REPORTS prognostic value in patients with locally advanced breast cancer. [J Natl Cancer Inst 83:111-116,1991]
Pierre Verrelle* Frederic Meissonnier, Yvette Fonck, Viviane Feillel, Claude Dionet, Fabrice Kwiatkowski, Robert Plagne, Jacques Chassagne
In 20 women with breast carcinoma, 17 of whom had locally advanced cancer and 3 of whom had confirmed metastases, the expression of P-glycoprotein was evaluated before the start of a chemotherapy regimen that included multidrug resistance-related drugs. With the use of the C494 monoclonal antibody in an avidin-biotin-immunoperoxidase technique, P-glycoprotein was detected in 17 of 20 tumor samples. Results were expressed in a semiquantitative manner, taking into account the number of positive tumor cells (N index) and the specific staining intensity (I index). The 17 patients with nonmetastatic cancer were followed from the first cycle of chemotherapy to cancer recurrence; subsequent to six cycles of chemotherapy, all of these patients except one were rendered clinically disease-free through surgery and/or radiation. The end point was defined as either local/regional recurrence or metastasis. Strong P-glycoprotein-positive staining in a majority of tumor cells (the N + / I + phenotype) was significantly correlated with no initial response to chemotherapy (P < .02) and with a shorter progression-free survival (P < .02). Thus, the pretreatment evaluation of P-glycoprotein expression may be of Vol. 83, No. 2, January 16, 1991
Intrinsic or acquired resistance to various chemotherapeutic agents has been a major problem in cancer treatment. One of the phenomena associated with chemoresistance of tumor cells is the expression of the multidrug resistant (MDR) phenotype (7). The relationship between the MDR phenotype and a 170kd membrane glycoprotein (usually referred to as P-170 or P-glycoprotein) has been well established (2-4), and measurement of the level of P-glycoprotein expression could be a valuable tool for guiding chemotherapy. In breast cancer cell lines or tissue samples, P-glycoprotein expression has been investigated specifically with electrophoretic methods such as the detection of gene amplification by Southern blot analysis or of gene overexpression by Northern and Western blotting (5). In contrast to results reported for breast cancer cell lines (6), there has not been any evidence of P-glycoprotein overexpression in primary breast tumor samples (5). However, these biochemical techniques are not effective in detecting a small number of P-glycoprotein-positive tumor cells within tissue samples containing a large population of P-glycoprotein-negative cells (5). Only a limited number of studies using in situ sensitive assays have reported examination of P-glycoprotein expression at the single-cell level in clinical breast tumor specimens (7-9); in these studies, a minority of positive cells were detected by an immunohistochemical method using a P-glycoprotein-specific monoclonal antibody (MAb). We therefore undertook an immunohistochemical study to assess the presence of P-glycoprotein-positive tumor cells within breast carcinoma samples. Using a murine MAb (C494 MAb) specific for P-glycoprotein, we tested biopsy specimens obtained from
Patients and Methods Patients Twenty women with breast cancer (mean age, 58 years; range, 28 to 75 years), 17 of whom had locally advanced disease and three of whom had metastatic disease, were entered in this study from October 1987 to May 1989. The clinical characteristics of the patients are summarized in Table 1. Chemotherapy and Initial Evaluation of Patient Responses Sixteen patients with nonmetastatic breast cancer were treated with six cycles of AVCF chemotherapy (doxorubicin [Adriamycin], vincristine, cyclophosphamide, and fluorouracil); methotrexate was added after the third cycle if no satisfactory local response was achieved. One patient with nonmetastatic cancer (No. 15) was treated with only three cycles of the AVCF regimen followed by a local treatment (surgical
Downloaded from http://jnci.oxfordjournals.org/ at University of California, San Francisco on November 20, 2014
Clinical Relevance of Immunohistochemical Detection of Multidrug Resistance P-Glycoprotein in Breast Carcinoma
20 untreated patients. To assess the reactivity of the C494 MAb with breast tumor cells, we obtained these specimens just before the first course of chemotherapy (regimen included two MDR-related drugs, doxorubicin and vincristine). Four patients were re-evaluated after chemotherapy (two after three courses and two after six courses).
Received June 15, 1990; revised October 12, 1990; accepted October 18, 1990. Supported by the Federation Nationak des Centres de Lutte Contre le Cancer. P. Verrdle, F. Meissonnier, C Dionet (Service de Radiotherapie), Y. Fbnck (Laboratoire d'Anatomie PathotogiqueX V. Feillel (Service des Methodes Physiques), F. Kwiatkowski (Service de BiostatistiquesX R. Plagne, J. Chassagne (Laboratoire d'Immunologie et de Cancerologie), Centre Jean Perrin, ClermontFerrand Cede*, France. We thank Mrs G. Bargoin for excellent technical assistance. 'Correspondence to: Pierre Verrelle, MD, Service de Radiotherapie, Centre Jean Perrin, Place Henri Dunant, BP 392,63011 Clermont-Ferrand Cedex, France.
REPORTS 111
Table 1. Clinical characteristics of patients with breast cancer Tumor staging* TNM Stage
Patient Age, y
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
74 61 59 53 61 59 63 51 54 65 47 50 59 65 64 58 75 59 61 28
T3NlbMl T4NlbMl T4N3M1 T4NlbM0 T4NlbM0 T2N3M0 T2NlbM0 T2N0M0 T4NlbM0 T3NlbM0 T2NlbM0 T4N3M0 T2NlbM0 T4N2M0 T4NlbM0 T4NlbM0 T4N2M0 T2NlbM0 T4NlbM0 T4NlbM0
Histologic parameters! Type Grade
IV IV IV Illb Illb Illb II II Illb Ilia II Illb 11 Illb Illb Illb Illb II Illb Illb
IDC IDC IDC IDC IDC ILC IDC IDC IDC IDC IDC IDC IDC IDC IDC IDC IDC ILC ILC IDC
II II II II II _ II II/III II/III II II II II III II III II — _ II
•International Union Against Cancer classification before 1988 (10). fTumors were classified according to the World Health Organization histological typing of breast tumors (//). IDC = invasive ductal carcinoma. ILC = invasive lobular carcinoma. — = not graded.
resection and radiation therapy). In the three patients with metastatic disease (Nos. 1, 2, and 3), chemotherapy was continued after six cycles. Immediately following three and six cycles of chemotherapy and before further treatment, patients were evaluated for tumor and node responses to chemo-
therapy through clinical examination, mammography, and breast ultrasonography (Table 2). Responders were defined as patients with a complete response (CR) or a partial response (PR: >50% regression from the maximum diameter of the initial tumor); nonresponders were patients with a minor
Extent of metastasis
Pretreatment P-170 evaluation N*
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Ml Ml Ml MO MO MO MO MO MO MO MO MO MO MO MO MO MO MO MO MO
It
Tumor and node responses after Three cycles}: Sixcydes| Tumor Node Tumor Node PR MR Prog CR CR PR PR PR PR MR MR MR MR MR NC NC NC NC Prog Prog
CR CR NC CR CR CR CR CR CR CR CR MR NC NC PR NC NC NC Prog Prog
CR MR Prog CR CR CR CR PR PR MR MR PR PR Prog -§ MR NC NC MR NC
CR CR Prog CR CR CR CR CR CR CR CR PR NC NC -§ NC NC NC MR Prog
•Number of positive cells: N— = no positive cells; N(+) = coexistence of negative and positive cells; and N+ = 75% or more positive cells. tSpecific staining intensity: I— = no staining; I(+) = weak staining; and 1+ = strong staining. JTumor and node responses were evaluated after three and six cycles of chemotherapy and before subsequent surgery and/or radiation therapy. CR = completeresponse;PR = partial response; MR = minor response; NC = no change; and Prog = local progression. §The tumor of patient 15 was resected after three cycles. 112
Following chemotherapy, all 17 patients who were determined to have nonmetastatic cancer at the beginning of the studyreceivedlocal treatment (ie, surgical resection and/or radiation therapy) that subsequently rendered 16 of them clinically disease-free. (Patient 19 was discovered to have lung metastases following chemotherapy and radiation.) Complete responders to chemotherapy received radiation only; all other patients with nonmetastatic cancer received surgery followed by radiation. Partial responders received either conservative or radical surgery (ie, either segmental mastectomy with axillary node dissection and examination of margins or modified radical mastectomy) followed by radiation therapy. The surgery and/or radiation therapy occurred after the evaluation of initial responses to chemotherapy (Table 2) and after the follow-up study starting point. Patients with metastatic disease received radiation therapy as well as prolonged chemotherapy. Method for Obtaining Immunocytochemical Specimens
Table 2. Initial P-gh/coprotein evaluation and local response to chemotherapy Patient No.
Treatment Following Chemotherapy
Tumor tissue samples were obtained from all 20 patients under local anesthesia by biopsy (Tru-Cut; Travenol Laboratories, Inc, Deerfield, 111) just before the first cycle of chemotherapy. Biopsy wasrepeatedin two patients (Nos. 3 and 15) after the third cycle and in two others (Nos. 13 and 16) after the sixth cycle of chemotherapy. Tumor specimens were immediately frozen in liquid nitrogen until they were processed. Immunoperoxidase Staining Procedure Details ofkhe immunohistochemical method used were described elsewhere (12). Frozen sections were labeled by a triple-layer immunoperoxidase technique (Vectastain ABC Kit, Vector Laboratories, Burlingame, Calif). The MAbs are listed below; the peroxidase substrate, 3-aminoethyl carbazole, gave a red coloring to the stained cells. Tissue samples were characterized by their reactivity with MAbs. First, a series of reagents, including antibodies raised Journal of the National Cancer Institute
Downloaded from http://jnci.oxfordjournals.org/ at University of California, San Francisco on November 20, 2014
No.
response (MR:
Pierre Verrelle* Frederic Meissonnier, Yvette Fonck, Viviane Feillel, Claude Dionet, Fabrice Kwiatkowski, Robert Plagne, Jacques Chassagne
In 20 women with breast carcinoma, 17 of whom had locally advanced cancer and 3 of whom had confirmed metastases, the expression of P-glycoprotein was evaluated before the start of a chemotherapy regimen that included multidrug resistance-related drugs. With the use of the C494 monoclonal antibody in an avidin-biotin-immunoperoxidase technique, P-glycoprotein was detected in 17 of 20 tumor samples. Results were expressed in a semiquantitative manner, taking into account the number of positive tumor cells (N index) and the specific staining intensity (I index). The 17 patients with nonmetastatic cancer were followed from the first cycle of chemotherapy to cancer recurrence; subsequent to six cycles of chemotherapy, all of these patients except one were rendered clinically disease-free through surgery and/or radiation. The end point was defined as either local/regional recurrence or metastasis. Strong P-glycoprotein-positive staining in a majority of tumor cells (the N + / I + phenotype) was significantly correlated with no initial response to chemotherapy (P < .02) and with a shorter progression-free survival (P < .02). Thus, the pretreatment evaluation of P-glycoprotein expression may be of Vol. 83, No. 2, January 16, 1991
Intrinsic or acquired resistance to various chemotherapeutic agents has been a major problem in cancer treatment. One of the phenomena associated with chemoresistance of tumor cells is the expression of the multidrug resistant (MDR) phenotype (7). The relationship between the MDR phenotype and a 170kd membrane glycoprotein (usually referred to as P-170 or P-glycoprotein) has been well established (2-4), and measurement of the level of P-glycoprotein expression could be a valuable tool for guiding chemotherapy. In breast cancer cell lines or tissue samples, P-glycoprotein expression has been investigated specifically with electrophoretic methods such as the detection of gene amplification by Southern blot analysis or of gene overexpression by Northern and Western blotting (5). In contrast to results reported for breast cancer cell lines (6), there has not been any evidence of P-glycoprotein overexpression in primary breast tumor samples (5). However, these biochemical techniques are not effective in detecting a small number of P-glycoprotein-positive tumor cells within tissue samples containing a large population of P-glycoprotein-negative cells (5). Only a limited number of studies using in situ sensitive assays have reported examination of P-glycoprotein expression at the single-cell level in clinical breast tumor specimens (7-9); in these studies, a minority of positive cells were detected by an immunohistochemical method using a P-glycoprotein-specific monoclonal antibody (MAb). We therefore undertook an immunohistochemical study to assess the presence of P-glycoprotein-positive tumor cells within breast carcinoma samples. Using a murine MAb (C494 MAb) specific for P-glycoprotein, we tested biopsy specimens obtained from
Patients and Methods Patients Twenty women with breast cancer (mean age, 58 years; range, 28 to 75 years), 17 of whom had locally advanced disease and three of whom had metastatic disease, were entered in this study from October 1987 to May 1989. The clinical characteristics of the patients are summarized in Table 1. Chemotherapy and Initial Evaluation of Patient Responses Sixteen patients with nonmetastatic breast cancer were treated with six cycles of AVCF chemotherapy (doxorubicin [Adriamycin], vincristine, cyclophosphamide, and fluorouracil); methotrexate was added after the third cycle if no satisfactory local response was achieved. One patient with nonmetastatic cancer (No. 15) was treated with only three cycles of the AVCF regimen followed by a local treatment (surgical
Downloaded from http://jnci.oxfordjournals.org/ at University of California, San Francisco on November 20, 2014
Clinical Relevance of Immunohistochemical Detection of Multidrug Resistance P-Glycoprotein in Breast Carcinoma
20 untreated patients. To assess the reactivity of the C494 MAb with breast tumor cells, we obtained these specimens just before the first course of chemotherapy (regimen included two MDR-related drugs, doxorubicin and vincristine). Four patients were re-evaluated after chemotherapy (two after three courses and two after six courses).
Received June 15, 1990; revised October 12, 1990; accepted October 18, 1990. Supported by the Federation Nationak des Centres de Lutte Contre le Cancer. P. Verrdle, F. Meissonnier, C Dionet (Service de Radiotherapie), Y. Fbnck (Laboratoire d'Anatomie PathotogiqueX V. Feillel (Service des Methodes Physiques), F. Kwiatkowski (Service de BiostatistiquesX R. Plagne, J. Chassagne (Laboratoire d'Immunologie et de Cancerologie), Centre Jean Perrin, ClermontFerrand Cede*, France. We thank Mrs G. Bargoin for excellent technical assistance. 'Correspondence to: Pierre Verrelle, MD, Service de Radiotherapie, Centre Jean Perrin, Place Henri Dunant, BP 392,63011 Clermont-Ferrand Cedex, France.
REPORTS 111
Table 1. Clinical characteristics of patients with breast cancer Tumor staging* TNM Stage
Patient Age, y
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
74 61 59 53 61 59 63 51 54 65 47 50 59 65 64 58 75 59 61 28
T3NlbMl T4NlbMl T4N3M1 T4NlbM0 T4NlbM0 T2N3M0 T2NlbM0 T2N0M0 T4NlbM0 T3NlbM0 T2NlbM0 T4N3M0 T2NlbM0 T4N2M0 T4NlbM0 T4NlbM0 T4N2M0 T2NlbM0 T4NlbM0 T4NlbM0
Histologic parameters! Type Grade
IV IV IV Illb Illb Illb II II Illb Ilia II Illb 11 Illb Illb Illb Illb II Illb Illb
IDC IDC IDC IDC IDC ILC IDC IDC IDC IDC IDC IDC IDC IDC IDC IDC IDC ILC ILC IDC
II II II II II _ II II/III II/III II II II II III II III II — _ II
•International Union Against Cancer classification before 1988 (10). fTumors were classified according to the World Health Organization histological typing of breast tumors (//). IDC = invasive ductal carcinoma. ILC = invasive lobular carcinoma. — = not graded.
resection and radiation therapy). In the three patients with metastatic disease (Nos. 1, 2, and 3), chemotherapy was continued after six cycles. Immediately following three and six cycles of chemotherapy and before further treatment, patients were evaluated for tumor and node responses to chemo-
therapy through clinical examination, mammography, and breast ultrasonography (Table 2). Responders were defined as patients with a complete response (CR) or a partial response (PR: >50% regression from the maximum diameter of the initial tumor); nonresponders were patients with a minor
Extent of metastasis
Pretreatment P-170 evaluation N*
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Ml Ml Ml MO MO MO MO MO MO MO MO MO MO MO MO MO MO MO MO MO
It
Tumor and node responses after Three cycles}: Sixcydes| Tumor Node Tumor Node PR MR Prog CR CR PR PR PR PR MR MR MR MR MR NC NC NC NC Prog Prog
CR CR NC CR CR CR CR CR CR CR CR MR NC NC PR NC NC NC Prog Prog
CR MR Prog CR CR CR CR PR PR MR MR PR PR Prog -§ MR NC NC MR NC
CR CR Prog CR CR CR CR CR CR CR CR PR NC NC -§ NC NC NC MR Prog
•Number of positive cells: N— = no positive cells; N(+) = coexistence of negative and positive cells; and N+ = 75% or more positive cells. tSpecific staining intensity: I— = no staining; I(+) = weak staining; and 1+ = strong staining. JTumor and node responses were evaluated after three and six cycles of chemotherapy and before subsequent surgery and/or radiation therapy. CR = completeresponse;PR = partial response; MR = minor response; NC = no change; and Prog = local progression. §The tumor of patient 15 was resected after three cycles. 112
Following chemotherapy, all 17 patients who were determined to have nonmetastatic cancer at the beginning of the studyreceivedlocal treatment (ie, surgical resection and/or radiation therapy) that subsequently rendered 16 of them clinically disease-free. (Patient 19 was discovered to have lung metastases following chemotherapy and radiation.) Complete responders to chemotherapy received radiation only; all other patients with nonmetastatic cancer received surgery followed by radiation. Partial responders received either conservative or radical surgery (ie, either segmental mastectomy with axillary node dissection and examination of margins or modified radical mastectomy) followed by radiation therapy. The surgery and/or radiation therapy occurred after the evaluation of initial responses to chemotherapy (Table 2) and after the follow-up study starting point. Patients with metastatic disease received radiation therapy as well as prolonged chemotherapy. Method for Obtaining Immunocytochemical Specimens
Table 2. Initial P-gh/coprotein evaluation and local response to chemotherapy Patient No.
Treatment Following Chemotherapy
Tumor tissue samples were obtained from all 20 patients under local anesthesia by biopsy (Tru-Cut; Travenol Laboratories, Inc, Deerfield, 111) just before the first cycle of chemotherapy. Biopsy wasrepeatedin two patients (Nos. 3 and 15) after the third cycle and in two others (Nos. 13 and 16) after the sixth cycle of chemotherapy. Tumor specimens were immediately frozen in liquid nitrogen until they were processed. Immunoperoxidase Staining Procedure Details ofkhe immunohistochemical method used were described elsewhere (12). Frozen sections were labeled by a triple-layer immunoperoxidase technique (Vectastain ABC Kit, Vector Laboratories, Burlingame, Calif). The MAbs are listed below; the peroxidase substrate, 3-aminoethyl carbazole, gave a red coloring to the stained cells. Tissue samples were characterized by their reactivity with MAbs. First, a series of reagents, including antibodies raised Journal of the National Cancer Institute
Downloaded from http://jnci.oxfordjournals.org/ at University of California, San Francisco on November 20, 2014
No.
response (MR:
