Vol. 72 • No. I
LETTERS TO THE EDITOR
129
Estrogen-receptor Assay
Prior to April 1978, it was our procedure to select and freeze in liquid nitrogen 1-g specimens of human mammary carcinoma for estrogen receptor assay following histologic confirmation of carcinoma by frozen section. During the six-month period prior to April 1978, 14 biopsy specimens with mammary carcinoma were submitted for estrogen-receptor assay. Five (36%) of these specimens were positive. During the same interval, specimens from 16 outside laboratories, each of which submitted more than ten specimens, demonstrated an overall rate of positivity of 62.7%. In May 1978, we began to freeze 1 g of carcinoma tissue prior to the performance of frozen section for histologic confirmation. Specimens were obtained from the operating room immediately after excision, and grossly evaluated for the characteristics of mammary carcinoma. When the tissue was determined to be grossly malignant, a 1-g tissue block was immediately Received January 3, 1979; accepted for publication January 19, 1979. Address reprint requests to Dr. Bordin. Key words: Breast cancer; Estrogen receptor; Hormone receptor.
trimmed, blotted dry, and temporarily frozen at - 2 0 C in the cryostat; the tissue was transferred directly into liquid nitrogen within 10 min. These specimens were frozen to sub-0 C temperatures within 5 min of excision rather than the 10-15 min that had elapsed prior to adoption of this new procedure. During the interval May to October 1978, 23 mammary carcinomas were handled in this manner. Sixteen (70%) were positive. Of specimens from outside laboratories, 53.8% were positive during this six-month period. The increase in our rate of positivity is significant (x2 = 5.56; P < 0.05). There is too little emphasis in the literature upon the necessity for very rapid freezing of breast tissue to be submitted for estrogen-receptor assay. Keffer1 states that the tissue must be frozen "quickly," but gives no time interval. Muschenheim and co-workers'' indicate they select fresh mammarycarcinoma tissue for estrogen-receptor assay on the basis of the gross appearance and freeze it in liquid nitrogen prior to frozen-section diagnosis. The time interval between excision and freezing, however, is omitted in their paper. At least two laboratories'1"'5 utilize a protocol for the postoperative handling of mammary-carcinoma specimens that includes placement of the biopsy specimen directly into a beaker on ice (4 C). The time interval between chilling on ice and freezing in liquid nitrogen, however, is not stated. It is recommended that biopsy samples be frozen as quickly and as cold as possible, because estrogenreceptor proteins show time- and temperature-dependent degradation in vitro. The biopsy or mastectomy specimen should be transferred from the operating room to the pathology laboratory in a water-tight container on ice. The pathologist should examine the tissue and, when it is grossly malignant, a 1-g block should be selected, trimmed, blotted dry, and frozen immediately in liquid nitrogen (—195 C) or temporarily at cryostat temperature ( - 2 0 C). Such rapid freezing utilizing the cryostat until liquid nitrogen is available should increase the incidence
of positive assays for estrogen-receptor protein. Delayed appropriate freezing of breast-biopsy specimens may explain the reported 2 5-10% rates of falsenegative estrogen-receptor assays (estrogen receptor-negative tumor with clinical response to endocrine therapy). When a mammary carcinoma has only a low level of estrogen-receptor protein, i.e., a level near the limit of detectability of the particular assay, delayed or insufficient freezing may permit degradation of the estrogen-receptor protein to a level below the limit of detectability, rendering that specimen falsely "negative." To permit unnecessary degradation of estrogen-receptor protein may deprive the patient of potentially beneficial endocrine therapy.
GERALD M. BORDIN,
M.D.
Surgical Pathology Hospital of Scripps Clinic La Jolla. California 92037 MARCIA WILEY, MT(ASCP),
M.S.
Scripps Clinic Immunology Reference Laboratory La Jolla. California 92037 References 1. Keffer JH: Hormone-receptor assays and cancer of the breast. The pathologist's role. Am J Clin Pathol 70: 719-720, 1978 2. Lippman ME, Allegra JC: Receptors in breast cancer. N Engl J Med 299: 930-933, 1978 3. Muschenheim F, Furst JL. Bates HA: Increased incidence of positive tests for estrogen binding in'mammary carcinoma specimens transported in liquid nitrogen. Am J Clin Pathol 70: 780-782, 1978 4. Rosen PP. Menendez-Botet CJ, Nisselbaum JS, et al: Pathological review of breast lesions analyzed for estrogen receptor protein. Cancer Res 35: 3187-3194, 1975 5. Wittliff JL, Savlov ED: Estrogen-binding capacity of cytoplasmic forms of the estrogen receptors in human breast cancer, Estrogen Receptors in Human Breast Cancer. Edited by WL McGuire. PP Carbone, EP Vollmer. New York, Raven Press, 1975, pp 73-88
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 7, 2016
To the Editor: — Keffer' and Muschenheim and associates,' 1 in their discussions of human mammary carcinoma and the estrogen receptor assay, indicate the need for rapid, lowtemperature freezing of biopsy tissue submitted for this assay. Estrogen receptor protein is a thermolabile, unstable protein that loses its binding capacity when not frozen quickly at low temperature.2"'1 Muschenheim and coworkers'' document an improved rate of estrogen receptor positivity when biopsy specimens are frozen rapidly in liquid nitrogen. The authors further report that freezing with liquid nitrogen increased the rate of estrogen receptor positivity from 56 to 69.3% {P < 0.\). Our observations indicate that the shorter the interval between excision and freezing, either temporarily at cryostat temperatures ( - 2 0 C) or immediately in liquid nitrogen (-195 C), the greater the incidence of positive tests for estrogen-binding protein.
LETTERS TO THE EDITOR
129
Estrogen-receptor Assay
Prior to April 1978, it was our procedure to select and freeze in liquid nitrogen 1-g specimens of human mammary carcinoma for estrogen receptor assay following histologic confirmation of carcinoma by frozen section. During the six-month period prior to April 1978, 14 biopsy specimens with mammary carcinoma were submitted for estrogen-receptor assay. Five (36%) of these specimens were positive. During the same interval, specimens from 16 outside laboratories, each of which submitted more than ten specimens, demonstrated an overall rate of positivity of 62.7%. In May 1978, we began to freeze 1 g of carcinoma tissue prior to the performance of frozen section for histologic confirmation. Specimens were obtained from the operating room immediately after excision, and grossly evaluated for the characteristics of mammary carcinoma. When the tissue was determined to be grossly malignant, a 1-g tissue block was immediately Received January 3, 1979; accepted for publication January 19, 1979. Address reprint requests to Dr. Bordin. Key words: Breast cancer; Estrogen receptor; Hormone receptor.
trimmed, blotted dry, and temporarily frozen at - 2 0 C in the cryostat; the tissue was transferred directly into liquid nitrogen within 10 min. These specimens were frozen to sub-0 C temperatures within 5 min of excision rather than the 10-15 min that had elapsed prior to adoption of this new procedure. During the interval May to October 1978, 23 mammary carcinomas were handled in this manner. Sixteen (70%) were positive. Of specimens from outside laboratories, 53.8% were positive during this six-month period. The increase in our rate of positivity is significant (x2 = 5.56; P < 0.05). There is too little emphasis in the literature upon the necessity for very rapid freezing of breast tissue to be submitted for estrogen-receptor assay. Keffer1 states that the tissue must be frozen "quickly," but gives no time interval. Muschenheim and co-workers'' indicate they select fresh mammarycarcinoma tissue for estrogen-receptor assay on the basis of the gross appearance and freeze it in liquid nitrogen prior to frozen-section diagnosis. The time interval between excision and freezing, however, is omitted in their paper. At least two laboratories'1"'5 utilize a protocol for the postoperative handling of mammary-carcinoma specimens that includes placement of the biopsy specimen directly into a beaker on ice (4 C). The time interval between chilling on ice and freezing in liquid nitrogen, however, is not stated. It is recommended that biopsy samples be frozen as quickly and as cold as possible, because estrogenreceptor proteins show time- and temperature-dependent degradation in vitro. The biopsy or mastectomy specimen should be transferred from the operating room to the pathology laboratory in a water-tight container on ice. The pathologist should examine the tissue and, when it is grossly malignant, a 1-g block should be selected, trimmed, blotted dry, and frozen immediately in liquid nitrogen (—195 C) or temporarily at cryostat temperature ( - 2 0 C). Such rapid freezing utilizing the cryostat until liquid nitrogen is available should increase the incidence
of positive assays for estrogen-receptor protein. Delayed appropriate freezing of breast-biopsy specimens may explain the reported 2 5-10% rates of falsenegative estrogen-receptor assays (estrogen receptor-negative tumor with clinical response to endocrine therapy). When a mammary carcinoma has only a low level of estrogen-receptor protein, i.e., a level near the limit of detectability of the particular assay, delayed or insufficient freezing may permit degradation of the estrogen-receptor protein to a level below the limit of detectability, rendering that specimen falsely "negative." To permit unnecessary degradation of estrogen-receptor protein may deprive the patient of potentially beneficial endocrine therapy.
GERALD M. BORDIN,
M.D.
Surgical Pathology Hospital of Scripps Clinic La Jolla. California 92037 MARCIA WILEY, MT(ASCP),
M.S.
Scripps Clinic Immunology Reference Laboratory La Jolla. California 92037 References 1. Keffer JH: Hormone-receptor assays and cancer of the breast. The pathologist's role. Am J Clin Pathol 70: 719-720, 1978 2. Lippman ME, Allegra JC: Receptors in breast cancer. N Engl J Med 299: 930-933, 1978 3. Muschenheim F, Furst JL. Bates HA: Increased incidence of positive tests for estrogen binding in'mammary carcinoma specimens transported in liquid nitrogen. Am J Clin Pathol 70: 780-782, 1978 4. Rosen PP. Menendez-Botet CJ, Nisselbaum JS, et al: Pathological review of breast lesions analyzed for estrogen receptor protein. Cancer Res 35: 3187-3194, 1975 5. Wittliff JL, Savlov ED: Estrogen-binding capacity of cytoplasmic forms of the estrogen receptors in human breast cancer, Estrogen Receptors in Human Breast Cancer. Edited by WL McGuire. PP Carbone, EP Vollmer. New York, Raven Press, 1975, pp 73-88
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 7, 2016
To the Editor: — Keffer' and Muschenheim and associates,' 1 in their discussions of human mammary carcinoma and the estrogen receptor assay, indicate the need for rapid, lowtemperature freezing of biopsy tissue submitted for this assay. Estrogen receptor protein is a thermolabile, unstable protein that loses its binding capacity when not frozen quickly at low temperature.2"'1 Muschenheim and coworkers'' document an improved rate of estrogen receptor positivity when biopsy specimens are frozen rapidly in liquid nitrogen. The authors further report that freezing with liquid nitrogen increased the rate of estrogen receptor positivity from 56 to 69.3% {P < 0.\). Our observations indicate that the shorter the interval between excision and freezing, either temporarily at cryostat temperatures ( - 2 0 C) or immediately in liquid nitrogen (-195 C), the greater the incidence of positive tests for estrogen-binding protein.
