubated overnight with each primary antibody. Diaminobenzidine was used as the chromogen, and hematoxylin as the counterstain.

Table 1.

Results of immunohistochemistry

Patient no.	SCLC component (%)	Immunoreaction					Non‐SCLC component (%)	Immunoreaction					No. tumor embolism cells per slice † (%)
		CgA	SYN	NCAM	TTF‐1	p63		CgA	SYN	NCAM	TTF‐1	p63	SCLC	Ad	Sq
1	95	2+	3+	3+	3+	0	Ad, 5	1+	1+	1+	3+	0	30 (97)	1 (3)	–
2	80	3+	3+	3+	3+	0	Ad, 10	0	1+	1+	2+	1+	21 (84)	3 (12)	1 (4)
							Sq, 10	0	0	0	0	3+
3	70	1+	3+	3+	3+	0	Ad, 30	0	1+	0	3+	0	38 (93)	3 (7)	–
4	55	2+	3+	3+	3+	0	Ad, 45	0	0	1+	1+	0	24 (92)	2 (8)	–
5	35	3+	3+	3+	3+	0	Ad, 60	1+	1+	1+	3+	1+	17 (100)	0 (0)	0 (0)
							Sq, 5	0	1+	0	0	2+
6	5	Not done					Ad, 95	Not done					Not done

CgA, chromogranin‐A; NCAM, neural cell adhesion molecule; SCLC, small cell lung carcinoma; SYN, synaptophysin; TTF‐1, thyroid transcription factor‐1. Semiquantitative assessments of the percentage of positive tumor cells (0 = none, 1+ = 1–33%, 2+ = 34–66%, 3+ = 67–100%) were made. ^†We counted the number of lymph vessels with tumor embolisms confirmed by staining for D2‐40 for a representative slide.

Positive staining was defined as distinct linear membrane staining for neural cell adhesion molecule, cytoplasmic staining for chromogranin A and synaptophysin, and nuclear staining for TTF‐1 and p63. Immunostaining of each of the SCLC and non‐SCLC components was graded on a scale of 0–3+ according to the percentage of positive tumor cells (0 = none; 1+ = 1–33%; 2+ = 34–66%; 3+ = 67–100%). We then carried out immunohistochemical identification of lymph vessels with or without tumor embolisms for a representative slide.^{( 22 )} After independent evaluation by two of us (T. F. and K. T.), judgment consensus was obtained by joint viewing of the slides using a multiheaded microscope.

Analysis of EGFR mutational status.  In our previous study, we established a practical and precise non‐sequencing method for detecting EGFR mutations involving high‐resolution melting analysis (HRMA) using LCGreen I dye (Idaho Technology, Salt Lake City, UT, USA).^{( 23 )} First we screened for the EGFR mutations, DEL and L858R, using the HRMA method in formalin‐fixed paraffin sections obtained from surgically resected combined SCLC with Ad. Human genomic DNA (Roche Diagnostics, Basel, Switzerland) was used as a control sample with wild‐type EGFR. Second, we used 10% formalin‐fixed, paraffin‐embedded surgical specimens of primary combined SCLC from patients demonstrating DEL or L858R by HRMA, and the DNA was extracted from each of the SCLC and Ad components, respectively, the areas of which were clearly determined morphologically after laser capture microdissection (Arcturus Engineering, Mountain View, CA, USA) of the tumor tissue.^{( 24 )} Nested polymerase chain reaction (PCR) was carried out to amplify exons 19 and 21 of EGFR using previously described primers.^{( 17 )} The PCR products were electrophoresed on 2% agarose gels and subcloned into the TA vector (TOPO TA Cloning Kit, Invitrogen, Carlsbad, CA, USA), then the sequences were determined with M13 primers using an ABI Prism 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions.

Results

Clinical characteristics.  The clinical characteristics of the six patients are shown in Table 2. All patients were Japanese, aged between 62 and 77 years (mean 71.7 years). Five patients were male and one was female. Five patients were smokers whereas the remaining patient had never smoked. The median survival time of the six patients was 16.8 months (range 0.4–27.4 months); one patient died of heart failure 13 days after left pneumonectomy.

Table 2.

Clinical characteristic of the patients with combined small cell lung carcinoma (SCLC) with adenocarcinoma (Ad)

Patient no.	Age/Sex	ECOG PS	Smoking status	Smoking index	Tumor location	Size (mm)	Stage (cTNM)	Preoperative diagnosis	Surgical procedure
1	74/Male	0	Current	2160	Peripheral	31	IIb (210)	Unknown	RLL †
2	66/Male	0	Ever	900	Peripheral	38	IIb (210)	Unknown	RM/LL ‡
3	62/Female	0	Never	0	Peripheral	31	Ib (200)	SCLC	LUL
4	77/Male	1	Current	570	Peripheral	15	Ia (100)	Unknown	Left pneumonectomy
5	75/Male	0	Ever	1000	Peripheral	30	Ia (100)	Non‐SCLC	RUL
6	76/Male	0	Current	1120	Peripheral	28	Ia (100)	Ad	RUL

Smoking index: (number of cigarettes smoked per day) × years. Adjuvant chemotherapy: ^†cyclosphosphamide + doxorubicin + vincristine × 1 cycle. ^‡Cisplatin + etoposide × 1 cycle followed by cisplatin + irinotecan × 3 cycles. LUL, left upper lobectomy; RLL, right lower lobectomy; RM/LL, right middle and lower lobectomy; RUL, right upper lobectomy.

All six tumors were located in the peripheral portion of the lung. On clinical evaluation, three patients were staged as Ia (T1N0M0), one as Ib (T2N0M0) and two as IIb (T2N1M0). Preoperative pathological diagnoses were obtained in three patients and comprised one case each of SCLC, non‐SCLC and Ad.

Pathological findings.  Among six patients with combined SCLC with Ad, histological examination demonstrated that four had SCLC combined only with an Ad component (ratio of Ad in the tumor: 5, 30, 45 and 95%), whereas two had both Ad and Sq components (ratio of Ad/Sq: 10%/10% and 60%/5%, respectively). On pathological staging, one patient was staged as Ib (T2N0M0), one as IIa (T1N1M0), two as IIIa (T2N2M0) and two as IIIb (T4N1M0 and T4N2M0). In five of the six patients, the Ad components were observed in the peripheral part of the tumor showing a lepidic extension pattern, simulating bronchioloalveolar carcinoma. In the remaining one patient, Ad formed a minor component comprising approximately 5% of the tumor (Table 3). The Ad components in two patients showed a micropapillary growth pattern, whereas mucin production was not detected in any patient (Fig. 1a). The boundary between the SCLC and Ad components was not clear, and showed an indeterminate component that suggested gradual morphological transition from one to the other (Fig. 1b). In the two patients who also had combined Sq, the Sq component showed keratinization and was distinct from the SCLC component, but the border between the Ad and Sq components was unclear.

Table 3.

Histological findings of primary tumor and lymph node metastases, and epidermal growth factor receptor ( EGFR ) mutation

Patient no.	Stage (pTNM)	Ratio of each component (%)			Histological type of lymph node metastasis		BAC‐like extension	EGFR mutation
		SCLC	Ad	Sq	Mediastinal	Hilar
1	IIa (110)	95	5	0	Non †	SCLC	Absent	Wild type
2	IIIa (220)	80	10	10	SCLC	SCLC	Present	Wild type
3	IIIb (410)	70	30	0	Non †	Ad	Present	L858R
4	IIIb (420)	55	45	0	Ad	SCLC or Ad ‡	Present	Wild type
5	IIIa (220)	35	60	5	Ad	SCLC or Ad ‡	Present	Wild type
6	Ib (200)	5	95	0	Non†	Non †	Present	Wild type

†The patient had no mediastinal or hilar lymph node metastasis. ‡The patient had lymph node metastasis only from the SCLC component, and another lymph node showing metastasis only from the Ad component. Ad, adenocarcinoma; BAC, bronchioloalveolar carcinoma; hilar, hilar lymph node; L858R, mutation at codon 858 of EGFR; medical, mediastinum lymph node; pTNM, pathological TNM; SCLC, small cell lung carcinoma; Sq, squamous cell carcinoma.

Figure 1.

Combined small cell lung carcinoma (SCLC) with adenocarcinoma (Ad). (a) The periphery of this tumor consisted of a non‐mucinous bronchioloalveolar carcinoma‐like extension (patient no. 3). (b) The transitional zone between the SCLC and Ad components had poorly differentiated cells, shown by the immunohistochemical studies (patient no. 1). (c) D2‐40 with a membranous staining pattern of the lymph vessels. Tumor embolism of lymph vessels was confirmed by D2‐40 staining (patient no. 3). (c‐1) SCLC cell embolisms increased in number around the primary lesion. (c‐2) Ad cell embolisms invaded the lymph vessels.

The results of immunohistochemical studies carried out in five cases are shown in Table 1. The specimen from patient no. 6 was not available. All of the SCLC components showed positive staining for at least one neuroendocrine marker. In addition, the Ad components in all five patients examined showed positive staining for at least one neuroendocrine marker, although semiquantitative assessments of the percentage of positive Ad cells were lower than those for SCLC cells in the same tumor. Also, the Ad components showed positive staining for TTF‐1 in all five patients. TTF‐1 staining of the SCLC component tended to be similar to that of the Ad component in terms of the percentage of positive cells. p63 immunostaining served as a good marker of Sq differentiation.

Status of lymph node metastasis.  Five patients had pathologically confirmed hilar lymph node metastases, and three of them also had histologically proven mediastinal lymph node metastases, which had not been evident at the time of preoperative clinical evaluation (Table 3). Among these five patients with hilar lymph node metastases, two showed only SCLC in the metastatic lesion, one showed Ad only, and two showed SCLC or an Ad component that had developed separately in each lymph node. Among the three patients with mediastinal lymph node metastases, one had only SCLC in the nodes, and two had an Ad component only. Metastatic Ad components were found only in patients with a primary tumor in which Ad accounted for more than 30% of the total volume.

In the six patients, we identified tumor embolism of the lymph vessels immunohistochemically with D2‐40 staining. There were approximately 800–1000 lymph vessels in each of these tumors per representative slide. The major component invading the lymph vessels around the tumors was SCLC cells. Even in the two patients who had mediastinal lymph node metastases with an Ad component, the SCLC cells tended to spread to the lymph vessels rather than the Ad cells (Table 1).

EGFR mutational status.  First, we analyzed 10 surgically resected samples from six patients with combined SCLC and Ad by HRMA. Analysis of exon 19 demonstrated curves identical to those of the control (wild type) in all samples, as shown in Fig. 2a. In the analysis of exon 21, thorough melting curves were obtained for two samples from patient no. 3, showing a different curve from the control, whereas the other eight samples demonstrated curves identical to the control (wild type), as shown in Fig. 2b. The normal lung tissue from patient no. 3, who was a female non‐smoker, showed a wild‐type curve, and therefore we judged that this patient had L858R in exon 21 of EGFR.

Figure 2.

Results of high‐resolution melting analysis (HRMA). Adjusted melting curves obtained by HRMA of combined small cell lung carcinoma (SCLC) with primers designed to detect mutations in (a) exon 19 or (b) exon 21 of epidermal growth factor receptor (EGFR). Two samples from patient no. 3 were identified as containing the L858R mutations (↑). The DNA extracted from normal lung tissue of patient no. 3 was identified as wild type (not shown).

Next we confirmed this mutation in the SCLC and Ad components in patient no. 3. DNA was extracted from each SCLC and Ad component separately using laser capture microdissection or by manual microdissection, which was carried out for each clearly determined component on paraffin‐embedded sections. Sequence analysis of subcloned PCR products obtained from the separate components was carried out. Examination of both SCLC and Ad components showed an identical mutation (L858R) in exon 21 (Fig. 3), confirming the results obtained by HRMA.

Figure 3.

Results of DNA sequencing from patient no. 3. The tumor of patient no. 3 was microdissected into the small cell lung carcinoma (SCLC) and adenocarcinoma (Ad) components. (a) Sequence analysis of the subcloned polymerase chain reaction (PCR) products from the microdissected SCLC component. (b) Sequence analysis of the subcloned PCR products from the microdissected Ad component. The patient had a tumor with L858R of EGFR, which was in both the SCLC and Ad components.

Discussion

The present study using microdissected tumor tissue is the first to report a patient with combined SCLC with Ad showing the EGFR mutation in both the SCLC and Ad components. EGFR mutations, especially DEL and L858R, have been reported in Ad of the lung. These somatic mutations in the kinase domain of EGFR have been shown to be predictive molecular markers for sensitivity to kinase inhibitors such as gefitinib (Iressa; AstraZeneca, Osaka, Japan). However, these mutations have rarely been demonstrated in SCLC. To our knowledge, there have been two reported cases of metastatic SCLC harboring DEL in exon 19 of EGFR showing responsiveness to EGFR tyrosine kinase inhibitors.^{( 19} , ²⁰ , ^{25 )} Considering that the diagnosis of SCLC is often based on small biopsy specimens that may not be sufficiently representative of the total tumor, there is a possibility that any combined component may be overlooked.

In a clinical setting, the distinction of SCLC from non‐SCLC is critical because of major differences in management and prognosis between the two cancers. SCLC is well known to be more common in men and smokers, but so far SCLC with EGFR mutations has been detected only in female patients who have never smoked,^{( 19} , ^{20 )} as was the case in our present female patient. Thus it seems reasonable to suggest that in clinically unusual SCLC patients, for example those who are non‐smokers and female, showing peripheral nodular lesions and histological combination with Ad, EGFR mutation status should be analyzed because previous studies have shown that EGFR tyrosine kinase inhibitors are effective in patients with metastatic SCLC with EGFR mutations.

The present study is considerably informative with regard to the origin and histogenesis of SCLC. EGFR mutation is detected in patients with pre‐invasive adenocarcinomatous lesions such as atypical adenomatous hyperplasia and bronchioloalveolar carcinoma, which eventually progress to invasive lung Ad.^{( 26 )} In addition, EGFR mutations are also linked to Ad with a bronchioloalveolar carcinoma component.^{( 27 )} Thus it is suggested that EGFR mutation occurs and plays a critical role in the early developmental stage of lung Ad. The mutation is detected more frequently in Ad in female non‐smokers than in male smokers. In the present study, the only patient with SCLC harboring an EGFR mutation was female and a non‐smoker, and the combined Ad component also harbored the same mutation. Moreover, as mentioned above, the two SCLC patients with EGFR mutation reported previously were also female and non‐smokers. These findings imply that the mutations are an early genetic event in carcinogenesis of the lung and at least a certain proportion of SCLC may originate as a result of progression or transformation of Ad harboring EGFR mutation.

This phenomenon can also be linked to pathological features. The histological patterns of lymph node involvement showed that Ad components spread to mediastinal lymph nodes in the patients with hilar lymph node involved by SCLC or Ad component. Considering the status of tumor embolism of the lymph vessels observed using D2‐40 staining, SCLC cell embolisms, but not Ad, increase in number around primary lesion in these tumors. It is suggested that a common uncommitted stem cell might differentiate into each component after involvement in a lymph node. Furthermore, positive staining for TTF‐1, which is a highly specific immunohistochemical marker identifying carcinomas of pulmonary origin (especially non‐mucinous Ad and SCLC),^{( 28 )} was shown in the SCLC and Ad components, but not Sq. Previous studies have demonstrated TTF‐1 expression in 83–100% of SCLC, but low expression in Sq.^{( 29} , ^{30 )} These findings could be interpreted as being compatible with the hypothesis that SCLC and Ad originate from a common uncommitted stem (or precursor) cell originally expressing TTF‐1.^{( 31 )} It is possible to postulate that a fraction of SCLC possessing stem (or precursor) cell properties might have the potential to form an Ad component. In fact, in the present cases, there were some areas comprising morphologically indeterminate tumor cell components at the border of the SCLC and Ad components.

The rarity of patients with combined SCLC makes it difficult to determine the optimal management and biological characteristics of this tumor. However, the present findings suggest that the classical classification of lung cancer might provide insufficient management for a specified subpopulation in molecular targeted therapy. Although this retrospective study examined only a very limited number of lung carcinoma cases, we consider that the findings provide useful information for understanding the biology of this lung cancer and devising more effective forms of clinical management.