Radioisotopic localization of (90)Yttrium-ibritumomab tiuxetan in patients with CD20+ non-Hodgkin's lymphoma.

PURPOSE: (90)Yttrium-ibritumomab-tiuxetan (Zevalin) is an effective treatment for relapsed or refractory low-grade, follicular, or transformed B-cell NHL. The purpose of this study is to assess whether tissue and cellular localization of (90)Y-ibritumomab-tiuxetan determined by autoradiography and radioactivity localized to tumor tissue might enhance our understanding of the mechanism of action of radioimmunotherapy. METHODS: Eight eligible patients had CD20+ NHL, a bulky peripheral lymph node, and were scheduled for (90)Y-ibritumomab-tiuxetan treatment. 2-Deoxy-2-[F-18]fluoro-D: -glucose-positron emission tomography/computed tomography (FDG-PET/CT) was performed prior to treatment and at 12 weeks after therapy for assessment of response. Bone marrow, lymph node, and blood samples were collected 114 +/- 3 h after 14.8 MBq/kg (90)Y-ibritumomab-tiuxetan and processed for histology, scintillation counting, and microscopic autoradiography. RESULTS: Pericellular membrane localization of (90)Y-ibritumomab-tiuxetan to lymphoma cells was observed by autoradiography in the involved areas of lymph node with absence of significant localization in histologically normal sections of bone marrow. Pericellular radioactivity and the highest quantitative radioactivity were observed in lymph node samples of responding patients. CONCLUSIONS: (90)Y-ibritumomab-tiuxetan localizes to the surface membrane of CD20+ lymphoma cells in affected lymph nodes. The patients with the highest quantitative concentration of radioactivity to the lymph node as determined by scintillation counting were observed to have a clinical and FDG-PET/CT response.

Actinomycin D: inhibition of respiration and glycolysis.

Actinomycin D inhibited respiration and anaerobic glycolysis of human leukemic leukocytes and lowered the adenosine triphosphate content of the cells. Inhibitory effects on respiration and on RNA synthesis could not be dissociated from one another over a wide range of drug concentrations. Actinomycin D also impaired protein synthesis, probably by decreasing the availability of adenosine triphosphate and by inhibiting messenger RNA.

Immunohistochemical analyses of estrogen receptor in endometrial adenocarcinoma using a monoclonal antibody.

Immunohistochemical localization of estrogen receptor (ER) using specific monoclonal anti-human estrogen receptor antibody, H222, with an immunoperoxidase technique was performed on fresh frozen tissue derived from 100 endometrial adenocarcinomas. Immunohistochemical evaluation incorporated both intensity and distribution of staining. In all cases, H222 localized in the nucleus of target cells. A significant quantitative relationship was shown between histological score (H-Score) and the biochemical analysis of ER content in tissue homogenates (r = 0.65, P = 0.00001). Excellent sensitivity (92%) and specificity (93%) were observed for the comparison of H-Score to the biochemical assay. Significant ER localization was present in stromal and myometrial elements, component H-Score of which correlated weakly with component H-Scores of malignant epithelial elements. Divergent receptor localization in stromal and myometrial versus malignant epithelial elements suggests that biochemical assays of endometrial carcinoma specimens may not reflect cancer-relevant receptor content. The data presented here suggest that the immunoassay of ER using H222 monoclonal antibody provides additional histochemical information to complement conventional analyses of endometrial adenocarcinomas.

Immunocytochemical analysis of estrogen receptors as a predictor of prognosis in breast cancer patients: comparison with quantitative biochemical methods.

Biochemical quantitation of estrogen receptors has been used to predict prognosis in breast cancer. Immunocytochemical analysis of estrogen receptors correlates with biochemical analysis but has very few follow-up studies in the literature to validate it as a prognostic indicator. 257 patients were followed for up to 10 years (median, 6.2 years) after primary surgical treatment. Estrogen receptor analysis using both biochemical and immunocytochemical techniques was performed on their tumor specimens. Patients with positive estrogen receptor values had longer survival than patients with negative values. This was demonstrated by both methods in the first 5 years of follow-up but only by immunochemistry after 5 years. The relationship between estrogen receptor status and disease-free interval was less strong than with survival. This study demonstrates that immunocytochemical estrogen receptor analysis was of prognostic significance.

Immunohistochemical expression of CA 125 in endometrial adenocarcinoma: correlation of antigen expression with metastatic potential.

Immunohistochemical localization of CA 125 using murine monoclonal antibody OC 125 was performed on fresh frozen tissue from 44 endometrial adenocarcinomas and 26 benign endometria. Immunohistochemical evaluation incorporated both intensity and distribution of staining (CA 125 HSCORE). Thirty-seven cancers (84%) and 23 benign endometria (88%) expressed immunohistochemically detectable CA 125. Staining was confined to epithelial cells and was present both on the cell membrane and in the cytoplasm. Among the 44 endometrial cancers, CA 125 HSCORE did not correlate with histological grade, depth of myometrial invasion or estrogen/progesterone receptor levels. Following surgical staging, 13 patients (30%) were found to have extrauterine metastatic disease. The median CA 125 HSCORE of patients with metastatic disease (2.25) was significantly higher than that of patients with disease confined to the uterus (0.6) (P less than 0.001). In addition, high CA 125 HSCORE also correlated with the presence of lymph node metastasis (P less than 0.001). The results of this study suggest that high CA 125 expression by endometrial adenocarcinomas is associated with increased metastatic potential.

Use of a monoclonal anti-estrogen receptor antibody in the immunohistochemical evaluation of human tumors.

A monoclonal antibody to human estrogen receptor protein (H222 Sp gamma), amplified via immunoperoxidase techniques, was used in the analysis of estrogen receptor in 452 breast carcinomas, 100 endometrial carcinomas, and 15 melanomas. Immunohistochemical evaluation incorporated both intensity and distribution of staining (HSCORE). Quantitative estrogen receptor content was determined by dextran-coated charcoal analysis and sucrose density gradient analysis. In all cases H222 Sp gamma localized in the nucleus of target cells. A semiquantitative correlation existed between HSCORE and biochemical assays for breast and endometrial tissues. The sensitivities and specificities for HSCORE as compared to the biochemical assays ranged from 80 to 95% and from 74 to 94%, respectively. HSCORE correlated with tumor grade for breast and endometrial carcinoma. Immunohistochemical evaluation showed no specific staining in melanomas. The data suggest that immunohistochemical receptor localization provides information complementary to standard biochemical assays in the tissues studied.

Localization and in vitro specificity of histone acetylation.

Histones of isolated calf thymus chromatin incubated in the presence of [14C]acetyl-CoA are radioactively acetylated, at specific residues. Incubation of calf thymus chromatin with [14C]acetate, however, gives no radioactive acetylation. The distribution of the radioactivity among the histone fractions corresponds well to that observed after incubation of calf thymus nuclei with [14C]acetate. The most striking observation is the complete absence of acetylation of histone F1 in both experiments. The specific activity of histones acetylated in vitro is much greater than that of histones acetylated in calf thymus nuclei, most likely reflecting a smaller degree of dilution of the labelled acetate donor. Investigation of the specificity of acetylation of each histone fraction reveals that for each histone fraction, specific peptides are acetylated in vitro which correspond to the histone peptides acetylated in calf thymus nuclei. Histones F2a1 and F2a2, however, also demonstrate additional minor sites of acetylation in the in vitro studies.

Comparison of a novel assay for breast cancer mucin to CA15-3 and carcinoembryonic antigen.

PURPOSE: To compare the sensitivity and specificity of an automated microparticle enzyme immunoassay (MEIA) for breast cancer mucin (IMx BCM; Abbott Laboratories, North Chicago, IL) to that of CA15-3 and carcinoembryonic antigen (CEA) for detecting and monitoring breast cancer. MATERIALS AND METHODS: IMxBCM was compared to assays of CA15-3 and CEA in 630 serum specimens from healthy women, and from women with breast cancer, other malignancies, benign breast conditions, or other benign diseases. RESULTS: Analysis of the log-transforms for the three markers in all specimens showed a high correlation of IMxBCM with CA15-3 (r = .78), but not with CEA (r = .25). Based on a receiver-operating-characteristics (ROC)-curve analysis for any given specificity, IMxBCM was found to be a more sensitive marker than either CA15-3 or CEA for distinguishing 105 women with advanced or metastatic breast cancer from 89 healthy women (P = .003 and P = .04, respectively), from 98 women with benign breast conditions (P = .02 and P = .002), or from 191 women with benign diseases (P = .03 and P less than .0001). At 95% specificity, the sensitivities of IMxBCM, CA15-3, and CEA for detecting advanced or metastatic breast cancer were 69%, 51%, and 30%, respectively. Serial serum samples (n = 177) were analyzed in 20 additional metastatic breast cancer patients with measurable disease. Serial IMxBCM levels corresponded with the clinical course of disease in 80%, CA15-3 in 65%, and CEA in 60% of the 20 patients. CONCLUSIONS: Increased sensitivity of IMxBCM, despite a high correlation with CA15-3, suggests that IMxBCM and CA15-3 may recognize distinct epitopes on the same molecule. Although further research is indicated, IMxBCM may provide a promising marker in the clinical management of breast cancer patients.

Hormone responsiveness of a transplantable rat chondrosarcoma. II. Evidence for in vivo hormone dependence.

We have previously demonstrated that the Swarm rat chondrosarcoma responds to GH-dependent serum factors in vitro by increasing amino acid transport and macromolecular synthesis. The question of in vivo hormone dependence was evaluated by studying the growth of the tumor in hypophysectomized rats. Tumor-bearing hypophysectomized rats were treated with saline, T4, cortisone, bovine GH (bGH), or combinations of these hormones. Tumor growth was assessed in terms of tumor weight. Histological appearance was studied to ascertain the viability of the tumors and the relative contributions of cellularity vs. cartilage matrix to the weight of the chondrosarcoma. The weight of tumors grown in saline-treated hypophysectomized rats was less than 10% of the weight attained by tumors grown in normal rats for a comparable period of time. There was a greater relative cellularity in tumors grown in normal and hormone-treated hypophysectomized rats compared to that in tumors grown in saline-treated hypophysectomized rats. Tumors from saline-treated hypophysectomized rats had an atrophic appearance. Treatment of the hypophysectomized rats with bGH or cortisone increased the rate of tumor growth 5- to 6-fold and restored the histological appearance toward that of tumors grown in normal rats. A greater rate of tumor growth was effected by treatment with a combination of bGH and cortisone. T4 by itself did not stimulate chondrosarcoma growth in the hypophysectomized rat. Withdrawal of bGH and cortisone treatment from hypophysectomized rats after tumors were hormone stimulated caused a loss of the apparent exponential rate of growth observed in hypophysectomized rats treated through the full 38-day observation period. These data indicate that bGH and cortisone act in concert to stimulate the growth of the Swarm rat chondrosarcoma in vivo and that the tumor is hormone dependent.

Immunohistochemical analysis of human uterine estrogen and progesterone receptors throughout the menstrual cycle.

Estrogen receptors (ER) and progesterone receptors (PgR) were studied immunohistochemically using specific antireceptor monoclonal antibodies in uterine tissue samples from 33 women in various stages of the menstrual cycle. Immunohistochemical localization was quantified as to intensity of staining and tissue distribution in glandular epithelium, stroma, and myometrium, and the results were compared with those of standard ligand binding assays. In all samples ER and PgR localized within the nuclei of target cells. The maximal concentrations of ER and PgR occurred in the mid- to late proliferative phase of the menstrual cycle. ER content declined throughout the secretory phase. In contrast, PgR content underwent unexpectedly complex and dyssynchronous fluctuations during the secretory phase of the menstrual cycle. Specifically, the glandular epithelium had diminished PgR content, while the stroma and myometrium maintained a significant PgR content. PgR and perhaps ER are not concordant in different cell types within the uterus. Segregation of function through alteration of receptor content may be an important mechanism in steroid-dependent growth and differentiation of target tissues.

The evaluation of estrogen receptor in primary breast carcinoma by computer-assisted image analysis.

A monoclonal antibody prepared against estrogen receptor has been shown to be specific and sensitive for the detection of estrogen receptor in human breast lesions by use of immunohistochemical methods. Two hundred selected cases of primary breast carcinoma were assayed for estrogen receptor content by biochemical and immunohistochemical procedures. Quantitative evaluation was by biochemical, immunohistochemical, and automated computer-assisted image analysis using the Cell Analysis System's CAS/100 machine (Lombard, IL). Quantitative estrogen receptor content was determined by dextran-coated charcoal analysis and sucrose density gradient analysis. Immunohistochemical evaluation incorporated both intensity and distribution of staining, yielding a subjective score, histologic score (HSCORE). An objective quantitation, also incorporating intensity and distribution of staining, was done by computer-assisted image analysis, quantitative immunocytochemical score (QIC SCORE). HSCORE analysis was done with and without methyl green counterstain with no loss of sensitivity. Comparison of QIC SCORE with the biochemical and immunohistochemical analysis of the tissues examined revealed excellent sensitivities and specificities. These data suggest that automated image analysis provides an effective qualitative and quantitative means of evaluating estrogen receptor content in human breast cancers.

Sex steroid receptor concentration in breast carcinoma tissue: effect of devascularization during mastectomy.

The accurate determination of sex steroid receptors at the time of mastectomy (MX) for breast carcinoma is important for the determination of subsequent therapy of patients who develop metastases in inaccessible sites. The estrogen (E) and progesterone (P) receptor (R) proteins are heat labile, and measured levels may be vulnerable to alterations once the tumor is devascularized. To evaluate potential differences in ER and PR determinations in tumor tissue acquired at biopsy as compared with tumor from the MX specimen, quantitative analyses of ER (21 patients) and PR (17 patients) were performed on dual samples acquired from the initial biopsy (BX) and the subsequent MX specimen. Receptor concentrations were determined both by sucrose density gradient analysis and titration analysis, and results were expressed as fmol/mg cytosol protein. ER values were classified as receptor-rich (greater than 10 fmol/mg), intermediate (3 to 10 fmol/mg), or receptor-poor (less than 3 fmol/mg); PR values greater than 3 fmol/mg were considered positive. ER BX values were found to be rich or intermediate in 18 patients. When compared with BX values, MX ER values were quantitatively unchanged in 11 patients, lower (MX less than BX) in four patients, and higher in three patients (MX greater than BX). In no patient was the BX ER rich or intermediate and the concomitant MX ER poor. In two patients the PR value was "positive" at BX but "negative" at MX. Accordingly, malignant tissue from a pre-MX biopsy specimen is preferred for receptor analysis although it is apparent that tumor tissue from a properly handled MX specimen is satisfactory for the determination of ER status for clinical purposes.

Biochemical correlates of morphologic differentiation in human breast cancer.

In 115 breast carcinoma tissues, histologica grade and cell cytosol concentrations of estrogen receptor (ER) and progesterone receptor (PR) and two breast cyst fluid proteins (gross cystic disease fluid protein [GCDFP-15] and nonreceptor progesterone-binding protein [PBP]) were deterMined. Higher levels (expressed as femtomoles per milligram of protein) of ER (128 +/- 28 versus 11 +/- 1, P less than 0.001) and PR (82 +/- 16 versus 3 +/- 1, P less than 0.001) were found in grade 1 (well-differentiated) carcinomas as compared with grade 3 (poorly differentiated) carcinomas. Similarly, higher concentrations (expressed as nanograms per milligram of cytosol protein) of GCDFP-15 (2110 +/- 840 versus 210 +/- 40, p less than 0.001) and PBP (4920 +/- 1200 versus 370 +/- 60, P less than 0.001) were found in grade 1 as compared with grade 3 carcinomas. Tumor cytosols that contained low levels of both cyst proteins (less than 225 ng/mg GCDFP-15 and less than 750 ng/mg PBP) had a high incidence of grade 3 (35 of 46, 78%) or grade 2 (15 of 46, 33%) histologic findings and had a high incidence of receptor-negative specimens (27 of 52, 52%). Based on these cutoff levels, grade 2 lesions were subdivided into a "high" cyst protein group, which had ER and PR levels similar to grade 1 tumors (93.1 +/- 26.7 for ER and 84.7 +/- 32.4 for PR, P greater than 0.3), and a "low" group, which had receptor values similar to grade 3 carcinomas (14.1 +/- 5.3 for ER and 9.1 +/- 5.2 for PR, P less than 0.3). Although the mean cytosol content of carcinoembryonic antigen (CEA) was significantly higher in malignant tissues (125 +/- 27 ng/mg cytosol protein) than in benign tissues (4.8 +/- 1 ng/mg cytosol protein), the CEA content was not significantly different between grades 1 and 3 tumors.

Quantitative analysis of the calcium-sensing receptor messenger RNA in parathyroid adenomas.

BACKGROUND: In primary hyperparathyroidism, hypercalcemia fails to suppress adequately secretion of parathyroid hormone by the parathyroid gland, which may result from failure of the cell-surface calcium receptor (CaR) to sense calcium correctly. Quantification of mRNA concentrations should provide important information on the role of expression of Call in primary hyperparathyroidism. METHODS: We have developed a quantitative reverse transcriptase-polymerase chain reaction assay with a competitive template (CaR-M). Amplified cDNAs for CaR and CaR-M are quantified, and the concentration of CaR mRNA is determined from the ratio of CaR-M/CaR versus known CaR-M concentrations. RESULTS: In parathyroid adenomas (n = 12) the CaR mRNA was 19.2 +/- 2.4 (mean +/- SE) fg/ng total RNA (range, 7.4 to 32.8 fg/ng). Extracellular ionized calcium levels ranged from 1.38 to 1.74 mmol/L (normal 1.19 to 1.31 mmol/L) and parathyroid hormone from 69 to 345 pg/ml (normal, 14 to 65 pg/ml). In spite of the wide variability in CaR expression in the various adenomas, there was no correlation between mRNA and either extracellular ionized calcium (r2 = 0.013) parathyroid hormone levels (r2 = 0.001). Normal human parathyroid glands gave values of 8.0 and 16.6 fg/ng, whereas normal bovine parathyroid glands had a mean of 20 +/- 0.6 fg/ng (n = 4). CONCLUSIONS: There is no apparent relationship between CaR mRNA levels in adenomas and preoperative Ca and PTH levels. Our findings suggest that defective Ca sensing in adenomas may involve post-translational modification or signal transduction distal to the receptor. Our highly sensitive assay for CaR mRNA should prove useful in examining further the role of CaR in Ca sensing in parathyroid tissue.

Clinical correlates of estrogen- and progesterone-binding proteins in human endometrial adenocarcinoma.

Seventy-eight endometrial carcinoma specimens from 74 patients were evaluated for estrogen (E2R) and progesterone (PrR) receptors by multiconcentration saturation and sucrose density gradient analysis. The clinical stage and histologic grade of the tumors and the patients' clinical course were compared to the qualitative and quantitative evaluations of E2R and PrR. Although no correlation was seen between receptor content and extent of disease, tumors with significant levels of E2R and PrR tended to be less aggressive than those in which neither receptor was detected. The histologic grade correlated with a combination of estrogen and progesterone receptor content. In 92% of the patients there was a concordance between the presence or absence of E2R and PrR in the tumor and a clinical response to progestin therapy. The significance of these findings in relationship to the presence of multiple forms of steroid binding is discussed.

Hormone-resistant endometriosis following total abdominal hysterectomy and bilateral salpingo-oophorectomy: correlation with histology and steroid receptor content.

Endometriosis is rare after hysterectomy and oophorectomy for conditions unrelated to endometriosis. We present a case of delayed development of aggressive, hormone-resistant endometriosis temporally remote from hysterectomy and oophorectomy performed for chronic pelvic inflammatory disease. Treatment with depo-medroxyprogesterone acetate resulted in continued growth of the retroperitoneal endometrioma and necessitated posterior exenteration because of the endometrioma's location. Estrogen and progesterone receptor levels were measured to clarify why this woman's endometriosis was resistant to hormone therapy. Despite administration of large amounts of depo-medroxyprogesterone acetate, the progesterone receptor content was elevated while the estrogen receptor content was undetectable. Why this patient developed this particular type of aggressive endometriosis is unclear, but the lack of down-regulation of progesterone receptors in response to high-dose progestin therapy may indicate an alteration in basic regulatory and cellular processes within the endometriotic implant.

Endometrial cancer: histologic correlates of immunohistochemical localization of progesterone receptor and estrogen receptor.

Progesterone and estrogen receptor localization, using monoclonal anti-receptor antibodies JZB39 and H222, was studied in 105 endometrial adenocarcinomas. Immunohistochemical evaluation incorporated both intensity and distribution of staining. Both anti-progesterone receptor and anti-estrogen receptor localized in the nucleus of target cells. Significant levels of progesterone receptor and estrogen receptor were seen localized in stromal and myometrial elements that diverged from the malignant epithelial component. Analyses of endometrial adenocarcinomas with anti-progesterone receptor and anti-estrogen receptor antibodies correlated with histologic differentiation. The ability to define divergent receptor populations in stromal and myometrial elements versus malignant epithelial elements indicates that immunohistochemical assay of progesterone and estrogen receptor provides information complementary to that from conventional quantitative ligand binding assays.

Immunohistochemical analysis of estrogen and progesterone receptors in endometriosis: comparison with normal endometrium during the menstrual cycle and the effect of medical therapy.

Estrogen receptors (ER) and progesterone receptors (PgR) in 19 endometriotic implants from 16 normally cycling and hormonally treated women were measured using immunohistochemical techniques and compared with 34 samples of normal intrauterine endometrium. Endometriotic implants contained specific ER and PgR in both glandular epithelium and stroma. In contrast to intrauterine endometrium, receptor content among implants was noted to be more heterogeneous, and did not undergo predictable changes in response to endogenous hormones. In the endometriotic implants of patients treated with hormonal therapy, there were significant decreases in ER and PgR in both the glands and stroma relative to untreated patients. These data imply that endometriosis is unpredictable in its response to the cyclic hormonal milieu in terms of ER and PgR, but retains the ability to respond to hormonal suppression over a prolonged interval.

Absence of estrogen receptor in human melanoma as evaluated by a monoclonal antiestrogen receptor antibody.

Controversy regarding the presence of estrogen receptor proteins in human melanomas persists despite extensive investigations on this subject. While apparent high-affinity binding has been observed using dextran-coated charcoal assays, several other characteristics of receptor protein have not been observed. The production of free water on incubation of tritiated estradiol (labeled in the C2 position) with melanoma cytosols suggests the possibility that the apparent binding observed is due to phenomena other than specific receptor-steroid interactions. Melanomas from 15 patients were evaluated for the presence of estrogen receptor using immunocytochemical techniques with a monoclonal antibody directed against the human estrogen receptor protein (H222 Sp gamma). Immunohistochemical evaluation included intensity and distribution of staining. None of the 15 cases demonstrated specific immunohistologic reactivity with the anti-receptor antibody. Control breast and uterine tissue confirmed the specificity and sensitivity of the methods. These results suggest that the apparent estrogen-binding capacity of human melanoma tissues is the result of interactions other than with estrogen receptor, and reaffirm the need to investigate alternate steroid protein interactions, such as catechol estrogen formation, in studying sex steroid influences on human melanoma.

Reduced Cd2+ accumulation and elevated metallothionein levels in a Cd2+ and Zn2+-resistant clonal CHO-K1 cell line.

A clonal cell line, R40F, was selected from a heterogenous population of Cd2+ and Zn2+-resistant CHO-K1 cells. These R40F cells demonstrated resistance to 120- and 4-fold higher concentrations of Cd2+ and Zn2+, respectively, than did wild type CHO-K1 cells. When cultured in the presence of low concentrations of Cd2+ (0.5-1.0 microM), the accumulation of intracellular Cd2+ in R40F cells appears to be significantly less than in wild type cells. Since R40F cells maintained in medium containing high concentrations of Cd2+ (200 microM) retain levels of Cd2+ equivalent to the intracellular concentration observed in wild type cells exhibiting cytotoxicity, it is assumed that reduced Cd2+ transport alone is unlikely to account for the resistance to Cd2+ toxicity. Exposure of R40F cells to non-toxic (2 microM or 100 microM) or toxic (200 microM) Zn2+ levels resulted in an accumulation of Zn2+ equal to, or greater than, that observed in the wild type cell. When compared to the basal level in uninduced wild type cells, metallothionein levels were elevated 14- and 23-fold, respectively, in R40F cells cultured in the presence of 0.5 microM Cd2+ and 100 microM Zn2+. These results are consistent with the hypothesis that R40F cells express Cd2+ and Zn2+ resistance as a consequence of a reduction in unbound intracellular Cd2+ levels and an elevation of metallothionein synthesis.