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Psychological stress is recognized as a factor that contributes to increased susceptibility to a number of diseases, including cancer. Psychological stress, via release of chemical mediators, can induce long-term changes in the organism resulting in an altered responsiveness of the organism to external carcinogens. The present investigation sought to evaluate the impact of stress on polycyclic aromatic hydrocarbon (PAH)-induced carcinogenesis. We utilized a repetitive restraint stress mouse model and the model PAH, 7,12-dimethylbenz[a]anthracene (DMBA). Restraint stress was applied three times a week for 6 weeks and DMBA was administered intra-gastrically once a week for 6 weeks and formation of skin, mammary and ovarian tumours was examined at 26 weeks by pathological analyses. The results indicate that stress accelerates the onset of tumour formation produced in response to DMBA and significantly influences the type and location of tumours. Taken together, these results demonstrate that stress enhances the carcinogenicity of DMBA. These results clearly indicate the need for further characterization of the impact of stress on carcinogen metabolism in oncology. Copyright © 2010 John Wiley & Sons, Ltd.

We have isolated from human breast cancers several mutations in the Helix 12 component of activation function 2 (AF-2) in the estrogen receptor alpha (ERalpha). We used a novel approach to detect changes in the hormone-binding domain of ERalpha, based on the evidence that antiestrogens, such as 4-hydroxytamoxifen (ZOHT) and ICI 182,780, block the function of ERalpha by binding and folding the AF-2 transcriptional domain in a way that inhibits its association with coactivator proteins. We have identified a Helix 12 mutation, M543V, which leads to greater ERalpha transcription with ZOHT and other antiestrogens (including 1,1-dichloro-2,2,3-triarylcyclopropanes, DTACs) than with 17-beta estradiol (E2). We also found an independent mutation at the same position, M543I, which did not show this inverted ligand phenotype. In comparison to further Helix 12 mutations made in vitro, it appears that relative hydrophobicity of the amino acid side chains on the inner face of Helix 12 is key to maintaining the transcriptionally active, agonist conformation with bound E2. This active conformation can be induced, resulting in increased transcription, by adding excess p160 coactivator AIB1 in transcriptional assays with E2-bound receptors, while the ZOHT-bound receptors were not further activated by AIB1. Other experiments show that the cross talk between ERalpha and AP-1 protein from AP-1-binding sites is not dependent on Helix 12 integrity. We show that two alleles containing a proline substitution in Helix 12 that inactivate AF-2 function of ERalpha at EREs have little negative effect on function through AP-1 elements, supporting a prominent role for the N-terminal AF-1 of ERalpha in AP-1/ERalpha transcriptional cross talk.

Sentinel lymph node procedures are intended to provide sufficient sensitivity for the detection of metastases to allow the surgeon to forgo full axillary dissection if the sentinel nodes are found to be free of metastases. Two cases are presented that illustrate the complexities of pathologic assessment of sentinel lymph nodes, and demonstrate the need for improved techniques to overcome operator-dependent pitfalls. A combination of hematoxylin and eosin stained step sections, cytokeratin immunohistochemistry, and reverse transcriptase-polymerase chain reaction provide for improved sensitivity with excellent specificity.

A 75-year-old man with relapsed follicular non-Hodgkin lymphoma confined to a solitary lung mass was treated with radioimmunotherapy (RIT) using yttrium 90-ibritumomab tiuxetan. Imaging with positron emission tomography/computed tomography showed a complete response 3 months after RIT. Thirteen months after RIT, his positron emission tomography/computed tomography scan showed a fluorodeoxyglucose-avid infiltrate in the area of the previous lung mass. Bronchoscopy revealed the area to be obstructed with fibrosis, and cytologic washings and brushings did not show lymphoma. The patient remains asymptomatic, and the fluorodeoxyglucoseavid pulmonary infiltrate was unchanged 19 months after RIT. In view of the lack of respiratory symptoms or progressive imaging abnormalities, we believe radiation fibrosis is the most likely etiology. Radiation-induced lung injury after therapy with yttrium 90 was previously reported in the setting of intraarterial microspheres used to treat inoperable hepatic tumors. This is the first case in which radiation-induced radiographic changes are reported after RIT for lymphoma.

11q13 amplification occurs in a wide variety of tumors, including almost half of oral squamous cell carcinomas (OSCC) where it has been correlated with a poor outcome. In this study, we compiled 3.6 Mb of DNA sequence in the 11q13 amplicon core and refined the physical map of the amplicon. In the process, we determined the genomic structure and normal tissue expression patterns of two recently identified genes, TAOS2/TMEM16A and MRGF, which reside in the amplicon core. We then quantified DNA copy number and mRNA expression of all genes in the 11q13 amplicon in cell lines and primary tumors from OSCC. With the exception of FGF3, FGF4, FGF19, and MRGF, all genes were overexpressed in most tumors with genomic amplification. Furthermore, we found that the expression of genes in the amplicon appeared to be highly coordinated, making it difficult to determine which gene or genes are driving amplification. However, in nonamplified primary tumors, three genes, TAOS2/TMEM16A, OCIM, and TPCN2, are frequently overexpressed, whereas CCND1 and EMS1 are not. These results suggest that in addition to CCND1 and EMS1, other important genes also may be target genes driving 11q13 amplification. We hypothesize that 11q13 amplification may be driven by a cassette of genes that provide growth or metastatic advantage to cancer cells. This is supported by the finding that the human 11q13 amplicon core is syntenic to mouse chromosomal band 7F5, which is frequently amplified in chemically induced murine OSCC. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat

In breast cancer and other malignancies, involvement of regional lymph nodes is a strong prognostic indicator and greatly influences staging and clinical management.1–4 One benefit from the implementation of sentinel lymph node biopsy (SLNB) techniques is the identification of metastatic foci of cancer in 10% to 15% of patients that would have been previously staged as node-negative (pN0) by conventional methods.5,6 This improved sensitivity is attributed to both the addition of immunohistochemical staining (IHC) and to an increase in sampling volume.5,6 Many of these additional positive nodes contain only micrometastatic foci of tumor. However, the clinical significance of micrometastatic disease identified by SLNB techniques is highly controversial.7–11 Nonetheless, SLNB techniques are now widely used in breast cancer and melanoma and are being applied with increasing frequency to other tumors, including colorectal, oropharyngeal, prostate, lung, and other solid organ cancers.12–15 Another clear advantage to the SLNB technique is that in the majority of breast cancer patients, SLNB safely avoids axillary lymph node dissection (ALND) and the associated morbidity when the SLN is negative for metastatic disease.16 However, rapid, frozen-section analysis of SLN for metastasis is only 50% to 70% sensitive for the detection of metastasis compared with the permanent histologic sections and IHC of the same lymph node,17,18 and complete analysis of the SLNB specimen currently requires extensive preparation and time-consuming review.8 Even in experienced hands, 10% of SLNB specimens are later found to contain metastases.19 As a result, these patients have required a second surgical procedure to complete the ALND. This is clearly an undesirable algorithm for the patient, the healthcare provider, and the healthcare payer, and this dilemma has contributed to a new controversy; if the SLNB is positive, does a completion ALND confer a therapeutic or staging benefit to the patient? Until ongoing multicenter trials determine whether completing the ALND when the SLNB is positive for metastasis benefits the patient, it is clear that the time required to accurately evaluate SLNB specimens has significant implications.20 It must also be recognized that, even with adequate time, the accurate histologic analysis of lymph nodes for metastatic disease is challenging, and discordance in the interpretation of these materials is a well-known clinical problem. Indeed, a recent study found alarming disparity among pathologists in the analysis of SLNB specimens.21 Furthermore, protocols for SLNB specimen analysis vary widely between healthcare centers.22 Thus, current methods of lymph node analysis clearly lack standardization, are dependent in part on subjective criteria, and are subject to human error. We and others have shown that a real-time, quantitative RT-PCR (QRT-PCR) analysis of lymph nodes can be more accurate than conventional histologic analysis in predicting prognosis for solid organ malignancies,23,24 and we have previously reported our development of a rapid QRT-PCR procedure that can be completed (including RNA isolation) in less than 25 minutes.25 Importantly, a revolutionary tool for molecular-based assays called the GeneXpert (Cepheid, Sunnyvale, CA) is in the final stages of development. This instrument fully automates sample preparation, RNA isolation and purification, and QRT-PCR in a single-use, cartridge-based format that removes the major obstacles limiting routine use of molecular-based assays. Thus, we aimed to determine if a rapid, fully automated, internally controlled QRT-PCR assay performed by a prototype GeneXpert instrument could produce equivalent results to histologic techniques of SLNB analysis, including IHC. This assay could produce significant benefits to patients and healthcare providers by definitively analyzing lymph nodes using objective criteria in a time frame that allows intraoperative use, reduces patient psychologic distress, and improves standardization between healthcare centers.

A novel class of pure antiestrogens, 1,1-dichloro-2,2,3-triarylcyclopropanes (DTACs), lack estrogenic activity in a mouse uterotrophic assay and inhibit the growth of estrogen-sensitive MCF-7 breast cancer cells (Day et al., 1991). Here, reporter assays were used to evaluate the effects of the DTACs on estrogen receptor alpha (ERalpha)-mediated transcription from either classic estrogen-response elements (EREs) or nonclassic AP-1 elements. Among the DTACs tested, only the compounds with smaller aromatic substituents, BDRM72 and BDRM81, displayed weak agonist activity on EREs. Their activity was less than that observed for the ER partial agonist, 4-hydroxytamoxifen (ZOHT). In competition experiments, the DTACs blocked estradiol-stimulated transcription from an ERE in a dose-dependent manner and were more effective inhibitors than ZOHT. Each of the DTACs was significantly less active than ZOHT or the pure antiestrogen ICI 182,780 (faslodex) in stimulating transcription from nonclassic AP-1 elements in the presence of ERalpha. DTACs did not modulate either basal or TPA (12-O-tetradecanoylphorbol-13-acetate)-stimulated transcription from an AP-1 element in the absence of ERalpha, indicating that they are not nonspecific inhibitors of transcription and that ERalpha is the drug target. Glutathione S-transferase pull-down assays were used to examine whether DTACs alter the interaction between ERalpha and the p160 coactivator, GRIP1. BDRM35, which has the same dimethylaminomethoxy and phenolic moieties as ZOHT, reduced binding by more than 50%. Thus, disruption of p160 coactivator recruitment by ERalpha may represent one mechanism by which DTACs function as antiestrogens. BDRM35 also suppresses estradiol induction of endogenous target genes c-myc and cyclin D1 in MCF-7 breast cancer cells.

Antiestrogens block the function of estrogen receptor (ER) by binding and misfolding the AF-2 transcriptional activation region in the ligand-binding domain, inhibiting or altering its association with coactivator proteins. We describe a novel assay uniquely configured to identify aberrations in this function that may lead to antiestrogen resistance. The identification of mutations of ER that affect its function is important to current breast cancer therapies. Standard methods to detect these mutations are cumbersome and the number of described mutations is limited, reflecting this difficulty. Conventional ER analysis in the clinic demonstrates the presence of antigenic determinants of the receptor protein or estrogenic ligand binding without reflection on the critical ability of the liganded receptor to interact with transcription cofactors. Here, we describe the use of estrogenic regulation of a site-specific recombinase activity, measuring deletion of a color marker gene via FLP-ER fusion proteins, to detect functional changes in ER protein folding that affects the site where cofactors interact. The assay provides a method to readily detect single amino acid changes in ER, some with biologically important consequences. Without such a functional assay as described, phenotypic changes are likely to remain undetected and under-evaluated. It is probable that some human tumors have antihormone resistance resulting from ER mutations that either block antihormone binding or transmit antihormone binding as a positive transcriptional signal via cofactor interaction. An assay to evaluate functional ER will lead to better predictive tests of treatment modalities.

Breast tumor staging and treatment response is primarily determined by tumor size. Presently there are four techniques to measure breast cancer size: (1) palpations, (2) mammography, (3) ultrasonography, and (4) MR imaging. This article reviews the applications and limitations of each technique and the improvements MR imaging can provide.

90Yttrium-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 90Y-ibritumomab–tiuxetan determined by autoradiography and radioactivity localized to tumor tissue might enhance our understanding of the mechanism of action of radioimmunotherapy. Eight eligible patients had CD20+ NHL, a bulky peripheral lymph node, and were scheduled for 90Y-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 90Y-ibritumomab-tiuxetan and processed for histology, scintillation counting, and microscopic autoradiography. Pericellular membrane localization of 90Y-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. 90Y-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.

Background: One evolving approach to improved prognostication of cancer patients is the identification of previously occult disease by use of quantitative reverse transcription-PCR. Surprisingly, no systematic analysis of potential mRNA markers for colorectal cancer has been reported. We therefore performed an extensive mRNA marker survey for colorectal cancers.Methods: We identified potential markers through literature and database searches. We analyzed all markers by quantitative reverse transcription-PCR on a limited set of primary tumors and benign lymph nodes. Selected markers were further evaluated on a larger tissue set with positive lymph nodes.Results: We evaluated 43 markers and undertook further analysis of 6 in the secondary screening. Five gene markers—CDX1, carcinoembryonic antigen (CEA), CK20, TACSTD1, and Villin1 (VIL1)—provided perfect classification of lymph node status.Conclusions: Several mRNA markers are capable of providing exceptionally accurate characterization of lymph node status in colorectal cancer. An automated, multimarker, quantitative reverse transcription-PCR assay for characterization of lymph nodes from colorectal cancer patients may be useful for improved staging and therapeutic decision making in colorectal cancer.

Background: PCR-based assays can improve clinical care, but they remain technically demanding and labor-intensive. We describe a new instrument, the GeneXpert®, that performs automated nucleic acid isolation, reverse transcription, and fluorescence-based quantitative PCR in ∼35 min.Methods: Yield and integrity of RNA isolated on the GeneXpert were compared with Qiagen-based extraction for parallel samples (5-μm frozen tissue sections). The reproducibility of automated RNA isolation, reverse transcription, and quantitative PCR was determined by replicate (n = 10) analysis of 10 tissues, using duplex (target and endogenous control) reverse transcription-PCR reactions for two gene combinations. The GeneXpert was then used to perform rapid analysis of lymph nodes from melanoma, breast cancer, and lung cancer patients and analysis of melanoma metastatic to the lung, primary lung adenocarcinoma, and healthy lung tissue.Results: On the GeneXpert, RNA was recovered in slightly over 6 min, and the yield was ∼70% of that from parallel Qiagen reactions. The RNA integrity was comparable to that of Qiagen-isolated RNA as determined by gel electrophoresis. For the melanoma samples, the 95% prediction interval for the ΔCt for a new measurement was ±1.54 cycles, and for breast cancer samples, the interval for a newly observed ΔCt was ±1.40 cycles. GeneXpert assays successfully detected the presence of metastatic melanoma, breast cancer, and lung cancer in lymph nodes and also differentiated among metastatic melanoma, lung adenocarcinoma, and healthy lung.Conclusions: RNA yield and integrity on the GeneXpert are comparable to benchtop methods. Reproducibility of the GeneXpert data is similar to that seen with manual methods in our hands but may need improvement for some applications. The GeneXpert can perform RNA isolation, reverse transcription, and quantitative PCR in ∼35 min and could therefore be used for intraoperative testing when applicable.

This observational study involves the evaluation of a cohort of patients who had undergone a modified radical mastectomy for primary breast cancer and then underwent a contralateral subcutaneous mastectomy at the time of reconstruction. The study extends clinical follow-up to a minimum of 10 years, methodically reviews the histology of the primary mastectomy and contralateral mastectomy materials and compares the cohort to several control groups treated 'conventionally' without contralateral subcutaneous mastectomy. Initial evaluation of the cohort demonstrates a remarkable disease free survival compared to similar patients with similar tumors not exposed to contralateral subcutaneous mastectomy. The survival advantage is observed to be maximal the closer the second procedure was performed to the primary mastectomy, mitigating against the result being due to time to procedure selection bias. The study of the contralateral specimens for frequency and pattern of epithelial hyperplasia associated with contralateral cancer and the cataloguing of findings is being carried out to establish a resource for in-situ molecular studies on such unique material associated with whole gland evaluation.

For many solid carcinomas, high-resolution cross-sectional imaging has changed cancer staging, the evaluation of therapeutic response, the detection of recurrence, and even how therapy is selected and performed. Such imaging has not yet had similar effects on breast cancer. Evaluations of therapeutic response in breast carcinomas have been impeded by the current limited methods of evaluating breast tumor size and extent: clinical palpation, ultrasonography, and mammography. The use of magnetic resonance imaging (MRI) of the breast in the evaluation of breast tumors brings the advantages of high-resolution cross-sectional imaging to breast cancer staging and treatment evaluation and is likely to greatly enhance research efforts in this complex disease. MRI of the breast has evolved to be the most accurate noninvasive technique for local staging of breast cancer. MRI is most accurate in measuring tumor size and detecting multicentric disease. These staging characteristics affect the selection of therapy and initial determination of prognosis; therefore, MRI of the breast can change the assessment of fundamental parameters on which treatment is selected. Because clinical trials of new cancer treatments are predicated on proper and accurate characterization of the tumor, MRI also should affect how clinical trials are performed and evaluated.

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 CaR 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 (r 2 = 0.013) or parathyroid hormone levels (r 2 = 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.

Accurate measurement of the size of breast cancers becomes more important as breast cancer therapy advances. This study reports the accuracy of magnetic resonance imaging (MRI), ultrasonography and mammography for measuring the largest breast cancer diameter in comparison to the pathology measurement.Fourteen breast cancers were examined in 13 women with MRI, ultrasonography and mammography. The age range was 31-73 (mean 56). Six of the cancers were in premenopausal women. The MRI was performed with the intravenous injection of gadolinium based contrast agent and a three dimensional fast spoiled gradient echo sequence with fat suppression. The largest cancer diameter was measured with each imaging technique and compared to the largest cancer diameter measured at pathology.At pathological examination cancers ranged from 0.6 to 6 cm (mean 2.2) in largest diameter. MRI measurements had the highest correlation coefficient (r = 0.98) and the smallest standard error (0.34). Ultrasonography measurements had a correlation coeffient of r = 0.45 and a standard error of 0.78. Mammography measurements had a correlation coefficient of r = 0.46 and a standard error of 1.04.MRI was more accurate than ultrasonography and mammography in measuring the largest cancer diameters in this group of women. This was particularly evident for several larger cancers, and a postchemotherapy cancer.

An occult breast cancer was detected and wire localization performed using magnetic resonance imaging.

Introduction Possible environmental effects on the development of HER2+ breast cancer have received relatively little research attention. Here we used the well-established MMTV-neu mouse model, which overexpresses mammary ErbB2 (HER2-neu) and stochastically develops primary tumors over 6-12 months, to test the hypothesis that chronic exposure to environmental stressors may result in an earlier onset of tumors. Experimental Procedures Female mice (FVB/N-Tg(MMTVneu)202Mul/J) were purchased from Jackson Labs at age 4-5 weeks, housed in standard cages in an isolation chamber in a controlled-access animal room, and randomly assigned to one of four experimental exposure conditions in a factorial design that was maintained for the course of the study: 1) Restraint stress (RS) 90 min - 1x/wk (n=24); 2) Social disruption stress (SD) (via cage mate switching across home cages) 2x/wk (n=23); 3) RS&SD (n=22); 4) home cage control (HC)(n=20). Mice were visually inspected and palpated for tumor development 1x/wk (blinded as to treatment group). All mammary glands from each mouse were processed for histologic evaluation one week following positive tumor palpation. All palpated tumors were histologically confirmed by a pathologist's evaluation of H&E sections (blinded as to treatment group). Results Consistent with the study hypothesis, the results indicated that mice chronically exposed to the two environmental stressors (RS&SD) developed primary tumors at a significantly (p Conclusions Exposure to environmental stressors may speed the development of tumors in this transgenic model of HER2+ breast cancer. These novel findings suggest the importance of further investigation to determine the mechanisms responsible for these effects of exposure to environmental stressors. (Support: CA120795) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3270.

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.

Estrogen (ER) and progesterone (PgR) receptor values in 105 endometrial carcinomas were compared using immunocytochemical and standard biochemical techniques. Peroxidase-antiperoxidase staining for location of anti-ER (H222) and anti-PgR (JZB39) primary antibodies was used to generate a semiquantitative (HSCORE) assessment of receptor content in tissue components and the total specimen. Both total HSCORE and cancer component HSCORE correlated with log biochemical assay values for ER and PgR. Biochemical assay values, total HSCORE, and cancer component HSCORES all demonstrated internal correlations between ER and PgR levels. Correlation was somewhat closer for cancer component HSCORE values of ER and PgR than the values for total tissue HSCORE. When receptor content was analyzed by histologic grade, all three estimates of receptor status demonstrated a decreasing proportion of ER and PgR positive lesions with decreasing histologic differentiation; however, the proportion of receptor negative lesions in grade 3 lesions was much higher when using total HSCORE or cancer component HSCORE than when using biochemical assay values (P less than 0.005. Immunocytochemical techniques for localization of ER and PgR in endometrial carcinoma specimens may allow a more focused evaluation of the receptor content in the malignant elements than standard biochemical techniques.

2547 Background: Yttrium-90 ibritumomab tiuxetan (90Y IT, Zevalin) is effective treatment for relapsed or refractory low-grade, follicular, or transformed B-cell NHL although affected lymph nodes m...

Ytrrium 90 (90Y) ibritumomab tiuxetan (Zevalin®) is indicated for the treatment of relapsed or refractory low-grade, follicular, or transformed B-cell NHL at relapse or upon confirmation of refractory disease. Long-term responses in excess of 6 years have been observed following ibritumomab tiuxetan administration, underscoring the efficacy of this selective treatment modality. Dosing guidelines for 90Y ibritumomab tiuxetan were established in phase 1/2 trials and are dependent on body mass and platelet count, with the maximum recommended dose being 32 mCi. Biodistribution is evaluated by whole-body imaging prior to the delivery of the therapeutic dose using the gamma emitter indium 111 (111In) as the imaging radioimmunoconjugate. Current imaging methodology for the ibritumomab tiuxetan regimen has several limitations. First, the correlation between 111In ibritumomab tiuxetan dosimetry and either toxicity or tumor response is poor, and clinical parameters such as platelet count, patient weight, and percentage lymphomatous bone marrow involvement have been much more accurate. Further, while gamma (or PET/SPECT) imaging provides a visual evaluation of uptake in the blood pool and relevant organs, it cannot resolve biodistribution to the cellular level. This is particularly relevant for discriminating between radioisotope uptake in malignant and nonmalignant tissues. In order to assess the uniformity of cellular localization of 90Y ibritumomab tiuxetan, we performed autoradiographic analyses of lymph node tissue and bone marrow sampled after ibritumomab tiuxetan therapy. We also proposed to semi-quantify the energy doses delivered to lymphomatous tissue in an effort to better understand mechanisms of cellular sensitivity or resistance to this form of radioimmunotherapy. Following standard delivery of the ibritumomab tiuxetan regimen, bone marrow and lymph node tissues were sampled from a patient who presented with CD20+ NHL, bulky peripheral lymph nodes, and positive bone marrow involvement. Samples were collected 4 days after the administration of 90Y ibritumomab tiuxetan and immediately processed. Prepared sections were stained with hematoxylin and eosin (H&E) for histologic examination and then submitted for autoradiographic preparation with Kodak NBT-3 nuclear emulsion at 42o C, Kodak D-19 developer, and sodium thiosulphate fixative. Within lymph node tissue, radioisotope uptake was preferentially localized to lymphoma cells. An absence of significant localization in the histologically normal sections of bone marrow was also noted. The observed distribution patterns in the lymph node suggested that distribution of 90Y ibritumomab tiuxetan was localized to the cell membrane of lymphoma cells, with limited stromal and intravascular involvement. We are currently quantifying the density of radioisotope aggregation within malignant cells to assess whether a sufficient quantity of nuclide is recruited to achieve a crossfire effect on neighboring, unlabeled cells. Our results confirm that in vivo,90Y ibritumomab tiuxetan selectively targets tumor tissue with little binding to normal bone marrow and lymph node tissue, including stroma and vasculature. Additional patients with CD20+ NHL are being studied to verify the cellular localization pattern of 90Y ibritumomab tiuxetan.

The cytosolic estrogen receptor (CER) content of 1037 primary breast carcinomas was evaluated by sucrose density gradient analysis. Tumor specimens from premenopausal patients had significantly lower levels of CER (14.6 +/- 1.5 (mean +/- SEM) 8S binding fmole/mg protein) compared with carcinomas from postmenopausal patients (57.5 +/- 3.9 fmole/mg protein; p less than 0.001). The proportion of specimens with CER levels above threshold values of 3, 7, or 10 fmoles/mg protein were significantly higher for postmenopausal patients (72%, 63%, 59%, respectively) than for premenopausal patients (56%, 42%, 36%, p less than 0.001). When compared within half-decades, no statistically significant differences between premenopausal and postmenopausal patients were observed for mean, median, or rank sums of CER levels (p greater than 0.3). When patients were compared by half-decades, both mean and ranked sums of CER levels were significantly different (p less than 0.001). The proportion of specimens that demonstrated CER levels above a threshold value of 10 fmole/mg protein increased sequentially from a low of 13/51 (26%) for patients less than 35 years to a high of 60/81 (74%) for patients greater than 75 years.

The production of adrenocorticotropic hormone by non-pituitary carcinomas has been documented in several tumors, most frequently small cell carcinoma of the lung, islet cell carcinomas of the pancreas, thymomas and carcinoids. Electron microscopy of these tumors reveals typical membrane-limited "neurosecretory" granules. Confirmation of the granules as adrenocorticotropin (ACTH) requires the use of OsO4 as a primary fixative to give the characteristic cored granule appearance in conjunction with immunohistochemical demonstration of the hormone peptide. Because of the rarity of ectopic ACTH production by mammary carcinomas and the absence of appropriate ultrastructural studies in the two examples of such ectopic hormone production in the literature of which we are aware (1,2), we present biochemical and ultrastructural data from a carcinoma of the breast with apparent ACTH production.The patient had her primary tumor in the right breast in 1969. The tumor recurred as visceral and subcutaneous metastases in 1976 and again in 1977.

This study corroborates previous reports which suggested the efficacy of estrogen receptor (ER) analysis in predicting responses of patients with metastatic mammary carcinoma to hormonal therapeutic manipulation. The predictive value of multiconcentration titration and sucrose density gradient analyses of ERs and progesterone receptors (PRs) are compared. The predictive value of ER analyses can be improved by the discrimination of 8S versus 4S binding species or by the use of PR analysis in combination with ER analysis. The tumor-associated antigen, carcinoembryonic antigen (CEA), is evolving as an important quantitative aid in evaluating the clinical responses of patients receiving hormonal therapy.

An unusual site of metastasis for mammary carcinoma is presented with an examination of the tumor's histologic and ultrastructural features, as well as its estrogen and progesterone receptor protein content. The importance of careful examination of the tonsillar area is emphasized in this patient in whom the tonsillar metastasis represented the initial indication of breast tumor recurrence. Estrogen and progesterone receptor analyses are utilized both as an aid in identifying the origin of the metastatic tumor focus as well as an aid in selecting therapy.A case report and an ultrastructual study are published together to elucidate an unusual site of metastasis for mammary carcinoma. A 33-year-old American Indian mother presented with a left tonsillar mass; 11 months before she had undergone a right simple mastectomy for an apparently localized carcinoma of the breast. Biopsy material from the tonsillar mass contained carcinoma, and an axillary mass was subsequently excised and noted to contain similar tumor. After oophorectomy, the patient experienced remission of her bone pain and reduction in liver size. 5 figures form the bulk of the ultrastructural study. Histologically the tumor from the original mastectomy represented a medullary variant of infiltrating ductal carcinoma of the breast; the specimen was characterized by sheets of large bizarre, pleomorphic cells with hyperchromatic, pleomorphic nuclei and frequent mitosis. Only slight glandular differentiation of scirrhous reaction was apparent. The tonsillar biopsy contained several nodules of metastatic carcinoma with minimal adenomatous differentiation, bisicular nuclei, and prominent nuclei. Ultrastructurally, both metastatic foci of tumor were similar and characterized as adenocarcinomas with well-developed endoplasmic reticulum, cytoplasmic vacuolization, osmophilic granules, intracellular canaliculi with microvilli, numerous desmosomes, large nuclei with marginated nuclear chromatin, and enlarged nuceoli. Mitotic figures were abundant.

Basal cell carcinoma rarely metastasizes. There are over 130 reported cases, 70% of which involve lymph nodes. In many cases a large, chronic, neglected or inadequately treated basal cell carcinoma preceded the metastasis. We report a case in which a basal cell carcinoma of the cheek metastasized to cervical lymph nodes.

Cystosarcoma phyllodes is an unusual breast neoplasm that rarely metastasizes. Most series report chemotherapy, radiation, and hormonal therapy to be ineffective. Three patients were treated with cisplatin and etoposide combination chemotherapy with effective palliation in two patients. Radiation therapy was effective in controlling symptomatic metastasis in all three patients. Hormonal therapy was ineffective in two patients despite the presence of positive hormone receptors. Chemotherapy and radiotherapy may be more effective in the treatment of this tumor than has been reported, although there is no apparent role for hormonal therapy. Functional hormone receptors are probably not present.

SynopsisThe human breast undergoes profound changes during the menstrual cycle. The development of monocloiial antibodies to oestrogen-receptors (ER) allows study of receptor content and distribution in individual components of breast tissue. Forty-one specimens from premenopausal patients who underwent bilateral mastectomy for benign conditions were evaluated. The patients were categorised into early follicular, late follicular, early luteal, late luteal, and menstrual phases. An HSCORE, a semiquantitative evaluation of the intensity and distribution of receptor antigen localisation, was evaluated for the ductal and lobular epithelia as a tissue mean HSCORE. ER localised to the nuclei of the epithelium and was not observed in stromal elements. Receptor concentration was greater for ER in the follicular phase but this finding was not as striking as differences between lobules in a single breast and even between breasts. This heterogeneous expression between lobules was in contrast to the relatively uniform expression of receptor within a lobule of a given specimen.

Using direct immunofluorescence, lesions from 266 human breast specimens were studied for the presence of IgA, IgM, or IgG localization. The lesions included benign elements from 66 subcutaneous mastectomy specimens in which the absence of simultaneous breast malignancy was documented, primary breast carcinomas from 153 mastectomy specimens, and 47 biopsies containing metastatic breast cancer. A statistically significant association of IgA and IgM with benign lesions was contrasted to the association of IgG with malignant lesions. In both primary and metastatic lesions, IgG localization was associated with estrogen-receptor-poor primary cancers as compared with estrogen-receptorrich primary cancers. Among primary breast cancer patients, IgG localization in the tumor correlated with relative lymphopenia. A shorter disease-free interval was noted in association with IgG localization among the metastatic breast lesions. No statistically significant association between stage of disease and immunoglobulin presence was demonstrable. Moderate-to-severe intraductal epithelial hyperplasias were more often associated with immunoglobulin G localization than were other benign lesions. Cancer 48:69-75, 1981. EVERAL STUDIES have suggested a role of the host S immune response in the natural history of mammary car~inoma.~~,~~ The breast is a component of the secretory immune system, concentrating immunoglobulins in breast A differential concentration of immunoglobulins in benign vs. malignant lesions of the breast has been suggested.s,20,21 The accumulation of immunoglobulin A (IgA) and immunoglobulin M (IgM) in the mammary gland have been shown to be induced by hormones.23 In the present study the localization of IgA, IgG, and IgM observed in various breast lesions was compared with the estrogen receptor content of the tissues. Benign and malignant lesions were examined to determine if immunoglobulin localization may characterize primary and metastatic breast cancers as contrasted to benign breast lesions.

Confusion exists concerning the influence of pregnancy on survival in patients with malignant melanoma. To evaluate this problem a retrospective computer-aided study was performed of women in the child-bearing years treated for Stage I cutaneous melanoma at the Duke University Comprehensive Cancer Center. Fifty-eight women were identified who had melanoma arise during pregnancy (Group 1) and 43 patients were noted who became pregnant within 5 years of diagnosis of their melanoma (Group 2). Appropriate control groups matched for the clinical variables of age, primary site, and stage of disease and the pathologic variables of Clark's Level, tumor thickness, ulceration, and histologic type were selected from the cohort of 2938 melanoma patients seen at Duke. Actuarial survivals for Group 1 and 2 patients did not differ from their respective controls, although the small number of deaths in each group resulted in wide confidence intervals. When actuarial disease-free intervals were plotted, there was a significant difference beween women who had melanoma develop during pregnancy when compared to their controls (P = 0.04). In a multivariate regression analysis, after adjustment for the influence of the more significant prognostic factors for Stage 1 melanoma, including Clark's Level, ulceration, and tumor thickness, the effect of pregnancy on disease-free interval became more apparent (P = 0.02). No difference in actuarial disease-free interval was noted in the melanoma patients who elected to become pregnant within 5 years of diagnosis (P = 0.31). A multivariate regression analysis confirmed this finding. These data indicate that although an intercurrent melanoma during pregnancy has a worse prognosis than the control groups, once a woman has been diagnosed as having a cutaneous melanoma, a subsequent pregnancy has no effect on recurrence rate or survival. Cancer 55:1340-1344, 1985.

3 histochemical techniques for estrogen binding localization in tissue section were compared to sucrose density gradient analyses for estrogen receptor in 186 breast carcinomas. Apparent localization to epithelial elements was noted using 6-BSA-Fluor-CMO-17-beta-estradiol 17-BSA-Fluor-TSC-estrogen and polyestradiol phosphate/anti-estradiol antibody methods. The correlation between the histochemical methods and standard sucrose density gradient techniques was poor for the polyestradiol antibody method and the 6-BSA-Fluor-CMO-17-beta-estradiol technique. While an improved correlation was observed with the 17-BSA-Fluor-TSC-estrogen compound the compounds binding was not effectively blocked by preincubation with diethylstilbestrol. This failure to show convincing saturability of binding combined with problems of extraction of soluble receptor proteins in the aqueous incubation media and washes from the cells rendered permeable by cryostat sectioning indicates that several areas will require clarification before histochemical techniques can begin to be considered as a method for estrogen "receptor" analysis in the clinical evaluation of breast neoplasms. (Authors modified)

A subclone of Chinese hamster ovary cells, R40F, has been selected for its unusually high resistance to lethal concentrations of Cd and Zn. Although there is a 33% loss in Cd resistance when R40F cells are cultured in the absence of exogenous metals, the Zn resistance remains unaltered. These cells are 80% tetraploid and demonstrate an increased capacity for metallothionein protein synthesis. When compared to wild type cells cultured in the absence of exogenous metals, R40F cells exposed to 200 M Cd for 48 h exhibited an approximate 200-fold increase in metallothionein-I (MT-I) protein. A 32P-labeled mouse MT-I cDNA was employed in solution hybridization studies to measure the level of MT-I mRNA in wild type and R40F cells. Cd (0.5 M) induces MT-I mRNA about 2.5- and 5-fold in wild type and resistant Chinese hamster ovary cells, respectively. When optimally induced, the resistant cells have about 80-fold more MT-I mRNA than the sensitive cells. Southern blot analysis of HincII-cleaved DNA indicates that the MT-I gene is amplified approximately 60- to 75-fold in R40F cells.

The LH/hCG receptor content of porcine ovarian granulosa cells from 1- to 3-mm follicles can be increases to 4--5 times the preculture level during monolayer culture in serum-containing media supplemented with insulin and FSH. The binding of [125I]iodo-hCG declines during the first 2 days of culture, but then uniformly increases through 6 days, achieving a 14- to 15-fold increase relative to the 2-day level under optimal conditions. Analysis of receptor binding by autoradiography indicates that after 2 days, the number of cells specifically binding [125I]iodo-hCG increases significantly during culture, as does the intensity of binding on receptor-bearing cells. Granulosa cells in monolayer culture exhibit heterogeneous receptor induction, indicating that normalized [125I]iodo-hCG binding data cannot be used to estimate receptor concentration per cell. Receptor affinities in the initial and induced populations are identical. LH/hCG receptors induced in granulosa cells during culture are functional, as demonstrated by specific hCG-stimulated progesterone secretion. 17 beta-Estradiol produces a differential effect in vitro, generally increasing [125I]iodo-hCG binding with respect to FSH-induced levels but consistently depressing the subsequent hCG-stimulated steroidogenic response of cells bearing the induced receptor. The porcine granulosa cell monolayer system thus appears to be a useful model with which to study, in vitro, mechanisms of steroid and gonadotropin regulation of granulosa cell differentiation and overall follicular development.

In an attempt to clarify further the relationship between ovarian function and uterine leiomyomas, we investigated the cytoplasmic estrogen and progesterone receptor content in these uterine tumors. The study population consisted of 17 women of reproductive age at various stages of ovulatory menstrual cycles. Serum samples were analyzed for estrogen and progesterone content; tissue samples from endometrium, myometrium, and leiomyomas were analyzed for cytoplasmic estrogen and progesterone receptor content. The levels of cytoplasmic estrogen and progesterone receptor in leiomyomas demonstrated the same quantitative and qualitative cyclic changes throughout the menstrual cycle apparently in response to variations in circulating estrogen and progesterone as found in endometrium and myometrium.

Histologic review of 422 specimens from endometrial carcinoma submitted for biochemical cytosol estrogen and progesterone receptor analysis revealed that 16 (4.0%) contained no evidence of carcinoma on permanent histologic sections. An additional 11 (2.5%) contained focal carcinomas on permanent sections but had no evidence of malignancy in frozen sections of the tissue submitted for receptor analyses. Despite the paucity or even absence of malignancy, many of these specimens had significant cytoplasmic estrogen and progesterone receptors derived from endometrial and myometrial tissues. Rigorous histologic control of specimens from endometrial carcinomas submitted for biochemical steroid receptor analyses is necessary to establish valid clinical and histologic associations of steroid receptor content.

Several studies have demonstrated that mammographic screening of asymptomatic women results in a lower mortality rate where breast cancer is concerned. Often, screening mammograms reveal a nonpalpable radiographic abnormality and the diagnosis must be determined by an excisional biopsy after radiographic needle localization. The mammographic features associated with 179 nonpalpable breast abnormalities biopsied after radiographic needle localization were carefully characterized. There were 41 carcinomas (23%) in the series. The aim of this study was to determine which radiographic findings, if any, strongly portend the presence of either a malignant or benign lesion. Mammographic features that were commonly associated with malignancy include a change from a previous mammogram, a distortion of the surrounding architecture, the association of a soft tissue density and calcifications, and the presence of more than ten calcifications in the lesion. The radiographic abnormalities which were more commonly associated with benign disease include well-defined densities without calcifications, asymmetric densities without calcifications, and abnormalities consisting solely of a focus of mammographic calcifications that have fewer than ten concretions. The incidence of malignancy in lesions having these mammographic characteristics was only 5.5%. On the basis of these results alone, no firm threshold for biopsy can be recommended. The risks of deferring biopsy until there is worsening of the mammographic image remains to be determined.

The role of estrogens in the development of skin cancer is controversial. Sex steroids have a profound effect on the epidermis and epidermal appendages. Estradiol in pharmacologic doses has been reported to stimulate basal cell carcinoma in an animal model. Sex hormones act by means of a specific protein receptor. In this study we used a specific, highly sensitive monoclonal antibody to evaluate sex steroid receptors in human basal cell carcinoma. No estrogen or progesterone receptor protein was detected in the basal cell tumor, despite clear positive control tissues. We conclude that these sex steroid receptors are not present in significant amounts to mediate a direct effect in basal cell carcinoma.

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.

Fifty patients undergoing immediate reconstruction of the breast following modified radical mastectomy for carcinoma were characterized by their clinical findings and the pathologic features of their tumors. Evidence of nipple--areolar involvement by tumor was noted in five patients. This high incidence mitigates against nipple--areolar transplantation. In ten patients the breast contained multifocal tumor or intraductal carcinoma away from the primary tumor, thus emphasizing the importance of careful evaluation of the total tissue specimens. Four patients have had recurrence of tumor. The detection of the recurrence was not hindered by the presence of the prosthesis. In these four patients immediate reconstruction of the breast did not appear to adversely affect the natural history of the breast cancer. We suggest that when appropriate and thorough surgical extirpation followed by reconstruction is undertaken as a team effort among the general and plastic surgeons, pathologist and clinical psychologist, the optimal clinical and cosmetic results may be achieved. However, conclusive statements regarding the long-term implications of immediate reconstruction awaits additional follow-up.

Menstrual cycle dependent variations in cytosol levels of estrogen receptor (ER), progesterone receptor (PR), and two breast cyst fluid glycoproteins, gross cystic disease fluid protein (CDP) and nonreceptor progesterone binding protein (PBP), were demonstrated in epithelial-enriched “normal” breast tissues from 56 premenopausal women. Criteria for normalcy were: (1) normal menstrual history; (2) absence of drug use or debilitating disease; (3) normal adrenal, ovarian and pituitary function; and (4) no clinical, gross or histologic evidence of breast disease. Highest mean levels of CDP (8180 ± 2850 ng/mg protein) and PBP (17750 ± 6320 ng/mg protein) were noted during the follicular phase (days 8–14) of the menstrual cycle, while lowest levels of CDP (450 ± 260) and PBP (1810 ± 380) were observed during the luteal phase (days 15–20). Both CDP and PBP had peak values on days 10–12 with a smaller peak on days 20–23. The number of samples with ER > 3 fmole/mg protein (8/14; 57%) and mean ER level (7.5 ± 2.9 fmole/mg protein) were significantly higher during the proliferative phase (days 3–7) than during other menstrual phases. Maximum ER values occurred on days 5–8. The greatest number of samples with PR > 3 fmole/mg protein (5/15, 33%) and highest mean levels of PR (36 ± 27.8 fmole/mg protein) were noted during the follicular phase of the menstrual cycle. These data demonstrate that the normal human breast has menstrual cycle variations of CDP and PBP which may be useful as markers of sex steroid receptor integrity. These data also documented that appreciable ER levels were found during the proliferative phase with low levels at other times in the menstrual cycle.