Cytotoxic Chemotherapy Induces Cell Differentiation in
Small-Cell Lung Carcinoma By E. Brambilla, D. Moro, S. Gazzeri, P.Y. Brichon, H. Nagy-Mignotte, F. Morel, M. Jacrot, and C. Brambilla Despite the high response rates resulting from chemotherapy, the majority of small-cell lung cancer (SCLC) patients relapse with chemoresistant tumors. To analyze the phenotypic changes that are precursors of chemoresistant status, and to investigate the role of chemotherapy in these changes, tumor samples from 20 patients, taken before chemotherapy (etoposide, doxorubicin, and cyclophosphamide) and again at the onset of chemoresistance (after at least three courses of chemotherapy), were compared. The histologic changes were minor in 10 of 20 patients, as shown by an increase in cell size; they were major in 10 of 20 patients, with the appearance of mixed composite tumors in which neuroendocrine (NE), epidermoid, and glandular components were mixed. Major changes correlated with a good response to chemotherapy (P = .001). Ultrastructural studies showed an increase in neurosecretory granules and desmosomes, and a high frequency of multidirectional differentiation
(45%) when comparison was made with pretherapy samples (10%) (P < .01). Immunohistochemical (IH) analysis showed an increase in cytokeratin (CK) expression in treated patients, with a different labeling pattern and the expression of higher molecular weight CK. The expression of NE lineage markers (Leu 19, Sy 38, SL 11-14) remained stable, while that of NE differentiation markers (Leu 7, chromogranin) increased in the treated patients. The neuron-specific enolase (NSE) activity remained stable in treated SCLC. Large cells with a more differentiated phenotype and proliferative capacity (as shown by Ki 67 labeling), appeared to be characteristic of treated and secondary chemoresistant SCLC. The acquisition of a more complex phenotype, which correlates with primary response to therapy, implies a drug-induced differentiation in SCLC. J Clin Oncol 9:50-61. c 1991 by American Society of Clinical Oncology.
ESPITE increasing knowledge of the biologic
these treated patients, in contrast with the classic type of SCLC diagnosed from their pretreatment samples. 57 The aim of this study was to analyze the phenotypic changes that occur with the development of chemoresistance. As previous studies have dealt mainly with morphologic observations, we have analyzed phenotypic changes involved in antigenic expression to investigate the role of chemotherapy in the acquisition of these changes either through clonal selection, or induction of differentiation. Comparative studies of tumor samples from the same patients pre- and posttherapy, and of reference groups of untreated patients with large tumor samples, were carried out to assess the quality and frequency of the phenotypic heterogeneity observed after therapy.
properties of small-cell lung carcinomas (SCLC), this type of tumor remains a puzzling clinical and biologic problem. Clinical behavior and therapeutic response are difficult to predict because of tumor heterogeneity. 1 4 Although objective response to chemotherapy is obtained in 80% of patients, less than 10% survive more than 2 years. Despite this high response rate, patients die from progressive disease from relapse, or from the development of a "second lung cancer," with highly chemoresistant tumors. Data from a histologic review of autopsy material have shown the high frequency of mixed or composite tumors in
From the Department of Pathology and Electron Microscopy, Department of Respiratory Medicine, Department of Thoracic Surgery, and Department of Enzymology, Lung CancerResearch Group, Grenoble, France. Submitted November 29, 1989; acceptedJuly 12, 1990. Supported in part by grants from Institut National de la Sante et de la recherche Medicale-Paris,Association pour la Recherche sur le Cancer-Villejuif Paris, and Etablissement Public Regional Charbonieres,Lyons, France. Address reprint requests to C. Brambilla, MD, Service de Pneumologie CHRU BP 217 X. 38043 Grenoble Cedex, France. © 1991 by American Society of Clinical Oncology. 0732-183X/91/0901-0019$3.00/0
50
MATERIALS AND METHODS
Patients The study group consisted of 20 patients who had biopsies performed before and after the same chemotherapy treatment between 1984 and 1989. The diagnosis of SCLC was based on histologic examination of a bronchial biopsy and/or lymph node metastasis. All patients were given chemotherapy with the same drugs (cyclophosphamide, doxorubicin, and etoposide) followed by chest radiotherapy between the sixth and seventh cycles of chemotherapy. The posttherapy sample was obtained after at least three courses of chemotherapy, either on residual tumor (surgical lung
Journal of Clinical Oncology, Vol 9, No 1 (January), 1991: pp 50-61
Downloaded from ascopubs.org by UNIVERSITY of OTAGO on April 20, 2019 from 139.080.135.089 Copyright © 2019 American Society of Clinical Oncology. All rights reserved.
51
CELL DIFFERENTIATION IN SCLC resection) or to assess the tumor progression or relapse on a site (metastatic lymph node) before changing chemotherapy. The extent of disease was assessed by fiberoptic bronchoscopy, chest x-ray and computed tomographic (CT) scan, liver ultrasound, bone marrow aspiration, bone scintigraphy, and brain CT scan. Patients were classified according to the International Union Against Cancer (UICC) classification" as defined in Table 1. Response to treatment, after three courses of chemotherapy, was assessed by the same techniques as complete response (CR), partial response (PR), or nonresponsive (NR). Patients were considered as having major response (CR and PR with >50% regression of initial tumor volume) or minor response (NR and PR with < 50% regression of initial tumor volume). The reference groups included 110 pretherapy samples: 60 that had undergone electron microscopy (EM) analysis on 28 bronchial biopsies, 22 lymph node metastases, and 10 lung tumors; 30 (25 lymph node biopsies and five lung tumors) that had been studied immunohistochemically (IH), using a broad antibody panel on frozen sections, and 20 lung tumor resections that had been obtained before therapy with histologic analysis. Diagnosis and extension of the SCLC were assessed with the same procedures as in the study group. Histology Diagnosis of SCLC prior to treatment was established 9 according to the World Health Organization (WHO) classi-
fication criteria for the classic oat cell (0) and intermediate (I) cell types. The diagnoses of small- and large-cell types (S/L) were established according to the definition of Hirsch et al.2 They were subclassified, in the present study, into S/Ll, S/L2, S/L3, depending on an increasing percentage of the large-cell component: less than 20% in S/L1, between 20% and 40% in S/L2, from 40% to 60% in S/L3. Welldifferentiated neuroendocrine (WDNE) carcinomas'" were identical to atypical carcinoids. Glandular (G) or epidermoid (E) differentiation was identified using morphologic and histochemical criteria: acinar formation and/or mucin production (Alcian Blue and diastase-digested periodic acid-Schiff) for G, and intercellular bridges and/or keratinization for E differentiation. Tumor-cell size was measured by optical microscopy when electron microscopy could not be performed, using as an objective for sizing at 400 magnification. Electron Microscopy Samples of bronchial biopsies and surgical samples (lung and metastatic lymph nodes) were fixed in glutaraldehyde, postfixed in osmium tetroxide, and embedded in epoxyresin by classic techniques. Electron microscopic examination of ultrathin sections was performed on a Philipps 301 electron microscope (Bobigny, France). For cell sizing, the mean orthogonal diameter was measured on at least 50 cell sections. Neurosecretory granules (NSG) density was scored from one to four, according to the mean number of NSG per
Table 1. Clinical Data of the 20 Patients With SCLC Included in the Study Patient No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Pretherapy Sample Histology Origin N N N N B B N B B B B B B B B N B B B N
SL1 S L1 O I 0 I I 0 0 0 0 0 0 O I 0 0 I I I
Posttherapy Sample Histology Origin N N N N L L Adr L N N N N N L L L L L N N
SL3 S L2 S Ll SL1 IA S Li WD IA OE I I OWD I WDE IA OWDA OWD IA S L1 SL2
Disease Stage*
No. of Chemotherapy Cyclest
Type of Responset
Survival (months)
4 4 4 4 3 3 3 3 4 3 4 2 4 2 3 4 3 3 4 4
3 4 4 9 3 4 8 3 7 12 3 3 5 3 3 9 3 3 3 3
NR PR < NR CR PR > PR < PR > PR > PR > PR> PR < PR > PR < PR > PR > PR > CR PR > PR < NR
3 7.5 6.5 20 17 10 18 8.5 8 12 8 13 11.5 26 AI 15 31 3 12 2.5 4
Abbreviations: N, metastatic lymph node; NR, no response; B, bronchial biopsy; CR, complete response; L, lung tumor resection; PR >, partial response > 50%; Histology, histological classification; PR
Small-Cell Lung Carcinoma By E. Brambilla, D. Moro, S. Gazzeri, P.Y. Brichon, H. Nagy-Mignotte, F. Morel, M. Jacrot, and C. Brambilla Despite the high response rates resulting from chemotherapy, the majority of small-cell lung cancer (SCLC) patients relapse with chemoresistant tumors. To analyze the phenotypic changes that are precursors of chemoresistant status, and to investigate the role of chemotherapy in these changes, tumor samples from 20 patients, taken before chemotherapy (etoposide, doxorubicin, and cyclophosphamide) and again at the onset of chemoresistance (after at least three courses of chemotherapy), were compared. The histologic changes were minor in 10 of 20 patients, as shown by an increase in cell size; they were major in 10 of 20 patients, with the appearance of mixed composite tumors in which neuroendocrine (NE), epidermoid, and glandular components were mixed. Major changes correlated with a good response to chemotherapy (P = .001). Ultrastructural studies showed an increase in neurosecretory granules and desmosomes, and a high frequency of multidirectional differentiation
(45%) when comparison was made with pretherapy samples (10%) (P < .01). Immunohistochemical (IH) analysis showed an increase in cytokeratin (CK) expression in treated patients, with a different labeling pattern and the expression of higher molecular weight CK. The expression of NE lineage markers (Leu 19, Sy 38, SL 11-14) remained stable, while that of NE differentiation markers (Leu 7, chromogranin) increased in the treated patients. The neuron-specific enolase (NSE) activity remained stable in treated SCLC. Large cells with a more differentiated phenotype and proliferative capacity (as shown by Ki 67 labeling), appeared to be characteristic of treated and secondary chemoresistant SCLC. The acquisition of a more complex phenotype, which correlates with primary response to therapy, implies a drug-induced differentiation in SCLC. J Clin Oncol 9:50-61. c 1991 by American Society of Clinical Oncology.
ESPITE increasing knowledge of the biologic
these treated patients, in contrast with the classic type of SCLC diagnosed from their pretreatment samples. 57 The aim of this study was to analyze the phenotypic changes that occur with the development of chemoresistance. As previous studies have dealt mainly with morphologic observations, we have analyzed phenotypic changes involved in antigenic expression to investigate the role of chemotherapy in the acquisition of these changes either through clonal selection, or induction of differentiation. Comparative studies of tumor samples from the same patients pre- and posttherapy, and of reference groups of untreated patients with large tumor samples, were carried out to assess the quality and frequency of the phenotypic heterogeneity observed after therapy.
properties of small-cell lung carcinomas (SCLC), this type of tumor remains a puzzling clinical and biologic problem. Clinical behavior and therapeutic response are difficult to predict because of tumor heterogeneity. 1 4 Although objective response to chemotherapy is obtained in 80% of patients, less than 10% survive more than 2 years. Despite this high response rate, patients die from progressive disease from relapse, or from the development of a "second lung cancer," with highly chemoresistant tumors. Data from a histologic review of autopsy material have shown the high frequency of mixed or composite tumors in
From the Department of Pathology and Electron Microscopy, Department of Respiratory Medicine, Department of Thoracic Surgery, and Department of Enzymology, Lung CancerResearch Group, Grenoble, France. Submitted November 29, 1989; acceptedJuly 12, 1990. Supported in part by grants from Institut National de la Sante et de la recherche Medicale-Paris,Association pour la Recherche sur le Cancer-Villejuif Paris, and Etablissement Public Regional Charbonieres,Lyons, France. Address reprint requests to C. Brambilla, MD, Service de Pneumologie CHRU BP 217 X. 38043 Grenoble Cedex, France. © 1991 by American Society of Clinical Oncology. 0732-183X/91/0901-0019$3.00/0
50
MATERIALS AND METHODS
Patients The study group consisted of 20 patients who had biopsies performed before and after the same chemotherapy treatment between 1984 and 1989. The diagnosis of SCLC was based on histologic examination of a bronchial biopsy and/or lymph node metastasis. All patients were given chemotherapy with the same drugs (cyclophosphamide, doxorubicin, and etoposide) followed by chest radiotherapy between the sixth and seventh cycles of chemotherapy. The posttherapy sample was obtained after at least three courses of chemotherapy, either on residual tumor (surgical lung
Journal of Clinical Oncology, Vol 9, No 1 (January), 1991: pp 50-61
Downloaded from ascopubs.org by UNIVERSITY of OTAGO on April 20, 2019 from 139.080.135.089 Copyright © 2019 American Society of Clinical Oncology. All rights reserved.
51
CELL DIFFERENTIATION IN SCLC resection) or to assess the tumor progression or relapse on a site (metastatic lymph node) before changing chemotherapy. The extent of disease was assessed by fiberoptic bronchoscopy, chest x-ray and computed tomographic (CT) scan, liver ultrasound, bone marrow aspiration, bone scintigraphy, and brain CT scan. Patients were classified according to the International Union Against Cancer (UICC) classification" as defined in Table 1. Response to treatment, after three courses of chemotherapy, was assessed by the same techniques as complete response (CR), partial response (PR), or nonresponsive (NR). Patients were considered as having major response (CR and PR with >50% regression of initial tumor volume) or minor response (NR and PR with < 50% regression of initial tumor volume). The reference groups included 110 pretherapy samples: 60 that had undergone electron microscopy (EM) analysis on 28 bronchial biopsies, 22 lymph node metastases, and 10 lung tumors; 30 (25 lymph node biopsies and five lung tumors) that had been studied immunohistochemically (IH), using a broad antibody panel on frozen sections, and 20 lung tumor resections that had been obtained before therapy with histologic analysis. Diagnosis and extension of the SCLC were assessed with the same procedures as in the study group. Histology Diagnosis of SCLC prior to treatment was established 9 according to the World Health Organization (WHO) classi-
fication criteria for the classic oat cell (0) and intermediate (I) cell types. The diagnoses of small- and large-cell types (S/L) were established according to the definition of Hirsch et al.2 They were subclassified, in the present study, into S/Ll, S/L2, S/L3, depending on an increasing percentage of the large-cell component: less than 20% in S/L1, between 20% and 40% in S/L2, from 40% to 60% in S/L3. Welldifferentiated neuroendocrine (WDNE) carcinomas'" were identical to atypical carcinoids. Glandular (G) or epidermoid (E) differentiation was identified using morphologic and histochemical criteria: acinar formation and/or mucin production (Alcian Blue and diastase-digested periodic acid-Schiff) for G, and intercellular bridges and/or keratinization for E differentiation. Tumor-cell size was measured by optical microscopy when electron microscopy could not be performed, using as an objective for sizing at 400 magnification. Electron Microscopy Samples of bronchial biopsies and surgical samples (lung and metastatic lymph nodes) were fixed in glutaraldehyde, postfixed in osmium tetroxide, and embedded in epoxyresin by classic techniques. Electron microscopic examination of ultrathin sections was performed on a Philipps 301 electron microscope (Bobigny, France). For cell sizing, the mean orthogonal diameter was measured on at least 50 cell sections. Neurosecretory granules (NSG) density was scored from one to four, according to the mean number of NSG per
Table 1. Clinical Data of the 20 Patients With SCLC Included in the Study Patient No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Pretherapy Sample Histology Origin N N N N B B N B B B B B B B B N B B B N
SL1 S L1 O I 0 I I 0 0 0 0 0 0 O I 0 0 I I I
Posttherapy Sample Histology Origin N N N N L L Adr L N N N N N L L L L L N N
SL3 S L2 S Ll SL1 IA S Li WD IA OE I I OWD I WDE IA OWDA OWD IA S L1 SL2
Disease Stage*
No. of Chemotherapy Cyclest
Type of Responset
Survival (months)
4 4 4 4 3 3 3 3 4 3 4 2 4 2 3 4 3 3 4 4
3 4 4 9 3 4 8 3 7 12 3 3 5 3 3 9 3 3 3 3
NR PR < NR CR PR > PR < PR > PR > PR > PR> PR < PR > PR < PR > PR > PR > CR PR > PR < NR
3 7.5 6.5 20 17 10 18 8.5 8 12 8 13 11.5 26 AI 15 31 3 12 2.5 4
Abbreviations: N, metastatic lymph node; NR, no response; B, bronchial biopsy; CR, complete response; L, lung tumor resection; PR >, partial response > 50%; Histology, histological classification; PR
