Ann Surg Oncol DOI 10.1245/s10434-014-3642-5
ORIGINAL ARTICLE – THORACIC ONCOLOGY
Clinicopathology and Genetic Profile of Synchronous Multiple Small Adenocarcinomas: Implication for Surgical Treatment of an Uncommon Lung Malignancy Mong-Wei Lin, MD1,5, Chen-Tu Wu, MD2,5, Shuenn-Wen Kuo, MD3, Yih-Leong Chang, MD2,5, and Pan-Chyr Yang, MD, PhD4,5 Department of Surgery, National Taiwan University Hospital Hsin-Chu Branch, Hsinchu, Taiwan; 2Department of Pathology, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan; 3 Department of Surgery, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan; 4Department of Internal Medicine, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan; 5Graduate Institute of Pathology, National Taiwan University College of Medicine, Taipei, Taiwan 1
ABSTRACT Purpose. Synchronous multiple small adenocarcinomas are detected more frequently than in the past; however, the genetic profile, treatment, and prognosis of patients remain unclear. For treatment decisions and prognostic applications, we evaluated epidermal growth factor receptor (EGFR), p53, and KRAS somatic mutations in synchronous multiple small lung adenocarcinomas. Methods. The presence of EGFR, p53, and KRAS somatic mutations was determined in 64 synchronous multiple lung adenocarcinomas B2 cm in maximal dimension. Mutational analysis was performed on DNA extracted from paraffin-embedded tumors. Results. Five-year disease-free survival (DFS) was 86.1 %, and overall survival was 95.8 %. EGFR, p53, and KRAS mutations were detected in 41 (64.1 %), 8 (12.5 %), and 4 (6.3 %) patients, respectively. The high frequency of genetic mutations resulted in a high discrimination rate of tumor clonality (68.8 %; 44/64) in the study group. Fourteen (31.8 %) patients were assessed as having the same clonality, whereas 30 (68.2 %) patients had different
Electronic supplementary material The online version of this article (doi:10.1245/s10434-014-3642-5) contains supplementary material, which is available to authorized users. Ó Society of Surgical Oncology 2014 First Received: 21 December 2013 Y.-L. Chang, MD e-mail: [email protected]
clonality, which further supported the concept of field cancerization. Multivariate analysis showed lymph node metastasis (p = 0.003) and smoking (p = 0.011) were significantly correlated with tumor relapse. Surgical method, clonality, and tumor location were not correlated with tumor relapse. Conclusions. Whether these tumors are different or the same clonal, sublobar resection of each lesion can achieve long-term DFS and is the treatment of choice for synchronous multiple small lung adenocarcinomas. Patients with lymph node metastasis are at risk of relapse and adjuvant chemotherapy is indicated.
The occurrence of multiple primary cancers within the same organ, such as the breast and ovary, has been widely reported. Although relatively uncommon, lung cancer can present as multiple tumors that are either synchronous or metachronous. The terms ‘synchronous’ and ‘metachronous’ extend a time-related connotation to the evolution of these multifocal tumors; however, it does not specify which tumors are clonal and therefore are related tumors, i.e. are descendants of a single clone, and which tumors are unrelated and therefore are of independent origin. Synchronous multiple primary lung cancers (MPLCs) were first reported in 1924 when Beyreuther identified two separate primary lung cancers in a patient with pulmonary tuberculosis.1 The incidence of synchronous multiple lung cancers in reported clinical series ranges from 1 to 7 %.2–4 Synchronous multiple lung cancers can either be MPLCs or intrapulmonary metastasis. MPLCs may develop
M.- W. Lin et al.
coincidentally. However, sometimes the entire lung epithelium is at risk for carcinogenesis due to similar exposure to carcinogens.3,5 This concept, referred to as field cancerization, may explain the development of MPLCs. The survival of patients with synchronous MPLCs is highly variable; the 5-year survival for all patients and for patients with stage I tumors ranges from 0 to 70 % and 0 to 79 %, respectively.6 Advances in high-resolution computed tomography (CT) imaging and the prevalence of CT screening for lung cancer detection in asymptomatic patients have increased the detection of synchronous multiple small lung tumors in peripheral lung tissue.6,7 Most tumors were B2 cm in maximal dimension, pure or mixed ground glass opacity (GGO) on CT, and diagnosed as adenocarcinomas.6,8–14 Few studies have been published on the outcome of synchronous multiple small lung adenocarcinomas. The reported 5-year survival ranges from 64 to 100 %,8–14 which is considerably better than the prognosis of MPLCs. The clinicopathologic characteristics, genetic profile, and prognosis of patients with synchronous multiple small lung adenocarcinomas still remain unclear. Toward developing useful genetic markers for clonality assessment and patient management, we took advantage of the highly frequent and early somatic mutations of epidermal growth factor receptor (EGFR), p53, and KRAS genes15–18 to define the clonal origins of synchronous multiple small lung adenocarcinomas in 64 patients. For potential diagnostic and management applications, the correlation between genetic mutations, clinicopathologic features, and disease-free survival (DFS) was determined.
METHODS Study Population A total of 64 patients with synchronous multiple small lung adenocarcinomas measuring B2 cm in maximal dimension who underwent complete surgical resection by our team at the National Taiwan University Hospital between January 2005 and June 2012 were included in the study. Written informed patient consent was obtained for tissue analysis at the time of tumor specimen procurement. The Research Ethics Committee of the National Taiwan University Hospital approved this study (project approval number 201110045RD). All synchronous multiple lung adenocarcinomas were incidentally identified by chest CT. Patients were not treated with prior EGFR-targeted therapy, neoadjuvant chemotherapy, or neoadjuvant radiotherapy. All patients received preoperative staging workups, including brain CT or magnetic resonance imaging, bone scan or positron emission tomography (PET), and abdominal CT. Patients with distant metastasis
and supraclavicular lymph node (N3) metastasis were excluded from the study. All patients had undergone complete resection of synchronous multiple lung tumors. The term ‘complete resection’ indicated that all the synchronous multiple small lung adenocarcinomas were resected in a one-stage operation. Patients with residual inoperable synchronous lung tumors were excluded from the study. The surgical methods were classified into two categories: sublobar resection (i.e. wedge resection and segmentectomy) and lobar resection (i.e. lobectomy and bilobectomy). Systemic lymph node dissection was performed in 58 patients, including hilar, interlobar, lobar, and mediastinal lymph nodes. The remaining six patients with peripheral ground glass lesions did not receive lymph node dissection. For 21 patients with synchronous bilateral lung tumors, bilateral tumor resections were both performed in a one-stage operation. All resected specimens were formalin-fixed and sectioned for microscopic examination after staining with hematoxylin-eosin. Lung cancer histology was classified according to the WHO pathology classification.19 Histological diagnosis and pathologic features, including tumor cell type, vascular invasion, visceral pleural invasion, and regional/mediastinal lymph node metastasis, were assessed by two independent pathologists (C-TW and Y-LC). The clinical data of enrolled patients retrieved from chart review included age, sex, smoking status, preoperative carcinoembryonic antigen (CEA) level, relapse, metastasis, and mortality. Patients were excluded if they had combined tumor histology other than adenocarcinoma. Patients with tumors [2 cm in maximal dimension were also excluded from the study. Mutational Analysis of Epidermal Growth Factor Receptor (EGFR), p53, and KRAS EGFR, p53, and KRAS genomic DNA was extracted from paraffin-embedded tumors using the QIAamp DNA Mini kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. Exons 18–21 of EGFR, exons 5–8 of p53, and exon 2 of KRAS were amplified using nested polymerase chain reaction (PCR) with specific primers, as described previously.20,21 The resultant PCR amplicons were purified using a Gel/PCR DNA Fragments Extraction kit (Geneaid Biotech Ltd, Taiwan) according to the manufacturer’s instructions. PCR amplicons were sequenced using the BigDye Terminator kit (Applied Biosystems, Foster City, CA, USA) and ABI Prism 3100 DNA Analyzer (Applied Biosystems) according to the manufacturer’s instructions. All sequencing reactions were performed in both forward and reverse directions by using tracings from at least two independent PCRs. Mutations
Synchronous Small Lung Adenocarcinomas
were also checked against the corresponding sequences from the non-neoplastic lung tissue DNA and single nucleotide polymorphism database. Statistical Analysis The correlation between various clinicopathologic variables and somatic mutations in EGFR, p53, and KRAS was analyzed using Fisher’s exact test. The effect of EGFR, p53, and KRAS on survival was tested using Kaplan–Meier survival plots and analyzed using the log-rank test. All tests were two-sided, and p \ 0.05 was considered significant. The Statistical Package for the Social Sciences version 10.0 software (SPSS Inc., Chicago, IL, USA) was used for all analyses. RESULTS Patient Characteristics Our team at the National Taiwan University Hospital performed 1,223 surgical lung resections in primary lung adenocarcinoma patients between January 2005 and June 2012. The study included 64 patients with a total number of 140 synchronous multiple lung adenocarcinomas measuring B2 cm in maximal dimension. The incidence was 5.2 %. Median follow-up time was 27 months, and the median age was 60 years (range 27–81 years). Of the 64 patients, 46 (71.9 %) patients were women, and only 6 (9.4 %) patients were smokers. Preoperative elevated CEA value was noted in 3 (4.7 %) patients. Fifty-three patients (82.8 %) had two synchronous multiple lung adenocarcinomas, whereas the remaining 11 patients (17.2 %) had more than two synchronous multiple lung adenocarcinomas. Twenty-three (35.9 %) patients had multiple tumors in a single lobe, whereas 20 (31.3 %) patients had tumors in separate lobes of unilateral lung field, and 21 (32.8 %) patients had tumors in bilateral lung fields. Most patients underwent sublobar resection (56.3 %; 36/64), including wedge resection and segmentectomy. Thirteen patients underwent lobar resection (20.3 %; 13/64), including lobectomy and bilobectomy. Lobar and sublobar resections were both performed in the remaining 15 (23.4 %) patients. Fourteen (21.9 %) and 8 (12.5 %) patients had all pure GGO lesions and all pure solid lesions on chest CT, respectively. The remaining patients had all mixed GGO lesions (14; 21.9 %) or heterogeneous lesions (28; 43.8 %). Regarding the largest tumor size, 24 (37.5 %) tumors were B1 cm and 40 (62.5 %) tumors were [1 cm and B2 cm. Vascular invasion and visceral pleural invasion were noted in two (3.1 %) patients and five (7.8 %) patients, respectively. Lymph node metastasis was noted in
four (6.3 %) patients; one patient had N1 metastasis and three patients had N2 metastasis. Relapses were recorded in eight cases, including four cases of bilateral pulmonary metastases and four cases of distant metastases. No local recurrence was noted. Disease-related mortality occurred in two patients. The 5-year DFS and overall survival (OS) of the study population was 86.1 and 95.8 %, respectively. Correlation of Genetic Mutations with Clinicopathologic Features and Clonality Assessment EGFR mutations were detected in 41 (64.1 %) patients. The majority of EGFR mutations were in exon 21 (56.1 %; 23/41) and exon 19 (48.7 %; 20/41). Female patients (71.7 %; 33/46; p = 0.049) had higher EGFR mutation rates compared with male patients (44.4 %; 8/18) (Table 1). For clonality assessments, the mutation status of synchronous multiple tumors was classified into four different distribution patterns: Group A, only one tumor with a mutation; Group B, two or more tumors with identical mutations; Group C, two or more tumors with different mutations; and Group D, tumors with no mutations. Of the 64 patients, 17 (26.6 %) and 24 (37.5 %) patients were classified as having the same clonality (Group B) and different clonality (Group A or C), respectively (Table 2). Clonality was not determined in 35.9 % (23/64) of patients because of the absence of EGFR mutations (Group D). p53 and KRAS mutations were detected in eight (12.5 %) and four (6.3 %) patients, respectively. p53 and KRAS mutations were not correlated with clinicopathologic characteristics (Table 1). Clonality of tumors was not evaluated in 87.5 % (56/64) and 93.8 % (60/64) of patients whose tumors had no detectable p53 and KRAS mutations, respectively (Table 2). Clonality Analysis Using Mutations in EGFR/p53/ KRAS Somatic mutations in EGFR, p53, and KRAS were combined for clonality assessment. This strategy increased the patient number for clonality analysis to 68.8 % (44/64), with EGFR/p53/KRAS mutations absent in only 31.3 % (20/64) of cases. Among the 44 cases suitable for clonality assessment, 14 cases (31.8 %) were classified as having the same clonality (electronic supplementary material [ESM] Fig. 1a), and 30 cases (68.2 %) were classified as having different clonality (ESM Fig. 1b). Clonality assessment was not significantly associated with clinicopathologic characteristics (Table 2). The different clonality rate in patients with synchronous multiple small adenocarcinomas in a single lobe, unilateral different lobes, and bilateral different lobes was 50 % (6/12),
M.- W. Lin et al. TABLE 1 Frequency of p53, EGFR, and KRAS mutations in relation to clinical variables and pathologic characteristics Parameters
No. of patients
p53 mutation
p Value
EGFR mutation
p Value
KRAS mutation
?
-
?
-
?
-
64
8
56
41
23
4
60
B65
39
5
34
26
13
1
38
[65
25
3
22
15
10
0.605
3
22
18
3
15
1
17
46
5
41
0.049
3
43
Positive
6
2
4
0
6
Negative
58
6
52
4
54
No. of patients
p Value
Age (years) 1.000
0.291
Sex Male Female Smoking status
0.676
0.159
8
10
33
13
5
1
36
22
0.406
1.000
1.000
Tumor site One lobe
23
3
20
12
11
0
23
[1 lobe, unilateral
20
3
17
15
5
1
19
[1 lobe, bilateral
21
2
19
0.899
14
7
0.282
3
18
12
12
2
22
0.699
29
11
0.106
2
38
3
1
0
4
34
20
4
50
0.152
Tumor size (cm) B1
24
2
22
[1
40
6
34
Positive
4
1
3
Negative
54
7
47
0.627
Lymph node metastasisa 0.457
1.000
1.000
EGFR epidermal growth factor receptor a
Six patients with pure ground glass opacity lesions did not undergo lymph node dissection
62.5 % (10/16), and 87.5 % (14/16), respectively. Two of the three patients (66.7 %) with lymph node metastasis and available clonality assessment data had different clonality. Correlation between Clinicopathologic Features and Disease-Free Survival The 5-year DFS of the 64 patients was 86.1 %. Univariate analysis identified five significant risk factors for relapse: age B65 years (p = 0.020), smoking (p = 0.011), presence of solid nodules on CT (p = 0.016), lymph node metastasis (p = 0.003), and same EGFR/p53/KRAS clonality (p = 0.024) (Table 3). Multivariate analysis using the Cox proportional hazard regression model identified smoking and lymph node metastasis as significant risk factors for relapse in patients with synchronous multiple small lung adenocarcinomas (Table 4, ESM Fig. 2). Notably, surgical method, clonality, and tumor location were not significantly associated with DFS in our study group. DISCUSSION Patients who present with multiple lung tumors remain a challenge to both clinicians and pathologists. In the 7th
edition of the TNM staging system guidelines,22 multiple lung cancers within the same lobe were downgraded from T4 to T3, whereas intrapulmonary metastasis in other lobes on the same side was downgraded from M1 to T4. Because of the increased use of CT imaging, synchronous multiple small lung tumors have been detected more frequently in the last decade, particularly in Asia.814 According to the few reported studies, most synchronous multiple small lung tumors were adenocarcinomas and pure or mixed GGO on CT, and had good prognosis.8–14 Less is known about the genetic profile and clinicopathologic characteristics of MPLCs. In the current study, we included 64 patients with surgically resected synchronous multiple small lung adenocarcinomas measuring B2 cm in maximal dimension. Most patients were female and non-smokers. Lymph node involvement was present in only 6.9 % of patients. The 5year DFS and OS were 86.1 and 95.8 %, respectively. The prognosis of these patients is similar to that of stage I nonsmall cell lung cancer (NSCLC) patients, therefore clinicians and pathologists should be very careful not to regard these patients as intrapulmonary metastasis and misinterpret the TNM stage. EGFR/p53/KRAS mutations were analyzed for clonality assessment. In our study, the EGFR mutation rates were high (64.1 %), whereas p53 mutations rates were low
Synchronous Small Lung Adenocarcinomas TABLE 2 Clinicopathologic characteristics of patients with the same clonality and different clonality between tumors (n = 44)a Parameters
p53 mutation
KRAS mutation
p53, EGFR, and/or KRAS mutation
SC
DC
SC
DC
SC
DC
SC
DC
3
5
17
24
0
4
14
30
B65
2
3
8
18
0
1
6
20
[65
1
2
9
6
0
3
8
10
Male
2
1
4
4
0
1
4
5
Female Smoking status
1
4
13
20
0
3
10
25
Positive
1
1
2
3
0
0
2
3
Negative
2
4
15
21
0
4
12
27
No. of patients
p Value
EGFR mutation p Value
p Value
p Value
Age (years) 1.000
0.102
NA
0.191
Sex 0.464
1.000
0.698
1.000
NA
NA
0.434
0.647
Tumor site One lobe
2
1
7
5
0
0
6
6
[1 lobe, unilateral
1
2
7
8
0
1
6
10
[1 lobe, bilateral
0
2
0.679
3
11
0.165
0
3
NA
2
14
6
6
0
2
5
8
1.000
11
18
0.507
0
2
NA
9
22
1
2
0
0
1
2
14
20
0
4
11
26
0.100
Tumor size (cm) B1
1
1
[1
2
4
0.724
Lymph node metastasisb Positive
0
1
Negative
3
4
1.000
1.000
NA
1.000
EGFR epidermal growth factor receptor, DC different clonality, SC same clonality, NA not applicable a
Patients with no EGFR/p53/KRAS mutation were deleted
b
Six patients with pure ground glass opacity lesions did not undergo lymph node dissection
(12.5 %). A high frequency of EGFR mutations has been reported in the early stage of pulmonary adenocarcinoma development.23 In contrast, p53 overexpression was significantly less frequent in non-invasive tumors.23 Our study population had multiple small lung adenocarcinomas B2 cm in maximal dimension, and the prognosis was as good as stage I NSCLCs. Therefore, the high EGFR and low p53 mutation rates in our study can be explained by the theory of EGFR mutations as early events associated with tumor initiation and p53 mutations as late events associated with tumor progression.23 KRAS mutations are one of the most common oncogenic events in human carcinomas of endodermal origin, occurring at high frequency in adenocarcinomas of lung, pancreatic, and colorectal origin.24,25 KRAS mutations have been detected in 5–15 % of East Asian patients with lung adenocarcinoma.26 The frequency of KRAS mutations was 6.3 % in the current study, which was similar to previous studies. In the current series, 30 patients (68.2 %; 30/44) showed different EGFR/p53/KRAS clonality despite having a similar histological type, and also presented with similar CT characteristics. Therefore, it may be unreliable to use histological type or imaging characteristics alone to
differentiate primary tumors from intrapulmonary metastasis. Clonality assessment can provide more information for further clarification of MPLCs. Synchronous multiple small lung adenocarcinomas with different EGFR/p53/ KRAS clonality can be regarded as MPLCs. However, it still lacks evidence that those with the same EGFR/p53/ KRAS clonality are definite intrapulmonary metastasis. Multivariate analysis showed that lymph node metastasis and smoking were significant factors for tumor relapse. Smoking as a significant predictor of poor prognosis after lung cancer surgery has been reported in several previous studies.27,28 The relationship of advanced lymph node status and poor prognosis has also been well documented, and lymph node status has become an important staging factor in the current TNM system.22 In patients with synchronous multiple lung cancers, a better 5-year OS (37– 52.5 %) has been reported in node-negative patients compared with node-positive patients (15.5–24 % %).4,29 However, the relationship between lymph node metastasis and prognosis in synchronous multiple small lung adenocarcinomas has not been reported. Our study is the first to report a more favorable 5-year DFS in node-negative patients compared with node-positive patients (91.8 vs.
M.- W. Lin et al. TABLE 3 Univariate survival analysis of clinicopathologic features in patients with synchronous multiple small lung adenocarcinomas Parameters
No. of patients
No. of relapses (%)/ 5-year DFS (%)
Total no. of patients
64
8 (12.5)/86.1
25 39
6 (24.0)/78.4 2 (5.1)/93.0
Male
18
3 (16.7)/82.5
Female
46
5 (10.9)/89.3
Positive
6
3 (50.0)/50.0
Negative
58
5 (8.6)/91.3
Normal
61
6 (9.8)/88.3
Abnormal
3
2 (66.7)/66.7
C3
11
1 (9.1)/90.0
=2
53
7 (13.2)/87.3
0.708
40 24
7 (17.5)/82.7 1 (4.2)/95.5
0.152
TABLE 3 continued Parameters
No. of patients
No. of relapses (%)/ 5-year DFS (%)
p Value
23
2 (8.7)/95.2
0.369
Mutation
8
2 (25.0)/75.0
Wild type
56
6 (10.7)/89.2
Mutation
4
0 (0.0)/100.0
Wild type
60
8 (13.3)/86.6
p Value Wild type p53 gene
Age (years) B65 [65
0.020
Sex 0.798
Smoking status 0.011
CEA
0.159
KRAS gene 0.527
CEA serum carcinoembryonic antigen, CT computed tomography, DFS disease-free survival, GGO ground glass opacity, EGFR epidermal growth factor receptor a
Six patients with pure ground glass opacity lesions did not undergo lymph node dissection
b
Patients with no EGFR/p53/KRAS mutation were deleted
0.056
Tumor number
Largest tumor size (cm) [1 cm B1 cm
TABLE 4 Multivariate survival analysis of clinicopathologic features in patients with synchronous multiple small lung adenocarcinomas Parameters
No. of No. of Hazard 95 % CI patients relapses ratio (%)
p Value
[65
39
2 (5.1)
0.006–1.325
0.080
B65
25
6 (24.0) 1.000 0.005–0.601
0.017
0.005–0.665
0.022
2 (10.0) 1.022
0.033–31.844
0.990
0.845–273.959 0.065
Tumor site One lobe
23
4 (17.4)/85.3
[1 lobe, unilateral
20
2 (10.0)/86.8
[1 lobe, bilateral
21
2 (9.5)/89.3
Pure GGO
14
0 (0.0)/100.0
Negative
6
3 (50.0) 0.055
Mixed type
42
4 (9.5)/88.7
Positive
58
5 (8.6)
1.000
Solid
8
4 (50.0)/62.5
0.058
Positive
2
1 (50.0)/0.0
Negative
62
7 (11.3)/89.3
Positive
5
1 (20.0)/50.0
Negative
59
7 (11.9)/89.0
Positive
4
3 (75.0)/37.5
Negative
54
4 (7.4)/91.8
Age (years) 0.828
CT characteristics
Smoking status
0.016
Lymph node metastasisa
Vascular invasion 0.092
Pleural invasion
Negative
54
4 (7.4)
Positive
4
3 (75.0) 1.000
Clonality assessment Wild type 20
0.358
Lymph node metastasisa
SC
14
4 (28.6) 15.216
DC
30
2 (6.7)
CT characteristics Pure GGO 0.003
Surgery Sublobar resection
36
2 (5.6)/96.3
Lobar ? sublobar
15
3 (20.0)/77.0
Lobar resection
13
3 (23.1)/75.0
Same
14
4 (28.6)/55.9
Different
30
2 (6.7)/92.5
41
6 (14.6)/82.5
EGFR gene
14
0 (0.0)
0.000
0.000
0.984
Mixed type 42
4 (9.5)
0.458
0.029–7.208
0.579
Solid
4 (50.0)
8
CI confidence interval, CT computed tomography, DC different clonality, GGO ground glass opacity, SC same clonality 0.252
Clonality assessmentb
Mutation
0.092
0.024
a
Six patients did not undergo lymph node dissection
37.5 %). This finding raises two importance issues. First, adjuvant therapy may be indicated for patients with lymph node metastasis. Second, preoperative mediastinal lymph node assessment (CT and/or PET) is important to rule out
Synchronous Small Lung Adenocarcinomas
lymph node metastasis in synchronous multiple small lung adenocarcinoma patients. Tumor location has been regarded as an important prognostic factor in synchronous multiple lung cancers. In the Martini and Melamed criteria, multiple lung cancers with a similar histological type and located in the same lobe were regarded as intrapulmonary metastasis.30 However, our study showed no relationship between tumor location and relapse. Furthermore, DFS was not significantly different according to surgical method or clonality. Therefore, no matter whether the tumors were the same or different clonal, sublobar resection for each lesion of synchronous multiple small lung adenocarcinomas is the treatment of choice to preserve more pulmonary parenchyma and achieve the same DFS. CONCLUSIONS This study showed the good prognosis of surgically resected synchronous multiple small lung adenocarcinomas. The status of this particular form of NSCLCs might be considered in the TNM staging system for more accurate prediction of patient prognosis. Sublobar resection of each lesion in patients with node-negative synchronous multiple small lung adenocarcinomas is associated with a DFS rate similar to that of stage I NSCLCs and is the treatment of choice. However, patients with lymph node metastasis have poor DFS and, therefore, sublobar resection treatment is not adequate in these patients and adjuvant chemotherapy should be considered. ACKNOWLEDGEMENT The authors would like to thank Professor Yung-Chie Lee (October 23, 1948 to December 30, 2010) for his substantial contribution to patient care and surgery. This study was funded by the National Taiwan University Hospital (NTUH 100N1713). CONFLICTS OF INTEREST Mong-Wei Lin, Chen-Tu Wu, Shuenn-Wen Kuo, Yih-Leong Chang, and Pan-Chyr Yang declare no conflicts of interest.
REFERENCES 1. Beyreuther H. Multipicate von carcinomen bei einem fall von sog: ‘Schnee- berger’ lungenkrebs mit tuberculose. Virchows Arch Pathol Anat. 1924;250:230-6. 2. Ferguson MK, DeMeester TR, DesLauriers J, Little AG, Piraux M, Golomb H. Diagnosis and management of synchronous lung cancers. J Thorac Cardiovasc Surg. 1985;89:378-85. 3. Chang YL, Wu CT, Lin SC, Hsiao CF, Jou YS, Lee YC. Clonality and prognostic implications of P53 and EGFR somatic aberrations in multiple primary lung cancers. Clin Cancer Res. 2007;13:52-8. 4. Chang YL, Wu CT, Lee YC. Surgical treatment of synchronous multiple primary lung cancers: experience of 92 patients. J Thorac Cardiovasc Surg. 2007;134:630-7.
5. Nelson MA, Wymer J, Clements N Jr. Detection of K-ras gene mutations in non-neoplastic lung tissue and lung cancers. Cancer Lett. 1996;103:115-21. 6. Kozower BD, Larner JM, Detterbeck FC, Jones DR; American College of Chest Physicians. Special treatment issues in lung cancer: ACCP evidence-based clinical practice guidelines (3rd edition). Chest. 2013;143:e369-e399s. 7. Detterbeck FC, Mazzone PJ, Naidich DP, Bach PB; American College of Chest Physicians. Screening for lung cancer: ACCP evidence-based clinical practice guidelines (3rd edition). Chest. 2013;143:e78-e92s. 8. Ebright MI, Zakowski MF, Martin J, et al. Clinical pattern and pathologic stage but not histologic features predict outcome for bronchioloalveolar carcinoma. Ann Thorac Surg. 2002;74:16406. 9. Mun M, Kohno T. Efficacy of thoracoscopic resection for multifocal bronchioloalveolar carcinoma showing pure ground-glass opacities of 20 mm or less in diameter. J Thorac Cardiovasc Surg. 2007;134:877-82. 10. Roberts PF, Straznicka M, Lara PN, Lau DH, Follette DM, Gandara DR, et al. Resection of multifocal non-small cell lung cancer when the bronchioloalveolar subtype is involved. J Thorac Cardiovasc Surg. 2003;126:1597-602. 11. Nakata M, Sawada S, Yamashita M, Saeki H, Kurita A, Takashima S, et al. Surgical treatments for multiple primary adenocarcinoma of the lung. Ann Thorac Surg. 2004;78:1194-9. 12. Carretta A, Ciriaco P, Melloni G, et al. Surgical treatment of multiple primary adenocarcinomas of the lung. Thorac Cardiovasc Surg. 2009;57:30-4. 13. Kim HK, Choi YS, Kim J, Shim YM, Lee KS, Kim K. Management of multiple pure ground-glass opacity lesions in patients with bronchioloalveolar carcinoma. J Thorac Oncol. 2010;5:20610. 14. Casali C, Rossi G, Marchioni A, et al. A single institution-based retrospective study of surgically treated bronchioloalveolar adenocarcinoma of the lung: clinicopathologic analysis, molecular features, and possible pitfalls in routine practice. J Thorac Oncol. 2010;5:830-6. 15. Mitsudomi T, Yatabe Y, Koshikawa T, et al. Mutations of the P53 tumor suppressor gene as clonal marker for multiple primary lung cancers. J Thorac Cardiovasc Surg. 1997;114:354-60. 16. Matsuzoa D, Hideshima T, Ohshima K, Kawahara K, Shirakusa T, Kimura A. Discrimination of double primary lung cancer from intrapulmonary metastasis by P53 gene mutation. Br J Cancer. 1999;79:1549-52. 17. Van Rens MT, Eijken EJ, Elbers JR, Lammers JW, Tilanus MG, Slootweg PJ. P53 mutation analysis for definite diagnosis of multiple primary lung carcinoma. Cancer. 2002;94:188-96. 18. Tang X, Shigematsu H, Bekele BN, et al. EGFR tyrosine kinase domain mutations are detected in histologically normal respiratory epithelium in lung cancer patients. Cancer Res. 2005;65:7568-72. 19. Travis WD, Brambilla E, Muller-Hermelink HK, et al. Pathology and genetics: tumours of the lung, pleura, thymus and heart. Lyon: IARC Press; 2004. 20. Linardou H, Dahabreh IJ, Kanaloupiti D, et al. Assessment of somatic k-RAS mutations as a mechanism associated with resistance to EGFR-targeted agents: a systemic review and metaanalysis of studies in advanced non-small-cell lung cancer and metastatic colorectal cancer. Lancet Oncol. 2008;9:962-72. 21. Sidransky D, Mikkelsen T, Schwechheimer K, Rosenblum ML, Cavanee W, Vogelstein B. Clonal expansion of P53 mutant cells is associated with brain tumour progression. Nature. 1992;355:846-7. 22. Goldstraw P. Staging manual in thoracic oncology. Denver: IASLC; 2009:85-89.
M.- W. Lin et al. 23. Yoo SB, Chung JH, Lee HJ, Lee CT, Jheon S, Sung SW. Epidermal growth factor receptor mutation and p53 overexpression during the multistage progression of small adenocarcinoma of the lung. J Thorac Oncol. 2010;5:964-9. 24. Malumbres M, Barbacid M. RAS oncogenes: the first 30 years. Nat Rev Cancer. 2003;3:459-65. 25. Bos JL. Ras oncogenes in human cancer: a review. Cancer Res. 1989;49:4682-9. 26. Sugio K, Ishida T, Yokoyama H, Inoue T, Sugimachi K, Sasazuki T. Ras gene mutations as a prognostic marker in adenocarcinoma of the human lung without lymph node metastasis. Cancer Res. 1992;52:2903-6.
27. Nakamura H, Haruki T, Adachi Y, Fujioka S, Miwa K, Taniguchi Y. Smoking affects prognosis after lung cancer surgery. Surg Today. 2008;38:227-31. 28. Sobue T, Suzuki T, Fujimoto I, Doi O, Tateishi R, Sato T. Prognostic factors for surgically treated lung adenocarcinoma patients, with special reference to smoking habit. Jpn J Cancer Res. 1991;82:33-9. 29. Okumura T, Asamura H, Suzuki K, Kondo H, Tsuchiya R. Intrapulmonary metastasis of non-small cell lung cancer: a prognostic assessment. J Thorac Cardiovasc Surg. 2001;122:24-8. 30. Martini N, Melamed MR. Multiple primary lung cancers. J Thorac Cardiovasc Surg. 1975;70:606-12.
ORIGINAL ARTICLE – THORACIC ONCOLOGY
Clinicopathology and Genetic Profile of Synchronous Multiple Small Adenocarcinomas: Implication for Surgical Treatment of an Uncommon Lung Malignancy Mong-Wei Lin, MD1,5, Chen-Tu Wu, MD2,5, Shuenn-Wen Kuo, MD3, Yih-Leong Chang, MD2,5, and Pan-Chyr Yang, MD, PhD4,5 Department of Surgery, National Taiwan University Hospital Hsin-Chu Branch, Hsinchu, Taiwan; 2Department of Pathology, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan; 3 Department of Surgery, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan; 4Department of Internal Medicine, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan; 5Graduate Institute of Pathology, National Taiwan University College of Medicine, Taipei, Taiwan 1
ABSTRACT Purpose. Synchronous multiple small adenocarcinomas are detected more frequently than in the past; however, the genetic profile, treatment, and prognosis of patients remain unclear. For treatment decisions and prognostic applications, we evaluated epidermal growth factor receptor (EGFR), p53, and KRAS somatic mutations in synchronous multiple small lung adenocarcinomas. Methods. The presence of EGFR, p53, and KRAS somatic mutations was determined in 64 synchronous multiple lung adenocarcinomas B2 cm in maximal dimension. Mutational analysis was performed on DNA extracted from paraffin-embedded tumors. Results. Five-year disease-free survival (DFS) was 86.1 %, and overall survival was 95.8 %. EGFR, p53, and KRAS mutations were detected in 41 (64.1 %), 8 (12.5 %), and 4 (6.3 %) patients, respectively. The high frequency of genetic mutations resulted in a high discrimination rate of tumor clonality (68.8 %; 44/64) in the study group. Fourteen (31.8 %) patients were assessed as having the same clonality, whereas 30 (68.2 %) patients had different
Electronic supplementary material The online version of this article (doi:10.1245/s10434-014-3642-5) contains supplementary material, which is available to authorized users. Ó Society of Surgical Oncology 2014 First Received: 21 December 2013 Y.-L. Chang, MD e-mail: [email protected]
clonality, which further supported the concept of field cancerization. Multivariate analysis showed lymph node metastasis (p = 0.003) and smoking (p = 0.011) were significantly correlated with tumor relapse. Surgical method, clonality, and tumor location were not correlated with tumor relapse. Conclusions. Whether these tumors are different or the same clonal, sublobar resection of each lesion can achieve long-term DFS and is the treatment of choice for synchronous multiple small lung adenocarcinomas. Patients with lymph node metastasis are at risk of relapse and adjuvant chemotherapy is indicated.
The occurrence of multiple primary cancers within the same organ, such as the breast and ovary, has been widely reported. Although relatively uncommon, lung cancer can present as multiple tumors that are either synchronous or metachronous. The terms ‘synchronous’ and ‘metachronous’ extend a time-related connotation to the evolution of these multifocal tumors; however, it does not specify which tumors are clonal and therefore are related tumors, i.e. are descendants of a single clone, and which tumors are unrelated and therefore are of independent origin. Synchronous multiple primary lung cancers (MPLCs) were first reported in 1924 when Beyreuther identified two separate primary lung cancers in a patient with pulmonary tuberculosis.1 The incidence of synchronous multiple lung cancers in reported clinical series ranges from 1 to 7 %.2–4 Synchronous multiple lung cancers can either be MPLCs or intrapulmonary metastasis. MPLCs may develop
M.- W. Lin et al.
coincidentally. However, sometimes the entire lung epithelium is at risk for carcinogenesis due to similar exposure to carcinogens.3,5 This concept, referred to as field cancerization, may explain the development of MPLCs. The survival of patients with synchronous MPLCs is highly variable; the 5-year survival for all patients and for patients with stage I tumors ranges from 0 to 70 % and 0 to 79 %, respectively.6 Advances in high-resolution computed tomography (CT) imaging and the prevalence of CT screening for lung cancer detection in asymptomatic patients have increased the detection of synchronous multiple small lung tumors in peripheral lung tissue.6,7 Most tumors were B2 cm in maximal dimension, pure or mixed ground glass opacity (GGO) on CT, and diagnosed as adenocarcinomas.6,8–14 Few studies have been published on the outcome of synchronous multiple small lung adenocarcinomas. The reported 5-year survival ranges from 64 to 100 %,8–14 which is considerably better than the prognosis of MPLCs. The clinicopathologic characteristics, genetic profile, and prognosis of patients with synchronous multiple small lung adenocarcinomas still remain unclear. Toward developing useful genetic markers for clonality assessment and patient management, we took advantage of the highly frequent and early somatic mutations of epidermal growth factor receptor (EGFR), p53, and KRAS genes15–18 to define the clonal origins of synchronous multiple small lung adenocarcinomas in 64 patients. For potential diagnostic and management applications, the correlation between genetic mutations, clinicopathologic features, and disease-free survival (DFS) was determined.
METHODS Study Population A total of 64 patients with synchronous multiple small lung adenocarcinomas measuring B2 cm in maximal dimension who underwent complete surgical resection by our team at the National Taiwan University Hospital between January 2005 and June 2012 were included in the study. Written informed patient consent was obtained for tissue analysis at the time of tumor specimen procurement. The Research Ethics Committee of the National Taiwan University Hospital approved this study (project approval number 201110045RD). All synchronous multiple lung adenocarcinomas were incidentally identified by chest CT. Patients were not treated with prior EGFR-targeted therapy, neoadjuvant chemotherapy, or neoadjuvant radiotherapy. All patients received preoperative staging workups, including brain CT or magnetic resonance imaging, bone scan or positron emission tomography (PET), and abdominal CT. Patients with distant metastasis
and supraclavicular lymph node (N3) metastasis were excluded from the study. All patients had undergone complete resection of synchronous multiple lung tumors. The term ‘complete resection’ indicated that all the synchronous multiple small lung adenocarcinomas were resected in a one-stage operation. Patients with residual inoperable synchronous lung tumors were excluded from the study. The surgical methods were classified into two categories: sublobar resection (i.e. wedge resection and segmentectomy) and lobar resection (i.e. lobectomy and bilobectomy). Systemic lymph node dissection was performed in 58 patients, including hilar, interlobar, lobar, and mediastinal lymph nodes. The remaining six patients with peripheral ground glass lesions did not receive lymph node dissection. For 21 patients with synchronous bilateral lung tumors, bilateral tumor resections were both performed in a one-stage operation. All resected specimens were formalin-fixed and sectioned for microscopic examination after staining with hematoxylin-eosin. Lung cancer histology was classified according to the WHO pathology classification.19 Histological diagnosis and pathologic features, including tumor cell type, vascular invasion, visceral pleural invasion, and regional/mediastinal lymph node metastasis, were assessed by two independent pathologists (C-TW and Y-LC). The clinical data of enrolled patients retrieved from chart review included age, sex, smoking status, preoperative carcinoembryonic antigen (CEA) level, relapse, metastasis, and mortality. Patients were excluded if they had combined tumor histology other than adenocarcinoma. Patients with tumors [2 cm in maximal dimension were also excluded from the study. Mutational Analysis of Epidermal Growth Factor Receptor (EGFR), p53, and KRAS EGFR, p53, and KRAS genomic DNA was extracted from paraffin-embedded tumors using the QIAamp DNA Mini kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. Exons 18–21 of EGFR, exons 5–8 of p53, and exon 2 of KRAS were amplified using nested polymerase chain reaction (PCR) with specific primers, as described previously.20,21 The resultant PCR amplicons were purified using a Gel/PCR DNA Fragments Extraction kit (Geneaid Biotech Ltd, Taiwan) according to the manufacturer’s instructions. PCR amplicons were sequenced using the BigDye Terminator kit (Applied Biosystems, Foster City, CA, USA) and ABI Prism 3100 DNA Analyzer (Applied Biosystems) according to the manufacturer’s instructions. All sequencing reactions were performed in both forward and reverse directions by using tracings from at least two independent PCRs. Mutations
Synchronous Small Lung Adenocarcinomas
were also checked against the corresponding sequences from the non-neoplastic lung tissue DNA and single nucleotide polymorphism database. Statistical Analysis The correlation between various clinicopathologic variables and somatic mutations in EGFR, p53, and KRAS was analyzed using Fisher’s exact test. The effect of EGFR, p53, and KRAS on survival was tested using Kaplan–Meier survival plots and analyzed using the log-rank test. All tests were two-sided, and p \ 0.05 was considered significant. The Statistical Package for the Social Sciences version 10.0 software (SPSS Inc., Chicago, IL, USA) was used for all analyses. RESULTS Patient Characteristics Our team at the National Taiwan University Hospital performed 1,223 surgical lung resections in primary lung adenocarcinoma patients between January 2005 and June 2012. The study included 64 patients with a total number of 140 synchronous multiple lung adenocarcinomas measuring B2 cm in maximal dimension. The incidence was 5.2 %. Median follow-up time was 27 months, and the median age was 60 years (range 27–81 years). Of the 64 patients, 46 (71.9 %) patients were women, and only 6 (9.4 %) patients were smokers. Preoperative elevated CEA value was noted in 3 (4.7 %) patients. Fifty-three patients (82.8 %) had two synchronous multiple lung adenocarcinomas, whereas the remaining 11 patients (17.2 %) had more than two synchronous multiple lung adenocarcinomas. Twenty-three (35.9 %) patients had multiple tumors in a single lobe, whereas 20 (31.3 %) patients had tumors in separate lobes of unilateral lung field, and 21 (32.8 %) patients had tumors in bilateral lung fields. Most patients underwent sublobar resection (56.3 %; 36/64), including wedge resection and segmentectomy. Thirteen patients underwent lobar resection (20.3 %; 13/64), including lobectomy and bilobectomy. Lobar and sublobar resections were both performed in the remaining 15 (23.4 %) patients. Fourteen (21.9 %) and 8 (12.5 %) patients had all pure GGO lesions and all pure solid lesions on chest CT, respectively. The remaining patients had all mixed GGO lesions (14; 21.9 %) or heterogeneous lesions (28; 43.8 %). Regarding the largest tumor size, 24 (37.5 %) tumors were B1 cm and 40 (62.5 %) tumors were [1 cm and B2 cm. Vascular invasion and visceral pleural invasion were noted in two (3.1 %) patients and five (7.8 %) patients, respectively. Lymph node metastasis was noted in
four (6.3 %) patients; one patient had N1 metastasis and three patients had N2 metastasis. Relapses were recorded in eight cases, including four cases of bilateral pulmonary metastases and four cases of distant metastases. No local recurrence was noted. Disease-related mortality occurred in two patients. The 5-year DFS and overall survival (OS) of the study population was 86.1 and 95.8 %, respectively. Correlation of Genetic Mutations with Clinicopathologic Features and Clonality Assessment EGFR mutations were detected in 41 (64.1 %) patients. The majority of EGFR mutations were in exon 21 (56.1 %; 23/41) and exon 19 (48.7 %; 20/41). Female patients (71.7 %; 33/46; p = 0.049) had higher EGFR mutation rates compared with male patients (44.4 %; 8/18) (Table 1). For clonality assessments, the mutation status of synchronous multiple tumors was classified into four different distribution patterns: Group A, only one tumor with a mutation; Group B, two or more tumors with identical mutations; Group C, two or more tumors with different mutations; and Group D, tumors with no mutations. Of the 64 patients, 17 (26.6 %) and 24 (37.5 %) patients were classified as having the same clonality (Group B) and different clonality (Group A or C), respectively (Table 2). Clonality was not determined in 35.9 % (23/64) of patients because of the absence of EGFR mutations (Group D). p53 and KRAS mutations were detected in eight (12.5 %) and four (6.3 %) patients, respectively. p53 and KRAS mutations were not correlated with clinicopathologic characteristics (Table 1). Clonality of tumors was not evaluated in 87.5 % (56/64) and 93.8 % (60/64) of patients whose tumors had no detectable p53 and KRAS mutations, respectively (Table 2). Clonality Analysis Using Mutations in EGFR/p53/ KRAS Somatic mutations in EGFR, p53, and KRAS were combined for clonality assessment. This strategy increased the patient number for clonality analysis to 68.8 % (44/64), with EGFR/p53/KRAS mutations absent in only 31.3 % (20/64) of cases. Among the 44 cases suitable for clonality assessment, 14 cases (31.8 %) were classified as having the same clonality (electronic supplementary material [ESM] Fig. 1a), and 30 cases (68.2 %) were classified as having different clonality (ESM Fig. 1b). Clonality assessment was not significantly associated with clinicopathologic characteristics (Table 2). The different clonality rate in patients with synchronous multiple small adenocarcinomas in a single lobe, unilateral different lobes, and bilateral different lobes was 50 % (6/12),
M.- W. Lin et al. TABLE 1 Frequency of p53, EGFR, and KRAS mutations in relation to clinical variables and pathologic characteristics Parameters
No. of patients
p53 mutation
p Value
EGFR mutation
p Value
KRAS mutation
?
-
?
-
?
-
64
8
56
41
23
4
60
B65
39
5
34
26
13
1
38
[65
25
3
22
15
10
0.605
3
22
18
3
15
1
17
46
5
41
0.049
3
43
Positive
6
2
4
0
6
Negative
58
6
52
4
54
No. of patients
p Value
Age (years) 1.000
0.291
Sex Male Female Smoking status
0.676
0.159
8
10
33
13
5
1
36
22
0.406
1.000
1.000
Tumor site One lobe
23
3
20
12
11
0
23
[1 lobe, unilateral
20
3
17
15
5
1
19
[1 lobe, bilateral
21
2
19
0.899
14
7
0.282
3
18
12
12
2
22
0.699
29
11
0.106
2
38
3
1
0
4
34
20
4
50
0.152
Tumor size (cm) B1
24
2
22
[1
40
6
34
Positive
4
1
3
Negative
54
7
47
0.627
Lymph node metastasisa 0.457
1.000
1.000
EGFR epidermal growth factor receptor a
Six patients with pure ground glass opacity lesions did not undergo lymph node dissection
62.5 % (10/16), and 87.5 % (14/16), respectively. Two of the three patients (66.7 %) with lymph node metastasis and available clonality assessment data had different clonality. Correlation between Clinicopathologic Features and Disease-Free Survival The 5-year DFS of the 64 patients was 86.1 %. Univariate analysis identified five significant risk factors for relapse: age B65 years (p = 0.020), smoking (p = 0.011), presence of solid nodules on CT (p = 0.016), lymph node metastasis (p = 0.003), and same EGFR/p53/KRAS clonality (p = 0.024) (Table 3). Multivariate analysis using the Cox proportional hazard regression model identified smoking and lymph node metastasis as significant risk factors for relapse in patients with synchronous multiple small lung adenocarcinomas (Table 4, ESM Fig. 2). Notably, surgical method, clonality, and tumor location were not significantly associated with DFS in our study group. DISCUSSION Patients who present with multiple lung tumors remain a challenge to both clinicians and pathologists. In the 7th
edition of the TNM staging system guidelines,22 multiple lung cancers within the same lobe were downgraded from T4 to T3, whereas intrapulmonary metastasis in other lobes on the same side was downgraded from M1 to T4. Because of the increased use of CT imaging, synchronous multiple small lung tumors have been detected more frequently in the last decade, particularly in Asia.814 According to the few reported studies, most synchronous multiple small lung tumors were adenocarcinomas and pure or mixed GGO on CT, and had good prognosis.8–14 Less is known about the genetic profile and clinicopathologic characteristics of MPLCs. In the current study, we included 64 patients with surgically resected synchronous multiple small lung adenocarcinomas measuring B2 cm in maximal dimension. Most patients were female and non-smokers. Lymph node involvement was present in only 6.9 % of patients. The 5year DFS and OS were 86.1 and 95.8 %, respectively. The prognosis of these patients is similar to that of stage I nonsmall cell lung cancer (NSCLC) patients, therefore clinicians and pathologists should be very careful not to regard these patients as intrapulmonary metastasis and misinterpret the TNM stage. EGFR/p53/KRAS mutations were analyzed for clonality assessment. In our study, the EGFR mutation rates were high (64.1 %), whereas p53 mutations rates were low
Synchronous Small Lung Adenocarcinomas TABLE 2 Clinicopathologic characteristics of patients with the same clonality and different clonality between tumors (n = 44)a Parameters
p53 mutation
KRAS mutation
p53, EGFR, and/or KRAS mutation
SC
DC
SC
DC
SC
DC
SC
DC
3
5
17
24
0
4
14
30
B65
2
3
8
18
0
1
6
20
[65
1
2
9
6
0
3
8
10
Male
2
1
4
4
0
1
4
5
Female Smoking status
1
4
13
20
0
3
10
25
Positive
1
1
2
3
0
0
2
3
Negative
2
4
15
21
0
4
12
27
No. of patients
p Value
EGFR mutation p Value
p Value
p Value
Age (years) 1.000
0.102
NA
0.191
Sex 0.464
1.000
0.698
1.000
NA
NA
0.434
0.647
Tumor site One lobe
2
1
7
5
0
0
6
6
[1 lobe, unilateral
1
2
7
8
0
1
6
10
[1 lobe, bilateral
0
2
0.679
3
11
0.165
0
3
NA
2
14
6
6
0
2
5
8
1.000
11
18
0.507
0
2
NA
9
22
1
2
0
0
1
2
14
20
0
4
11
26
0.100
Tumor size (cm) B1
1
1
[1
2
4
0.724
Lymph node metastasisb Positive
0
1
Negative
3
4
1.000
1.000
NA
1.000
EGFR epidermal growth factor receptor, DC different clonality, SC same clonality, NA not applicable a
Patients with no EGFR/p53/KRAS mutation were deleted
b
Six patients with pure ground glass opacity lesions did not undergo lymph node dissection
(12.5 %). A high frequency of EGFR mutations has been reported in the early stage of pulmonary adenocarcinoma development.23 In contrast, p53 overexpression was significantly less frequent in non-invasive tumors.23 Our study population had multiple small lung adenocarcinomas B2 cm in maximal dimension, and the prognosis was as good as stage I NSCLCs. Therefore, the high EGFR and low p53 mutation rates in our study can be explained by the theory of EGFR mutations as early events associated with tumor initiation and p53 mutations as late events associated with tumor progression.23 KRAS mutations are one of the most common oncogenic events in human carcinomas of endodermal origin, occurring at high frequency in adenocarcinomas of lung, pancreatic, and colorectal origin.24,25 KRAS mutations have been detected in 5–15 % of East Asian patients with lung adenocarcinoma.26 The frequency of KRAS mutations was 6.3 % in the current study, which was similar to previous studies. In the current series, 30 patients (68.2 %; 30/44) showed different EGFR/p53/KRAS clonality despite having a similar histological type, and also presented with similar CT characteristics. Therefore, it may be unreliable to use histological type or imaging characteristics alone to
differentiate primary tumors from intrapulmonary metastasis. Clonality assessment can provide more information for further clarification of MPLCs. Synchronous multiple small lung adenocarcinomas with different EGFR/p53/ KRAS clonality can be regarded as MPLCs. However, it still lacks evidence that those with the same EGFR/p53/ KRAS clonality are definite intrapulmonary metastasis. Multivariate analysis showed that lymph node metastasis and smoking were significant factors for tumor relapse. Smoking as a significant predictor of poor prognosis after lung cancer surgery has been reported in several previous studies.27,28 The relationship of advanced lymph node status and poor prognosis has also been well documented, and lymph node status has become an important staging factor in the current TNM system.22 In patients with synchronous multiple lung cancers, a better 5-year OS (37– 52.5 %) has been reported in node-negative patients compared with node-positive patients (15.5–24 % %).4,29 However, the relationship between lymph node metastasis and prognosis in synchronous multiple small lung adenocarcinomas has not been reported. Our study is the first to report a more favorable 5-year DFS in node-negative patients compared with node-positive patients (91.8 vs.
M.- W. Lin et al. TABLE 3 Univariate survival analysis of clinicopathologic features in patients with synchronous multiple small lung adenocarcinomas Parameters
No. of patients
No. of relapses (%)/ 5-year DFS (%)
Total no. of patients
64
8 (12.5)/86.1
25 39
6 (24.0)/78.4 2 (5.1)/93.0
Male
18
3 (16.7)/82.5
Female
46
5 (10.9)/89.3
Positive
6
3 (50.0)/50.0
Negative
58
5 (8.6)/91.3
Normal
61
6 (9.8)/88.3
Abnormal
3
2 (66.7)/66.7
C3
11
1 (9.1)/90.0
=2
53
7 (13.2)/87.3
0.708
40 24
7 (17.5)/82.7 1 (4.2)/95.5
0.152
TABLE 3 continued Parameters
No. of patients
No. of relapses (%)/ 5-year DFS (%)
p Value
23
2 (8.7)/95.2
0.369
Mutation
8
2 (25.0)/75.0
Wild type
56
6 (10.7)/89.2
Mutation
4
0 (0.0)/100.0
Wild type
60
8 (13.3)/86.6
p Value Wild type p53 gene
Age (years) B65 [65
0.020
Sex 0.798
Smoking status 0.011
CEA
0.159
KRAS gene 0.527
CEA serum carcinoembryonic antigen, CT computed tomography, DFS disease-free survival, GGO ground glass opacity, EGFR epidermal growth factor receptor a
Six patients with pure ground glass opacity lesions did not undergo lymph node dissection
b
Patients with no EGFR/p53/KRAS mutation were deleted
0.056
Tumor number
Largest tumor size (cm) [1 cm B1 cm
TABLE 4 Multivariate survival analysis of clinicopathologic features in patients with synchronous multiple small lung adenocarcinomas Parameters
No. of No. of Hazard 95 % CI patients relapses ratio (%)
p Value
[65
39
2 (5.1)
0.006–1.325
0.080
B65
25
6 (24.0) 1.000 0.005–0.601
0.017
0.005–0.665
0.022
2 (10.0) 1.022
0.033–31.844
0.990
0.845–273.959 0.065
Tumor site One lobe
23
4 (17.4)/85.3
[1 lobe, unilateral
20
2 (10.0)/86.8
[1 lobe, bilateral
21
2 (9.5)/89.3
Pure GGO
14
0 (0.0)/100.0
Negative
6
3 (50.0) 0.055
Mixed type
42
4 (9.5)/88.7
Positive
58
5 (8.6)
1.000
Solid
8
4 (50.0)/62.5
0.058
Positive
2
1 (50.0)/0.0
Negative
62
7 (11.3)/89.3
Positive
5
1 (20.0)/50.0
Negative
59
7 (11.9)/89.0
Positive
4
3 (75.0)/37.5
Negative
54
4 (7.4)/91.8
Age (years) 0.828
CT characteristics
Smoking status
0.016
Lymph node metastasisa
Vascular invasion 0.092
Pleural invasion
Negative
54
4 (7.4)
Positive
4
3 (75.0) 1.000
Clonality assessment Wild type 20
0.358
Lymph node metastasisa
SC
14
4 (28.6) 15.216
DC
30
2 (6.7)
CT characteristics Pure GGO 0.003
Surgery Sublobar resection
36
2 (5.6)/96.3
Lobar ? sublobar
15
3 (20.0)/77.0
Lobar resection
13
3 (23.1)/75.0
Same
14
4 (28.6)/55.9
Different
30
2 (6.7)/92.5
41
6 (14.6)/82.5
EGFR gene
14
0 (0.0)
0.000
0.000
0.984
Mixed type 42
4 (9.5)
0.458
0.029–7.208
0.579
Solid
4 (50.0)
8
CI confidence interval, CT computed tomography, DC different clonality, GGO ground glass opacity, SC same clonality 0.252
Clonality assessmentb
Mutation
0.092
0.024
a
Six patients did not undergo lymph node dissection
37.5 %). This finding raises two importance issues. First, adjuvant therapy may be indicated for patients with lymph node metastasis. Second, preoperative mediastinal lymph node assessment (CT and/or PET) is important to rule out
Synchronous Small Lung Adenocarcinomas
lymph node metastasis in synchronous multiple small lung adenocarcinoma patients. Tumor location has been regarded as an important prognostic factor in synchronous multiple lung cancers. In the Martini and Melamed criteria, multiple lung cancers with a similar histological type and located in the same lobe were regarded as intrapulmonary metastasis.30 However, our study showed no relationship between tumor location and relapse. Furthermore, DFS was not significantly different according to surgical method or clonality. Therefore, no matter whether the tumors were the same or different clonal, sublobar resection for each lesion of synchronous multiple small lung adenocarcinomas is the treatment of choice to preserve more pulmonary parenchyma and achieve the same DFS. CONCLUSIONS This study showed the good prognosis of surgically resected synchronous multiple small lung adenocarcinomas. The status of this particular form of NSCLCs might be considered in the TNM staging system for more accurate prediction of patient prognosis. Sublobar resection of each lesion in patients with node-negative synchronous multiple small lung adenocarcinomas is associated with a DFS rate similar to that of stage I NSCLCs and is the treatment of choice. However, patients with lymph node metastasis have poor DFS and, therefore, sublobar resection treatment is not adequate in these patients and adjuvant chemotherapy should be considered. ACKNOWLEDGEMENT The authors would like to thank Professor Yung-Chie Lee (October 23, 1948 to December 30, 2010) for his substantial contribution to patient care and surgery. This study was funded by the National Taiwan University Hospital (NTUH 100N1713). CONFLICTS OF INTEREST Mong-Wei Lin, Chen-Tu Wu, Shuenn-Wen Kuo, Yih-Leong Chang, and Pan-Chyr Yang declare no conflicts of interest.
REFERENCES 1. Beyreuther H. Multipicate von carcinomen bei einem fall von sog: ‘Schnee- berger’ lungenkrebs mit tuberculose. Virchows Arch Pathol Anat. 1924;250:230-6. 2. Ferguson MK, DeMeester TR, DesLauriers J, Little AG, Piraux M, Golomb H. Diagnosis and management of synchronous lung cancers. J Thorac Cardiovasc Surg. 1985;89:378-85. 3. Chang YL, Wu CT, Lin SC, Hsiao CF, Jou YS, Lee YC. Clonality and prognostic implications of P53 and EGFR somatic aberrations in multiple primary lung cancers. Clin Cancer Res. 2007;13:52-8. 4. Chang YL, Wu CT, Lee YC. Surgical treatment of synchronous multiple primary lung cancers: experience of 92 patients. J Thorac Cardiovasc Surg. 2007;134:630-7.
5. Nelson MA, Wymer J, Clements N Jr. Detection of K-ras gene mutations in non-neoplastic lung tissue and lung cancers. Cancer Lett. 1996;103:115-21. 6. Kozower BD, Larner JM, Detterbeck FC, Jones DR; American College of Chest Physicians. Special treatment issues in lung cancer: ACCP evidence-based clinical practice guidelines (3rd edition). Chest. 2013;143:e369-e399s. 7. Detterbeck FC, Mazzone PJ, Naidich DP, Bach PB; American College of Chest Physicians. Screening for lung cancer: ACCP evidence-based clinical practice guidelines (3rd edition). Chest. 2013;143:e78-e92s. 8. Ebright MI, Zakowski MF, Martin J, et al. Clinical pattern and pathologic stage but not histologic features predict outcome for bronchioloalveolar carcinoma. Ann Thorac Surg. 2002;74:16406. 9. Mun M, Kohno T. Efficacy of thoracoscopic resection for multifocal bronchioloalveolar carcinoma showing pure ground-glass opacities of 20 mm or less in diameter. J Thorac Cardiovasc Surg. 2007;134:877-82. 10. Roberts PF, Straznicka M, Lara PN, Lau DH, Follette DM, Gandara DR, et al. Resection of multifocal non-small cell lung cancer when the bronchioloalveolar subtype is involved. J Thorac Cardiovasc Surg. 2003;126:1597-602. 11. Nakata M, Sawada S, Yamashita M, Saeki H, Kurita A, Takashima S, et al. Surgical treatments for multiple primary adenocarcinoma of the lung. Ann Thorac Surg. 2004;78:1194-9. 12. Carretta A, Ciriaco P, Melloni G, et al. Surgical treatment of multiple primary adenocarcinomas of the lung. Thorac Cardiovasc Surg. 2009;57:30-4. 13. Kim HK, Choi YS, Kim J, Shim YM, Lee KS, Kim K. Management of multiple pure ground-glass opacity lesions in patients with bronchioloalveolar carcinoma. J Thorac Oncol. 2010;5:20610. 14. Casali C, Rossi G, Marchioni A, et al. A single institution-based retrospective study of surgically treated bronchioloalveolar adenocarcinoma of the lung: clinicopathologic analysis, molecular features, and possible pitfalls in routine practice. J Thorac Oncol. 2010;5:830-6. 15. Mitsudomi T, Yatabe Y, Koshikawa T, et al. Mutations of the P53 tumor suppressor gene as clonal marker for multiple primary lung cancers. J Thorac Cardiovasc Surg. 1997;114:354-60. 16. Matsuzoa D, Hideshima T, Ohshima K, Kawahara K, Shirakusa T, Kimura A. Discrimination of double primary lung cancer from intrapulmonary metastasis by P53 gene mutation. Br J Cancer. 1999;79:1549-52. 17. Van Rens MT, Eijken EJ, Elbers JR, Lammers JW, Tilanus MG, Slootweg PJ. P53 mutation analysis for definite diagnosis of multiple primary lung carcinoma. Cancer. 2002;94:188-96. 18. Tang X, Shigematsu H, Bekele BN, et al. EGFR tyrosine kinase domain mutations are detected in histologically normal respiratory epithelium in lung cancer patients. Cancer Res. 2005;65:7568-72. 19. Travis WD, Brambilla E, Muller-Hermelink HK, et al. Pathology and genetics: tumours of the lung, pleura, thymus and heart. Lyon: IARC Press; 2004. 20. Linardou H, Dahabreh IJ, Kanaloupiti D, et al. Assessment of somatic k-RAS mutations as a mechanism associated with resistance to EGFR-targeted agents: a systemic review and metaanalysis of studies in advanced non-small-cell lung cancer and metastatic colorectal cancer. Lancet Oncol. 2008;9:962-72. 21. Sidransky D, Mikkelsen T, Schwechheimer K, Rosenblum ML, Cavanee W, Vogelstein B. Clonal expansion of P53 mutant cells is associated with brain tumour progression. Nature. 1992;355:846-7. 22. Goldstraw P. Staging manual in thoracic oncology. Denver: IASLC; 2009:85-89.
M.- W. Lin et al. 23. Yoo SB, Chung JH, Lee HJ, Lee CT, Jheon S, Sung SW. Epidermal growth factor receptor mutation and p53 overexpression during the multistage progression of small adenocarcinoma of the lung. J Thorac Oncol. 2010;5:964-9. 24. Malumbres M, Barbacid M. RAS oncogenes: the first 30 years. Nat Rev Cancer. 2003;3:459-65. 25. Bos JL. Ras oncogenes in human cancer: a review. Cancer Res. 1989;49:4682-9. 26. Sugio K, Ishida T, Yokoyama H, Inoue T, Sugimachi K, Sasazuki T. Ras gene mutations as a prognostic marker in adenocarcinoma of the human lung without lymph node metastasis. Cancer Res. 1992;52:2903-6.
27. Nakamura H, Haruki T, Adachi Y, Fujioka S, Miwa K, Taniguchi Y. Smoking affects prognosis after lung cancer surgery. Surg Today. 2008;38:227-31. 28. Sobue T, Suzuki T, Fujimoto I, Doi O, Tateishi R, Sato T. Prognostic factors for surgically treated lung adenocarcinoma patients, with special reference to smoking habit. Jpn J Cancer Res. 1991;82:33-9. 29. Okumura T, Asamura H, Suzuki K, Kondo H, Tsuchiya R. Intrapulmonary metastasis of non-small cell lung cancer: a prognostic assessment. J Thorac Cardiovasc Surg. 2001;122:24-8. 30. Martini N, Melamed MR. Multiple primary lung cancers. J Thorac Cardiovasc Surg. 1975;70:606-12.
