1284 Neuroendocrine Differentiation Is an Independent Prognostic Factor in Chemotherapy-Treated Nonsmall Cell Lung Carcinoma Jeffrey T. Schleusener, M.D.*' Henry D. Tazelaar, M.D.' Sin-ho JUng, Ph.D.2 Stephen S. Cha, M.s.~ Peter J. Cera, M.D? Jeffrey L. Myers, M.D.' Edward T. Creagan, M.D? Richard M. Goldberg, M.D? Robert F. Marschke, Jr., M.D? Departments of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota. Department of Cancer Center Statistics, Mayo Clinic, Rochester, Minnesota. Department of Pathology, Geisinger Clinic, Geisinger Medical Center, Danville, Pennsylvania. Department of Medical Oncology, Mayo Clinic, Rochester, Minnesota. Division of Hematology and Medical Oncology, Mayo Clinic Scottsdale, Scottsdale, Arizona. BACKGROUND. Neuroendocrine differentiation can be identified in 10-30% of patients with nonsmall cell lung carcinoma (NSCLC) by immunohistochemical or electron microscopic techniques. However, its clinical significance is not well established. METHODS. Tumors from 107 patients with Stage IIIA, IIIB, and IV NSCLC treated with cisplatinletoposide with or without hydrazine in the North Central Cancer Treatment Group and Mayo Clinic protocols were analyzed immunohistochemically with antibodies to chromogranin A (CGA), Leu 7 (CD 57), and synaptophysin (SY). These results were compared with clinical outcomes. RESULTS. Keratin AElIAE3, used as a control, was positive in 99.1% of cases; 34.6% had positive staining for at least 1 neuroendocrine marker, and 11.3% had positive staining for 2 or more markers. CGA was positive in 4.7%, Leu 7 in 18.7%, and SY in 24.3% of cases. A significant increase in survival was seen in patients with tumors expressing any one neuroendocrine marker or any combination of neuroendocrine markers ( P 5 0.01). There was no correlation between the presence of neuroendocrine differentiation and either response to chemotherapy or time to disease progression (P> 0.3), nor was there any correlation between chemotherapy response, time to progression, or survival with staining intensity or percent of cells positive per case. CONCLUSIONS. Neuroendocrine differentiation may be of prognostic significance in patients with advanced stage NSCLC treated with chemotherapy. Cancer 1996; 77:1284-91. 0 1996 American Cancer Society. KEYWORDS Nonsmall cell lung carcinoma, neuroendocrine, chromogranin, synaptophysin, Leu 7, small cell lung carcinoma. L This study was supported in part by the North Central Cancer Treatment Group, NIH CA 25224. Address for reprints: Henry D. Tazelaar, M.D., Department of Laboratory Medicine and Pathology, Mayo Clinic, 200 First Street, SW, Rochester, MN 55905. Received June 21, 1995; revision received September 25,1995; accepted December 21,1995. 0 1996 American Cancer Society ung cancer is the leading cause of cancer-related death in the United States and is responsible for 33% of cancer-related deaths in men and 24% in women. An estimated 158,700 patients will have died of lung carcinoma in 1996,' and it is estimated that 2 million patients will be diagnosed worldwide in the year 2000. Despite recent therapeutic advances, the overall 5-year survival rate has improved little in the past 30 years and remains at a dismal 14%. Tumor stage is the most important prognostic factor; 5-year survival rates for patients with Stage I disease are about 50%, but are less than 5% for patients with Stage IIIB or N disease.' Histologic subtyping shows only a weak correlation with outcome when considered as an independent prognostic variable. The World Health Organization classifies lung cancer into four main histologic types: small cell lung carcinoma (SCLC), squamous cell carcinoma, adenocarcinoma, and large cell c a r ~ i n o m a .The ~ most important distinction Neuroendocrine Expression in Lung Cancer/Schleusener et at. 1285 TABLE 1 TABLE 2 Histologic Types of Lung Carcinoma Studied and Frequency of Neuroendocrine Differentiation Percentage of Cases of Nonsmd Cell Lung Carcinoma Reacting with a Panel of Immunohistochemical Neuroendocrine Markers Frequency of Histologic type Adenorarcinoma Squamous ceU carcinoma Large cell carcinomainonsmall ceU carcinoma, not otherwise specified Adenosquamous carcinoma histologic type No. of cases (%) % showing neuroendocrine differentiation (%) 62 (58) 22 (21) 18 (17) 5 (5) stain % Stain % 41 Keratin SY Leu 7 CGA 99.1 24.3 18.7 4.7 Leu 7 t SY SY t CGA Leu 7 t CGA Leu 7 t SY t CGA 9.3 2.8 2.8 1.9 33 SY: SvnaDtoDhVsin: CGA chromoeranin A 35 0 is between SCLC and the others, which can be considered together as nonsmall cell lung carcinoma (NSCLC).SCLC tends to be more chemo-and radiosensitive than NSCLC, yet it shows a more dismal overall survival. SCLCs are, in general, not amenable to surgical intervention, whereas surgery is the primary mode of therapy for Stage I and I1 NSCLC.’ Neuroendocrine lung tumors comprise a spectrum of epithelial neoplasms ranging from low grade carcinoid tumors to SCLC. Most SCLCs show neuroendocrine differentiation when studied by immunohistochemistry4 or electron micro~copy.~ A significant minority, approximately 10-30%, of NSCLCs also show neuroendocrine differentiation when similarly e~amined.~,’ Recent studies have shown varied and sometimes conflicting results regarding the therapeutic and prognostic significance of neuroendocrine differentiation in NSCLC.8-’6 The purpose of this study was to determine the relationship between neuroendocrine differentiation in NSCLC and response to chemotherapy, disease progression, and survival in patients with inoperable NSCLC who were entered into two cisplatinletoposide-based chemotherapy protocols. MATERIALS AND METHODS Patients All study patients were enrolled in North Central Cancer Treatment Group (NCCTG)protocol 89-24-5117and Mayo Clinic protocol 88-24-01. Protocol eligibility required histologically or cytologically proven, measurable, or evaluable Stage 111or IV NSCLC’ not amenable to surgical or radiation therapy. Patients who had received previous chemotherapy or significant radiation therapy were excluded. The NCCTG protocol randomized patients to receive cisplatinletoposide with or without hydrazine sulfate. The Mayo Clinic protocol utilized either continuous infusion or bolus cisplatinletoposide in its study design. Patients from both arms of the study had similar outcomes and were included in the present analysis. Two hundred seventy-seven patients (184 men and 93 women) ranging in age from 33 to 80 years were enrolled in the 2 protocols from 1988 to 1992. Of these, sufficient histologic or cytologic (cell block) material was available for pathologic review in 107 cases. Patient characteristics including age, sex, weight, performance status, disease stage, and the presence and location of metastases from the group with sufficient material for pathologic review were similar to patient characteristics of the larger group. Patient characteristics were also similar between the two protocols. Pathologic material was obtained via transbronchial biopsy, open lung biopsy or resection, or fine-needle aspiration or biopsy of a metastasis. Tissues were fixed in 10% buffered formalin and 5-p hematoxylin and eosinstained sections were reviewed to assess histologic subtype. Tissue blocks available ranged from 1 to 12 blocks per case (mean, 1.4 blocks). When more than one block was available, the two or three blocks containing the best preserved and most abundant tumor were selected for study. lmmunohistochemistry Paraffin sections were stained for a panel of neuroendocrine and epithelial markers including chromogranin A (CGA) (1:lOOO) (Boehringer-Mannheim, Indianapolis, IN), Leu 7 (1:5) (Becton-Dickinson,San Jose, CAI, synaptophysin (SY) (1:40),(ICN,Costa Mesa, CAI, and cytokeratin AEl/AE3 (1:lOO) (Boehringer-Mannheim, Indianapolis, IN) using the avidin-biotin peroxidase method.18Positive controls, including normal pancreas for CGA and SY, normal tonsil for Leu 7, and small intestine for cytokeratin and negative controls (consisting of patient tissues omitting the primary antibody) were used for all cases. Staining reactions were semiquantitatively assessed to include strength of reactivity (0: none; 1+: weak 2+: moderate; 3+: strong; and 4+: very strong) and percentage of tumor cells reacting (0:none; 1+: 1-25%; 2+: 26-50%; 3+: 5175%; and 4+: >75%). Reviewing pathologists were blinded to clinical outcomes in all cases. Clinical Outcomes Clinical outcomes measured included: (1) response to chemotherapy (50% reduction in measurable disease or 1286 CANCER April 1,1996 / Volume 77 / Number 7 FIGURE 1. lmmunohistochernical staining. Low grade squamous cell carcinoma with (a) 2(+) staining for synaptophysin and (b) 3(+) staining for Leu 7. (c) High grade squamous carcinoma with focal 4(+) staining for chromogranin A. (d) Large cell undifferentiated carcinoma with 4(+) staining for Leu 7. definite decrease in tumor size in evaluable disease); (2) time to disease progression (time from enrollment into treatment protocol to time when 2 25% increase in tumor size was detected); and (3) patient survival (time from enrollment to time of death). The presence of neuroendocrine differentiation was compared with clinical outcomes using Fisher's exact test and log rank test statistical methods. Multivariate analyses were also performed to fit a Cox proportional hazards model. A backward regression was used to find the most significant variables. The maximum likelihood estimate was obtained by adjusting the rest of the nonsignificant covariables. All P values were two-sided. RESULTS Sixty-six patients were men and 41 were women with a median age of 63 years (range, 33-80 years). Two patients had Stage IIIA disease, 14 had Stage IIIB disease, and 91 had Stage IV disease. Median survival for all patients was 257 days; median time to disease progression was 115 days. A response to chemotherapy was seen in 31.8% of patients. Sixty-two tumors (58%) were adenocarcinomas, 22 (21%) were squamous cell carcinomas, 18 (17%) were large cell undifferentiated carcinomas or NSCLC not otherwise specified, and 5 (5%) were adenosquamous carcinomas (Table 1).There were no cases that were classified as large cell neuroendocrine c a r ~ i n o m a 'present ~ in the study group. Examples of immunohistochemical staining results are shown in Figure 1. Because there proved to be no correlation between staining intensity or percent of positive tumor cells and the outcome variables, any degree of positivity was considered significant. Immunoreactiv- Neuroendocrine Expression in Lung Cancer/Schleusener et al. a 1287 b 800700 - 8 B')- 6 500- 0 400, P l3 .? 300- 5 200- v) B P-0.m I P-0.09 257 dmys Jlld 100 - d C 100 7 100 1 e B m P P E .- on e W e n P ._ D .u 0 E F FIGURE 2. Clinical outcomes versus neuroendocrine differentiation. (a) Survival versus neuroendocrine differentiation. (b) Survival versus individual neuoendocrine marker. (c) Response to chemotherapy versus neuroendocrine differentiation. (d) Response to chemotherapy versus individual neuroendocrine marker. ( f ) Time to disease progression versus individual neuroendocrine marker. (b and 1: open-ended bars indicate some patients are still alive at close of the study. CGA: chromogranin A; SY: synaptophysin). ity for at least 1 neuroendocrine marker was present in 37 (34.6%) cases; 12 cases (11.3%) were immunoreactive for 2 or more neuroendocrine markers. Table 1 shows the percentage of cases with neuroendocrine differentiation by histologic subtype. Table 2 shows the number of cases staining with each marker and each combination of markers. The presence of neuroendocrine differentiation sig- nificantly correlated with survival: 249d: all neuroendocrine markers negative versus 3 1 Id: 1 or more neuroendocrine markers positive, G = 0.01; and 2493: all neuroendocrine markers negative versus 464d: 2 or more neuroendocrine markers positive, P = 0.005 (Figs. 2A and 3). lmmunoreactivity for specific neuroendocrine markers, whether considered alone or together, was also significantly correlated with survival: 255d: CGA negative 1288 CANCER April 1,1996 / Volume 77 / Number 7 too- 80 - Bo- Survival (W 4020- Years from randomization UIcmmW1 FIGURE 3. Kaplan-Meier curve for survival versus neuroendocrine differentiation in nonsmall cell lung cancer. versus 569d: CGA positive, P = 0.04; 249d: Leu 7 negative versus 422d: Leu 7 positive, P = 0.006; 251d: SY negative versus 318d: SY positive, P = 0.02 (Fig. 2B). Time to disease progression was significantlyprolonged only in cases expressing both CGA and SY (112d: CGA and SY negative versus 386d: CGA and SY positive, P = 0.04). Otherwise there was no correlation between the presence of neuroendocrine differentiation and either response to chemotherapy or time to disease progression (Figs. 2C-F), disease stage, or tumor grade. In addition, in those cases with any neuroendocrine staining, combining the staining intensity score with the percentage of tumor cells reacting score showed no correlation with survival, response to chemotherapy, or time to disease progression. By multivariate analysis, immunoreactivity for any given neuroendocrine marker was an independent predictor of survival when considered together with age, sex, tumor grade, and stage. Performance status was also an independent predictor of survival (Table 3). DISCUSSION Immunohistochemical evidence of neuroendocrine differentiation was present in more than one-third of our cases of “ordinary” NSCLC and was associated with improved patient survival. Improved survival was seen when any single marker (CGA, Leu 7, or SY) or combination of markers was present. In addition, the majority of long term survivors had tumors with neuroendocrine differentiation. Interestingly, the effect of neuroendocrine differentiation on survival was not attributable to either a greater likelihood of response to chemotherapy, prolonged disease free interval, disease stage, or tumor grade. Carles et al.’ showed similar findings with respect to survival rates and chemotherapy response rates and suggested that response to chemotherapy may not be as important in predicting survival as was previously thought. They also suggested that stable, nonprogressive disease may be as important as chemotherapeutic response in predicting outcome. Other recent studies comparing clinical outcomes with neuroendocrine differentiation in chemotherapytreated NSCLC have shown conflicting results (Table 4). Carles et al.’ showed that the presence of neuron specific enolase (NSE) immunoreactivity correlated with an improved median survival of 3.5 months, but failed to show that neuroendocrine differentiation correlated with response to chemotherapy. Graziano et al.’ showed that, among patients who responded to chemotherapy, a 52week improvement in median survival was present in patients with tumors that had 2 or more neuroendocrine markers. This study also showed an increased response rate to chemotherapy in patients with tumors with NSE immunoreactivity (54%responders vs. 27% nonresponders, P = 0.04) and 2 or more positive neuroendocrine markers (38% responders vs. 0% nonresponders, P < 0.01). Ruckdeschel et al.15and Skov et a1.l’ both showed a significant trend toward response to chemotherapy in tumors with NSE expression, but no correlation with survival. Although there is some disagreement in the literature, most studies analyzing patients treated with surgery TABLE 3 Results of Multivariate Analysis Adjusted for Age, Sex, Stage, Grade, and Performance Status Varjable NE differentiation (21NE markers vs. 0 NE markers) Age, (yr) (2 60 vs. < 60) Sex (male vs. female) Grade (3, 4 vs. I, 2) Stage (lVvs. 111) Performance status (2 vs. 0, 1) N E neuroendocrine P values [chi square) 0.03 0.77 0.94 0.63 0.33 0.0003 95% confidence Risk ratio interval 0.61 1.07 1.02 0.84 1.32 2.64 0.39-0.95 0.70-1.63 0.65-1.59 0.40-1.74 0.75-2.33 1.62-5 Neuroendocrine Expression in Lung CancerEchleusener et al. 1289 TABLE 4 Summary of Previous Studies Examining Significance of Neuroendocrine Differentiation in Nonsmall Cell Lung Carcinoma Effect of NE differentiation on clinical outcomes Study Treatment Stage (No. of cases) NE markers used %I positive Skov et al? Chemotherapy (3 different combinations) IIIA. 25% IIIH, 27% IV. 48% NSE-lfi%: (>lo%?) 52%: (I-lO%-) CGA- 19%: (>I O % t ) 45%: (1-10%+) So correlation with survival Increased response to chemotherapy Improved survival No correlation uith response to chemotherapy I14 cases nf adenocarcinoma Carles et a,' 97 cases 01 SSCLC. chemotherapy (8 different combinations, cisplatin-based) Craziano et 52 cases 01 NSCLC Chemotherapy (4 cisplatin-based combinations) 1.ocoregional disease (39) Ilistant mets (43) Both (30) Sundaresan et a1.I359 cases of KSC1.C Surgical resection I(177) I1 (75) 111 (63) Graziano ct al." 260 cases of NSC1.C Surgical resection I (193) I1 (67) Linnoila e: al.' 23i cases of SSC1.C Surgical resection Sot stated Eibbelaar et al." 226 cases oi NSCK Berendsen et al. ' 111 cases of SSCLC Surgical resection Sot stated NSE-46% SY-23% CGA-2% IRU 7.2% 2 or more-12% NsE-409~ leu 7.31% CGA-6% 2 or more-19% NSE-57% CK BH-II% Bombesin-7% Neuro!ensin-10% CGA-7% SY-23% XCAM20% 2 or more-30% h'SE-7OI Leu 7-7.796 CGA-14.2% SY-I 1.2% 2 or more-23.5% SSE-46% CGA-2% L ~ 7.8% U Gastrin-releasing peptide-1% 2 or more-12% MAh 123C3 (NCM)-19.1% Resection-98 So resection-43 I II (70) I11 (71) MOC 1, 21, 32, 51, 91 % (> 50% cells) + - Improved survival Increased response to chemotherapy No correlation with survival Increased likelihood of higher disease stage and nodal metastases No correlation k\ith survival No correlation with survival So correlation with disease recurrence Decreased survival Ilccreased sunival YE: neurorridocrine; MI: neuron specific enolase GCA:chromogranin A; SY: synapmphysin; NSC1.C: nonsniall lung carcinoma; CK BE: creatinine lrinase B B NCAM neural ceii adhesion mokuie. only have indicated that neuroendocrine differentiation has no impact on survival (Table 4). Berendsen et al.'" used multivariate analysis to show that neuroendocrine differentiation in NSCLC had a negative impact on survival. Similarly, Sundaresan et al." showed that expression of neuroendocrine markers predicted for higher disease stage. IIowever. Linnoila et al.," Graziano et aI.,14 and Ruckdeschel et aI.,l5in larger series utilizing common antibodies for neuroendocrine differentiation, showed no correlation between neuroendocrine differentiation and survival in their surgically treated patients. We are aware of no series reporting a survival advantage for surgically treated NSCLC with neuroendocrine differentiation. Previous studies have identified neuroendocrine differentiation in approximately 10%-30% of NSCLCs." - 1 2 ~ 1 4 ~ 1 5 The frequency of neuroendocrine differentiation was simi- lar for all histologic tumor types in our study ('fable 1). Other seriesH~1".2" have shown that adenocarcinoma is more likely than squamous cell carcinoma to express neuroendocrine differentiation. The reasons for these discrepancies are unknown. Various terms have been applied to NSCI.Cs with neuroendocrine differentiation, including atypical endocrine tumors2' and large cell (undifferentiated) carcinoma with neuroendocrine differentiation." The term large cell neuroendocrine carcinoma is reserved for tumors that appear "neuroendocrine" by light microscopy (i.e., organoid, trabecular, andlor palisading growth pattern with coarse chromatin) and show immunohistochemical or ultrastructural evidence of neuroendocrine differentiation. Similar tumors have also been termed neuroendocrine carcinomas of intermediate differentiati~n.'~ 1290 CANCER April 1,1996 I Volume 77 I Number 7 Three commercially available markers of neuroendocrine differentiation were used in this study: CGA, Leu 7 , and SY. CGA is a high molecular weight acidic glycoprotein originally isolated from adrenal medulla. It is released along with neuroendocrine peptides through exocytosis from dense-core neurosecretory granules and its detection is directly correlated with the presence of these neurosecretory granules.'" Anti-Leu 7 (HNK- 1) antibody was initially established as a marker for natural killer cells but was shown to cross-react with various neuroendocrine tumors. It specifically recognizes myelin-associated glycoproteins and also cross-reacts with peripheral nerve^.^^^^^ SY is a transmembrane 38 kilodalton glycoprotein that has been isolated from presynaptic vesicles of neurons. In addition, SY is expressed in normal pancreatic islet cells and in a high percentage of neuroendocrine neoplasms.2fi We defined "neuroendocrine differentiation" as reactivity for any 1 marker because of the relatively specific nature of neuroendocrine markers used in this study. Other authors have defined "neuroendocrine differentiation" as having two or more positive neuroendocrine markers, although all these studies included NSE, which we avoided using because of its lack of specificity for neuroendocrine NSE has been positive in between 40-68% of cases of NSCLC from other ~ t ~ d i e ~ . "CGA ~"~ immunore'~~'~ activity was present in less than 5% of our cases; Leu 7 and SY expression was present in approximately 20% and 25% of our cases, respectively, similar to previous ~ t ~ d i Loy et al.3' found much higher rates of SY immunoreactivity in NSCLCs, but used a polyclonal SY antibody that may be less specific than the monoclonal antibody used in this study. 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