Kaira, Kyoichi MD*; Horie, Yoshiki MD*; Ayabe, Eriko MD*; Murakami, Haruyasu MD*; Takahashi, Toshiaki MD*; Tsuya, Asuka MD*; Nakamura, Yukiko MD*; Naito, Tateaki MD*; Endo, Masahiro MD†; Kondo, Haruhiko MD‡; Nakajima, Takashi MD§; Yamamoto, Nobuyuki MD*
Pulmonary pleomorphic carcinoma is rare and its incidence has ranged from 0.1 to 0.4% of all lung cancer.1 The recent World Health Organization histologic classification of lung tumors lists 5 subtypes of sarcomatoid carcinoma: pleomorphic carcinoma, spindle cell carcinoma, giant cell carcinoma, carcinosarcoma, and pulmonary blastoma.2 Pleomorphic carcinoma is defined as poorly differentiated adenocarcinoma, squamous cell carcinoma, or large cell carcinoma, containing spindle cell and/or giant cells or a carcinoma consisting of spindle and giant cells alone, with a sarcomatoid tumor component of at least 10%.2–4 Pulmonary pleomorphic carcinoma has a more aggressive clinical course than other non-small cell lung cancer (NSCLC), and the response to systemic chemotherapy is generally poor.3–5 Recent reports also described that pleomorphic carcinoma had a worse outcome than other NSCLC, and systemic therapy needed to be explored.5–7 However, the clinicopathological characteristics of pleomorphic carcinoma is not well known.
Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that is expressed in NSCLC. Approximately 70% of NSCLCs with EGFR mutations respond to EGFR-tyrosine kinase inhibitors.8 However, the information on the EGFR mutation status of pulmonary pleomorphic carcinoma is sparse. Moreover, we have only limited information on the glucose metabolism by 2-[18F]-fluoro-2-deoxy-d-glucose positron emission tomography (18F-FDG PET), or Ki-67 labeling index, as a proliferative marker of tumor cells. In the current study, therefore, we have retrospectively examined 17 patients with pulmonary pleomorphic carcinoma diagnosed by surgical resection and transbronchial lung biopsy.
PATIENTS AND METHODS
Between March 2004 and October 2008, we retrospectively analyzed 17 pulmonary pleomorphic carcinomas (0.8%) from a total of 2083 primary lung cancers at Shizuoka Cancer Center. Pleomorphic carcinoma was diagnosed according to the 2004 World Health Organization classification.2 A diagnosis was obtained based on the light microscopic findings, and it was confirmed based on immunohistochemical examinations. Pleomorphic carcinoma was defined as NSCLC containing at least 10% sarcomatoid components. The pathologic diagnosis was made by surgical resection or transbronchial lung biopsy. The study protocol was approved by the Institutional Review Board.
18F-FDG PET Imaging
Patients fasted for at least 4 hours before 18F-FDG PET examination. Patients received an intravenous injection of 200 to 250 MBq of fluoro-2-deoxy-d-glucose and then rested for approximately 1 hour before undergoing imaging. Image acquisition was performed using an Advance NXi PET scanner and Discovery PET/CT scanner (GE Medical Systems, Milwaukee, WI).9 Two-dimensional emission scanning was performed from the groin to the top of the skull. Acquired data were reconstructed by iterative ordered subset expectation maximization. To evaluate 18F-FDG accumulation, the tumor was first examined visually, and then the peak standardized uptake value (SUV) of the entire tumor was determined. The region of interest was manually drawn on the SUV images over the primary tumor. In this series, 16 patients underwent 18F-FDG PET.
EGFR Mutation, KRAS Mutation, and Ki-67 Labeling Index Analysis
We investigated EGFR gene mutations by peptide nucleic acid-locked nucleic acid polymerase chain reaction clamp, and the detailed protocol for this clamp method was published elsewhere.10 KRAS mutation was also detected by the assay using DNA extracted from tumor tissue and the detailed protocol published elsewhere.11
The detailed protocol for Ki-67 immunostaining was published elsewhere.12 Briefly, formalin-fixed and paraffin-embedded sections of resected specimens were dewaxed, rehydrated, trypsinized, and boiled in 0.01 mol/L citrate buffer for 20 minutes. For immunostaining, the murine monoclonal antibody MIB-1 (Dako, Denmark), specific for human nuclear antigen Ki-67, was used in a 1:40 dilution. The sections were lightly counterstained with hematoxylin. Sections of a normal tonsil were used as positive control for proliferating cells. A highly cellular area of the immunostained sections was evaluated. All epithelial cells with nuclear staining of any intensity were defined as positive. Approximately 1000 nuclei were counted on each slide. Proliferative activity was assessed as the percentage of MIB-1-stained nuclei (Ki-67 labeling index) in the sample.
We used the Response Evaluation Criteria in Solid Tumors to assess response to chemotherapy and/or radiotherapy.13 Response based on target (and nontarget lesions) was defined as follows: complete response, disappearance of all target (nontarget) lesions; partial response (PR), ≥30% reduction in size (or disappearance of one or more nontarget lesions); stable disease (SD), less than 30% decrease and less than 20% increase in size (or the persistence of one or more nontarget lesions); progressive disease (PD), more than 20% increase in size (or the appearance of new nontarget lesions and/or progression of existing nontarget lesions). The overall response was defined as the best response recorded from the start of treatment until disease progression or recurrence.
Overall survival time was determined as the time from the start of the treatment to death from any cause. Survival estimation was performed using Kaplan-Meier method and log-rank test. A p value less than 0.05 was considered indicative statistical significance. Fisher's exact test was used to examine the association of two categorical variables.
Patient characteristics is listed in Table 1. The median age of the patients was 72 years (range, 47–84 years). Thirteen patients were men and 4 were women. Performance status was 0 to 1 in 16 (94%) of 17 patients. Fourteen patients were smokers, and there were 9 patients with stage I–II and 8 patients with stage III–IV. Nine of 17 patients were diagnosed by resected primary tumor. Of the other 8 patients, 7 patients were diagnosed by bronchoscopic biopsy and one by surgical biopsy.
EGFR mutation and Ki-67 labeling index were investigated in all patients. Sixteen patients underwent 18F-FDG PET. As initial treatment, nine patients were treated with surgery, seven patients chemotherapy and/or radiotherapy, and one patient best supportive care.
EGFR Mutation, KRAS Mutation, and Ki-67 Labeling Index
EGFR mutation was observed in 3 (18%) of 17 patients. Two patient (patients 1 and 6) had an exon 19 (patient 6 had deletion 19 and T790M EGFR mutations) and the other patient (patient 16) had an exon 21. The association between EGFR mutation status and pathologic subtypes is summarized in Table 2. Adenocarcinoma (11 of 17, 65%) as carcinomatous elements was predominantly observed. All three patients with EGFR mutation had a pathologic feature of adenocarcinoma. In three mutated patients, the histologic pattern of adenocarcinoma in one patient (patient 16) indicated moderate differentiated adenocarcinoma with bronchioloalveolar carcinoma pattern, but that of the other patients (patients 1 and 6) was unknown. Next, we investigated independently EGFR mutations both in adenocarcinomaous and sarcomatoid components of these three mutated patients. In all cases, EGFR mutations were detected in adenocarcinomatous component, but not in sarcomatoid component. Moreover, KRAS mutations were also examined in these three mutated patients. KRAS mutations were not observed in all of these patients.
The median value of Ki-67 labeling index was 62% (range, 20–87%), and the value of 60% was chosen as the cutoff point. High expression (Ki-67 > 60%) was seen in 10 (59%) of 17 patients. The mean value of Ki-67 was significantly higher in stage III–IV (71 ± 3) than stage I–II (38 ± 4) (p < 0.001).
18F-FDG PET Findings
The SUVmax of the primary tumors in 16 patients ranged from 6.1 to 26.8 (median 19.3). The mean of the SUVmax demonstrated no significant difference between patients with stage III–IV (18.3 ± 2.2) and those with stage I–II (15.9 ± 3.4) (p = 0.552). Moreover, no significant difference was observed between patients with T1 or 2 (16.6 ± 2.5) and those with T3 or 4 (18.5 ± 3.0) (p = 0.653).
Clinical Outcome After Treatment
Of the nine patients who underwent surgery, six patients have no recurrence at the time of analysis. All patients were not treated with adjuvant therapy. One patient (patient 13) died of noncancer-related disease (pneumonia) from surgery. The other two patients (patients 3 and 16) developed recurrence after surgery. However, the patients died of cancer-related disease. Figure 1 shows overall survival curve in the nine patients with surgery and eight patients without surgery. The survival of patients without surgery demonstrated a significantly poor prognosis as compared with those with surgery (p = 0.0096). The median survival time was 8.5 months in patients who did not undergo surgery and was not reached in patients with surgery.
Table 3 shows the treatment results of chemotherapy in patients with advanced or relapsed disease. Two patients (patients 8 and 17) with stage III disease were treated with chemoradiotherapy and achieved partial disease. In six patients with advanced or relapsed disease, four patients had PD and two patients had SD after first-line chemotherapy. All the six patients had second-line chemotherapy. Four patients were treated with docetaxel or vinorelbine and achieved PD. One patient (patient 16) who had L858R mutation in exon 21 of EGFR and was treated with gefitinib achieved SD. One patient (patient 2) was treated with carboplatin plus gemcitabine and achieved partial response. However, two patients (patients 2 and 5) given third-line chemotherapy had PD.
Two patients (patients 1 and 6) who had EGFR exon 19 deletion mutation were not treated with gefitinib. One patient (patient 6) was treated with supportive care alone because of poor performance status, and the patient's survival time was 1.0 month. The other patient's survival time (patient 1) was 5.5 months.
This is a retrospective study to evaluate the clinicopathological features including EGFR mutation status in pulmonary pleomorphic carcinoma. EGFR mutation was observed in 3 (18%) of 17 patients. All patients with EGFR mutation had a carcinomatous element of adenocarcinoma. Of the three patients with EGFR mutation, one patient was treated with gefitinib and achieved SD. However, two patients were not treated with EGFR-tyrosine kinase inhibitor, and they had poor outcome. Moreover, the cell proliferation of tumor cells was significantly higher in patients with advanced stage than those with early stage. 18F-FDG uptake in the primary tumors tented to be high (median SUVmax, 19.3). However, no significant difference in 18F-FDG uptake was observed between early and advanced stage. The chemotherapy regimens commonly used for NSCLC were not effective for advanced pulmonary pleomorphic carcinoma.
Recently, Ushiki et al.14 described a case of pulmonary pleomorphic carcinoma with two different histologic types exhibiting EGFR mutations. Their case revealed that adenocarcinoma cells had an exon 19 deletion and sarcomatous cells had both the deletion 19 and 20 T790M EGFR mutations. The response to gefitinib in their case was small and transient. They speculated that tumor volume of adenocarcinoma was small or that the exon 19 deletion-positive cells were at a low frequency. Ito et al.15 described a case of pulmonary pleomorphic carcinoma treated with gefitinib, with L858R EGFR mutation. Their case exhibited partial response to gefitinib, and the pathologic subtype was adenocarcinoma combined with spindle cell. Moreover, Bae et al.6 also described that a case of pulmonary pleomorphic carcinoma with L858R EGFR mutation was treated with gefitinib and the response achieved SD. Only four cases including our present case had been described as the reported cases who had EGFR mutation and was treated with gefitinib. However, the incidence of EGFR mutation was unknown in pulmonary pleomorphic carcinoma. Although this study was small sample size, our results suggest that the incidence of EGFR mutation was approximately 20%, and EGFR mutations were observed in patients with a carcinomatous element of adenocarcinoma. In the three mutated patients, EGFR mutations were detected in adenocarcinomatous component, but not in sarcomatoid component. Moreover, KRAS mutations were not observed in all these patients. Our results were also consistent with the fact that NSCLC patients with EGFR mutations do not harbor KRAS mutation. As there was no evidence of EGFR mutations in sarcomatoid component, the response to gefitinib might be small and transient in this study. The other patients with EGFR mutation were not treated with gefitinib, therefore it is unclear whether gefitinib was effective for the two mutated patients. Recent study demonstrated that an adenocarcinoma component was found in 49% of pulmonary pleomorphic carcinoma.5 In this study, an adenocarcinoma component was observed in 11 (65%) of 17 patients, and the incidence seems to be high compared with previous study. Sartori et al. described that EGFR mutations were not observed in a small cohort of sarcomatoid carcinomas. This is consistent with our results.16 We compared our results with those published by Sartori et al.16 and found that male smokers with pleomorphic carcinoma from Japan have an higher rate of EGFR mutations than what one can see in a series of nonmucinous bronchioloalveolar carcinoma from never smoker Caucasian women. Further study should be investigated whether gefitinib is effective in pulmonary pleomorphic carcinoma with EGFR mutation.
Several researchers described that neither platinum-based nor nonplatinum-based chemotherapy was effective in patients with pulmonary pleomorphic carcinoma.6,15 Bae et al.6 reported that palliative chemotherapy was poor response in advanced pulmonary pleomorphic carcinoma, with short survival. Interestingly, however, the combination of carboplatin and gemcitabine in our study was effective in advanced pulmonary pleomorphic carcinoma. It is unknown why carboplatin and gemcitabine achieved partial response. In previous reports, advanced pulmonary pleomorphic carcinoma showed poor response to carboplatin or gemcitabine.6,15 In our study, whereas, platinum-based chemoradiotherapy was effective in stage III disease with pulmonary pleomorphic carcinoma. Because pulmonary pleomorphic carcinoma demonstrated an aggressive clinical course with a high relapsed rate after surgery, it is unclear whether combined chemoradiotherapy is effective in patients with unresectable stage III disease. Our results suggest that chemoradiotherapy commonly used for NSCLC should be administered to unresectable stage III disease with pleomorphic carcinoma.
Recently, Mochizuki et al.5 mentioned that pulmonary pleomorphic carcinoma has distinctive clinicopathological features and a poor prognosis compared with other NSCLC. They reported that massive coagulation necrosis was an independent prognostic factor for predicting a poor outcome. The coagulation necrosis was also described to be associated with a pathologic manifestation of rapidly proliferating tumor cells. In this study, the median value of Ki-67 labeling index was 62% and seemed to be higher than that of other NSCLC.11 Moreover, the mean value of Ki-67 was significantly higher in stage III–IV (71 ± 3) than stage I–II (38 ± 4). This is similar to the results of previous study.17 However, Pelosi et al.17 described that high Ki-67 labeling index correlated negatively with overall survival. Their study was also small sample size, and a meta-analysis indicated that the expression of Ki-67 is a factor of poor prognosis for survival in NSCLC.18 A large-scale study should be conducted to elucidate whether Ki-67 labeling index could be a prognostic factor for predicting a poor outcome in pulmonary pleomorphic carcinoma.
18F-FDG PET was useful for the diagnosis of lung cancer.19 Determination of malignant lesions with 18F-FDG PET is based on the glucose metabolism, and the overexpression of glucose transporter 1 (Glut1) in human cancer has been shown to be closely related to 18F-FDG uptake.20,21 Our results of 18F-FDG PET indicated that the SUVmax of pleomorphic carcinoma might be higher than that of other NSCLC. Ito's study was also similar to the results of our study. Previous study suggests that Glut1 expression was higher in undifferentiated adenocarcinoma than well-differentiated adenocarcinoma.20 In this study, we did not investigate the expression of Glut1 in pleomorphic carcinoma. Considering that the SUVmax of the primary tumors tended to be high in our study, the expression of Glut1 may be high in pulmonary pleomorphic carcinoma. High 18F-FDG uptake on PET might be helpful in the diagnosis of pleomorphic carcinoma.
One of the limitations of our study is that our population was small sample size, including the specimens obtained by transbronchial biopsy. However, only patients with an adequate specimen obtained during bronchoscopic biopsy were included. Another limitation is that the mutation of EGFR was not investigated before the administration of gefitinib or cytotoxic agents. Thus, two of three patients with EGFR mutations were not treated with gefitinib.
In conclusion, EGFR mutation was observed in 3 (18%) of 17 patients, which had a carcinomatous element of adenocarcinoma. Of the three mutated patients, the response of one patient given gefitinib was small and transient. Pulmonary pleomorphic carcinoma has an aggressive tumor associated with high cell proliferation. Palliative chemotherapy was almost poor response in advanced pulmonary pleomorphic carcinoma. Further investigation will be required to elucidate whether the use of a molecular targeting drug improve outcome for pulmonary pleomorphic carcinoma.
The authors thank M Abe for technical assistance in the analysis of EGFR and KRAS mutations.
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