Rosalyn D. Blumenthal

Expression patterns of CEACAM5 and CEACAM6 in primary and metastatic cancers

BackgroundMany breast, pancreatic, colonic and non-small-cell lung carcinoma lines express CEACAM6 (NCA-90) and CEACAM5 (carcinoembryonic antigen, CEA), and antibodies to both can affect tumor cell growth in vitro and in vivo. Here, we compare both antigens as a function of histological phenotype in breast, pancreatic, lung, ovarian, and prostatic cancers, including patient-matched normal, primary tumor, and metastatic breast and colonic cancer specimens.MethodsAntigen expression was determined by immunohistochemistry (IHC) using tissue microarrays with MN-15 and MN-3 antibodies targeting the A1B1- and N-domains of CEACAM6, respectively, and the MN-14 antibody targeting the A3B3 domain of CEACAM5. IHC was performed using avidin-biotin-diaminobenzide staining. The average score ± SD (0 = negative/8 = highest) for each histotype was recorded.ResultsFor all tumors, the amount of CEACAM6 expressed was greater than that of CEACAM5, and reflected tumor histotype. In breast tumors, CEACAM6 was highest in papillary > infiltrating ductal > lobular > phyllodes; in pancreatic tumors, moderately-differentiated > well-differentiated > poorly-differentiated tumors; mucinous ovarian adenocarcinomas had almost 3-fold more CEACAM6 than serous ovarian adenocarcinomas; lung adenocarcinomas > squamous tumors; and liver metastases of colonic carcinoma > primary tumors = lymph nodes metastases > normal intestine. However, CEACAM6 expression was similar in prostate cancer and normal tissues. The amount of CEACAM6 in metastatic colon tumors found in liver was higher than in many primary colon tumors. In contrast, CEACAM6 immunostaining of lymph node metastases from breast, colon, or lung tumors was similar to the primary tumor.ConclusionCEACAM6 expression is elevated in many solid tumors, but variable as a function of histotype. Based on previous work demonstrating a role for CEACAM6 in tumor cell migration, invasion and adhesion, and formation of distant metastases (Blumenthal et al., Cancer Res 65: 8809–8817, 2005), it may be a promising target for antibody-based therapy.

Combined gemcitabine and radioimmunotherapy for the treatment of pancreatic cancer.

MAb‐PAM4 is an anti‐MUC1 antibody that has been shown to be reactive with 85% of pancreatic adenocarcinomas with no reactivity with normal pancreas or other tissues. Initial clinical studies have shown excellent targeting with high tumor/nontumor ratios. Gemcitabine, an analog of deoxycytidine, is currently a frontline treatment for pancreatic cancer. Acting via a number of metabolic pathways, gemcitabine is also a powerful radiosensitizer. Combined‐modality, chemo/radiosensitization with gemcitabine and low dose 131I‐PAM4 radioimmunotherapy was performed to determine if a more effective treatment procedure could be developed. Athymic nude mice bearing large (1 cm3) CaPan1 human pancreatic tumors were given a single treatment cycle consisting of gemcitabine, 333 mg/m2 administered on Days 0, 3, 6, 9 and 12 by intraperitoneal injection, along with either 100 or 200 μCi, 131I‐PAM4 administered on Day 0 by intravenous injection. Gemcitabine did not interfere with the biodistribution of radiolabeled antibody. Specific tumor targeting was observed for 131I‐PAM4, with a tumor/blood radiation dose ratio of 2.6 over the first 14 days. Gemcitabine alone and low dose radioimmunotherapy alone, each had no affect upon tumor growth; no statistical differences were noted in comparison to the untreated group. When combined, however, a statistically significant (p = 0.0324), synergistic anti‐tumor effect was observed. Median survival time doubled for the combined treatment regimen compared to single modality treatment groups. The combined treatment modality was well tolerated by the mice. Our data show that combined gemcitabine with radioimmunotherapy may provide an improved alternative for the treatment of pancreatic cancer, achieving successful anti‐tumor effects with low toxicity.

Altered tumor vessel maturation and proliferation in placenta growth factor-producing tumors: Potential relationship to post-therapy tumor angiogenesis and recurrence

Cells in human tumor xenografts express similar levels of angiogenic growth factors before treatment. After radioimmunotherapy (RAIT) surviving tumor cells upregulate angiogenic growth factors, including placenta growth factor (PlGF), in a tumor‐specific pattern. To determine the role of post‐treatment PlGF expression on blood vessel recovery, tumor xenografts were assayed for post‐RAIT vessel density (CD34+), proliferation (PCNA+) and maturity (SMA+ pericytes/mural cells). To further analyze the role of PlGF in blood vessel formation, PlGF‐containing Matrigel implants were also assessed in a similar manner. The xenografts producing post‐treatment PlGF increased CD34+ microvessel density 2‐ to 4‐fold over untreated controls (p < 0.05) within 3 weeks of RAIT treatment. The proportion of mature microvessels (SMA+) decreased. Pericyte coverage and density of microvessels remained stable in the tumor that expressed neither PlGF nor VEGF after treatment. Hemoglobin content of PlGF‐containing Matrigel implants was 5.7‐fold that of anti‐PlGF/anti‐VEGF treated controls (Day 6, p < 0.03). The vessel density in PlGF‐implants averaged 36.8 ± 10.6/mm compared to 4.9 ± 6.5/mm2 in controls (p < 0.001). Vessels of PlGF‐implants were lined by vWF+ cells, which were mostly flt‐1+. These findings point to a role for PlGF in rapid restoration of tumor blood supply after treatment and thus, to enhanced likelihood of tumor regrowth. Likewise, the cells of primary human tumors that upregulate PlGF after treatment may be more likely to survive and form recurring tumors. Prevention of this angiogenic response to treatment may require administration of anti‐angiogenic therapy during, rather than after, treatment.

Methods and goals for the use of in vitro and in vivo chemosensitivity testing

Sensitive, specific, and accurate methods to assay chemosensitivity are needed to (1) screen new therapeutic agents, (2) identify patterns of chemosensitivity for different tumor types, (3) establish patterns of cross-resistance and sensitivity in treatment of naïve and relapsing tumors, (4) identify genomic and proteomic profiles associated with sensivity, (5) correlate in vitro response with preclinical in vivo effects and clinical outcomes for a particular therapeutic agent, and (6) tailor chemotherapy regimens to individual patients. Various methods are available to achieve these end points, including several in vitro clonogenic and proliferation assays, cell metabolic activity assays, molecular assay to monitor expression of markers for responsiveness, drug resistance, and for induction of apoptosis, in vivo tumor growth and survival assays in metastatic and orthotopic models, and in vivo imaging assays. The advantages and disadvantages of the specific assays are discussed. A summary of research questions related to chemosensitivity testing is also included.

Degranulating Eosinophils in Human Endometriosis

Degranulating eosinophils have been described in most endometrial cancers. We hypothesized that endometriosis (ectopic, nonneoplastic endometrial tissue) would be an appropriate model system for determining whether eosinophil degranulation is part of a specific immune response to endometrial cancer or if it is related to the more general phenomenon of tissue remodeling (wound healing) that is common to both disorders. To test this hypothesis, we performed immunohistochemistry and Western blotting to evaluate the presence of eosinophil peroxidase (a marker of eosinophil degranulation) in normal endometrium (n = 20) and endometriosis samples (n = 24) and to define the coexpression of three eosinophil chemoattractants: interleukin-5 (IL-5), eotaxin, and regulated on activator-normal T cell expressed and secreted (RANTES). There was focally intense deposition of eosinophil peroxidase in the fibrotic connective tissue and blood vessels of 21 of 24 human endometriosis specimens; two samples showed weak staining, and only one tissue was negative for eosinophil degranulation. None of the 10 normal proliferative endometrial specimens had evidence of eosinophil degranulation, and four of 10 secretory tissues stained only weakly for eosinophil peroxidase. The presence of degranulating eosinophils was also associated with the presence of eotaxin and IL-5 in some samples and with RANTES in others. We conclude that the abundant presence of degranulating eosinophils in the fibrous regions of endometriosis supports the interpretation that eosinophils are involved in general tissue remodeling and wound healing rather than a tissue-directed immune response.

Potential of Peroxisome Proliferator-Activated Receptor Gamma Antagonist Compounds as Therapeutic Agents for a Wide Range of Cancer Types

PPARγ is a therapeutic target that has been exploited for treatment of type II diabetes mellitus (T2DM) with agonist drugs. Since PPARγ is expressed by many hematopoietic, mesodermal and epithelial cancers, agonist drugs were tested and shown to have both preclinical and clinical anticancer activities. While preclinical activity has been observed in many cancer types, clinical activity has been observed only in pilot and phase II trials in liposarcoma and prostate cancer. Most studies address agonist compounds, with substantially fewer reports on anticancer effects of PPARγ antagonists. In cancer model systems, some effects of PPARγ agonists were not inhibited by PPARγ antagonists, suggesting noncanonical or PPARγ-independent mechanisms. In addition, PPARγ antagonists, such as T0070907 and GW9662, have exhibited antiproliferative effects on a broad range of hematopoietic and epithelial cell lines, usually with greater potency than agonists. Also, additive antiproliferative effects of combinations of agonist plus antagonist drugs were observed. Finally, there are preclinical in vivo data showing that antagonist compounds can be administered safely, with favorable metabolic effects as well as antitumor effects. Since PPARγ antagonists represent a new drug class that holds promise as a broadly applicable therapeutic approach for cancer treatment, it is the subject of this review.

Peroxisome proliferator-activated receptor-?? antagonists exhibit potent antiproliferative effects versus many hematopoietic and epithelial cancer cell lines

Peroxisome proliferator-activated receptor-&ggr; ligands have preclinical and clinical anticancer activity. Most studies in this area address agonists, with relatively few reports on anticancer effects of peroxisome proliferator-activated receptor-&ggr; antagonists. Thus, we evaluated the two pure peroxisome proliferator-activated receptor-&ggr; antagonists, T0070907 and GW9662, on a panel of hematopoietic and epithelial cell lines. The peroxisome proliferator-activated receptor-&ggr; antagonists and a reference agonist (pioglitazone) were tested in an in-vitro proliferation assay on a panel of seven hematopoietic and nine epithelial cancer cell lines, some of which are chemoresistant. Peroxisome proliferator-activated receptor-&ggr; expression was measured by immunoblotting, as was the effect of treatment with these agents on peroxisome proliferator-activated receptor-&ggr; levels. The effect of exogenous interleukin-6, an antiapoptotic cytokine, on growth inhibition was evaluated as well as the apoptotic effects of these drugs. The peroxisome proliferator-activated receptor-&ggr; antagonists showed significantly greater potency on all cell lines (IC50s of 3.2–29.7 versus 26.5–78.7 μmol/l for pioglitazone) and greater maximum growth inhibition. Peroxisome proliferator-activated receptor-&ggr; levels did not correlate with growth inhibition in this panel of cell lines. Combinations of peroxisome proliferator-activated receptor-&ggr; antagonists and the agonist actually showed schedule-dependent increases in growth inhibition. Exogenous interleukin-6 did not induce resistance to these agents. Both the antagonists and the agonist induced apoptosis, but only the former drugs showed caspase dependence. These two peroxisome proliferator-activated receptor-&ggr; antagonists have significantly more potent in-vitro antiproliferative effects versus hematopoietic and epithelial cancer cell lines. This effect does not correlate with peroxisome proliferator-activated receptor-&ggr; levels, suggesting alternative mechanisms or other targets of action. These findings support further translational studies to explore the mechanism of action and therapeutic potential of this class of agents.

Optimizing the use of combined radioimmunotherapy and hypoxic cytotoxin therapy as a function of tumor hypoxia

Combined radioimmunotherapy (RAIT) and hypoxic cytotoxin therapy (SR4233 or NLCQ‐1) have been evaluated with both modalities administered on the same day with only moderate improvement compared with the effects of RAIT alone. In a series of studies using oxygen electrodes, immunohistochemistry and radiotracers, we have demonstrated that RAIT induces a prolonged state of hypoxia in most tumors, without affecting the pO2 levels in normal tissues. Using serial microelectrode measurements through subcutaneous (s.c.) GW‐39 human colonic xenografts, we established that the median pO2 was unrelated to the initial size of the tumor, over a range of sizes from 1.0 to 4.0 cm. Fourteen days after mice were given a 240‐μCi dose of 131I‐MN‐14 anti‐carcinoembryonic antigen immunoglobulin G, their median pO2 declined from 26.1 ± 9.6 mmHg to 9.8 ± 3.9 mmHg (p < 0.001). Using the radiotracer 3H‐MISO that accumulates in hypoxic regions, uptake in GW‐39, LoVo and LS174T s.c. human colonic tumors increased 3.0‐ to 4.2‐fold from day 14 through day 28 post‐RAIT, but uptake of 3H‐MISO in CALU‐3 tumors remained unchanged after RAIT. Normal tissue (liver, kidney, lung) uptake of 3H‐MISO did not exhibit significant changes. The increase in tumor hypoxia was also demonstrated visually using anti‐PIMO staining of tumor sections. We postulated that sequential delivery of the 2 therapeutic agents, with the hypoxic cytotoxin given 2 weeks after RAIT when tumor pO2 levels were at their nadir, would improve the therapeutic response above either modality alone or above the 2 agents delivered on the same day. Tumor growth was compared in mice given either RAIT or cytotoxin alone, the combined treatment on the same day or with the cytotoxin delivered 14 days after RAIT. Tumor size on day 35 for RAIT‐treated and SR4233‐treated GW‐39 were 3.56 ± 0.40 and 7.98 ± 2.50 cm3. When RAIT + SR4233 were delivered on the same day, tumor size dropped to 2.78 ± 0.80 cm3. If RAIT was given on day 0 and SR4233 on day 14, size further declined further to 1.74 ± 0.32 cm3 (p < 0.05 compared with same day delivery). For LS174T, tumor size on day 28 for RAIT‐treated and SR4233‐treated tumors were 1.14 ± 0.36 cm3 and 3.65 ± 0.78 cm3, respectively. When RAIT + SR4233 were delivered on the same day, size was 0.51 ± 0.174 cm3. If RAIT was dosed on day 0 and SR4233 was given on day 14, tumor size was 0.13 ± 0.07 cm3 (p < 0.05). Similar results were obtained for LoVo, but not for CALU‐3 tumors. Another hypoxic cytotoxin, NLCQ‐1, was also more efficacious 2 weeks after RAIT, compared with same‐day dosing. Thus, information on tumor hypoxia after radioantibody therapy could be important for ascertaining a window of opportunity when the surviving tumor regions are most responsive to hypoxic cytotoxins.

Abnormal expression of the angiopoietins and Tie receptors in menorrhagic endometrium

OBJECTIVE To identify changes in expression of stimulatory and inhibitory factors when normal endometrium becomes menorrhagic. DESIGN Retrospective blinded immunohistologic study. SETTING Private research center. PATIENTS Premenopausal and postmenopausal women with non-menorrhagic and menorrhagic endometrium undergoing curettage or hysterectomy were selected. INTERVENTION(S) Samples of endometrium were obtained from all patients. MAIN OUTCOME MEASURE(S) Expression of angiopoietins 1 and 2, and vascular receptors Tie-1 and Tie-2 and endothelial nitric oxide synthase (eNOS). RESULT(S) Angiopoietin 1 (ANG-1) expression was similar in non-menorrhagic and menorrhagic endometrium. However, there was a significant increase in expression of ANG-2 and its receptor Tie-2 in menorrhagic tissues, which may be important in destabilizing the endometrial vasculature. Tie-1, responsible for endothelial integrity, was also increased in menorrhagic tissues, and is a likely compensatory mechanism for the existing vascular pathology. Expression of eNOS was also increased in menorrhagic women. CONCLUSION(S) Differences in the expression of ANG-2, Tie-2, Tie-1, and eNOS were found in menorrhagic endometrium, which may represent a new target for therapeutic intervention to correct menorrhagic conditions.

Characteristics of cyclic nucleotide dependent regulation of cytoplasmic E2 binders in cultured endometrial and breast cells

Estradiol binding levels have previously been shown to be increased or decreased by the addition of of cGMP or cAMP respectively to cellular homogenates prepared from human endometrial tissue or from the endometrial cell line HEC 1. Similar cyclic nucleotide dependent effects have now been demonstrated in homogenates prepared from a second endometrial cell line, HEC 50, and from two breast cancer cell lines, CG-5 and T47D. It was previously shown that ATP and Mg2+ are necessary for the generation or inactivation of E2 binding sites. The present study demonstrates that the concentration of K+ and of dithiothreitol can also affect these reactions.