Jose M. Esteban

Aromatase gene expression and its exon I usage in human breast tumors. Detection of aromatase messenger RNA by reverse transcription-polymerase chain reaction

The expression of aromatase in human breast tumors has been studied by the reverse-transcription polymerase chain reaction (RT-PCR) method on 70 breast tissue specimens. An RT-PCR analysis using two oligonucleotide primers derived from the exon II of the human aromatase gene revealed that aromatase mRNA was detected in all but three tissue specimens. Furthermore, primer-directed RT-PCR was performed to determine the exon I usage in aromatase mRNA in these breast tumor specimens. The analysis has revealed that exons I.3 and PII are the two major exon Is present in aromatase mRNA isolated from breast tumors, suggesting that promoters I.3 and II are the major promoters driving aromatase expression in breast cancer and surrounding adipose stromal cells. The RT-PCR analysis also detected two products, I.3A (334 bp in length) and I.3B (222 bp in length), when it was carried out using a primer derived from exon I.3 and a reverse primer derived from exon II. The nucleotide sequences of these products have been determined and indicate that I.3A contains a region which was previously thought to be an intron. In addition, RT-PCR analyses of RNA isolated from eight pairs of breast tumor and neighboring normal tissue specimens were performed to evaluate the exon I usage and the distribution of I.3A- and I.3B-containing aromatase RNA messages in breast tumor and neighboring normal tissues. The results suggest that I.3B- and I.3A-containing messages are mainly present in breast tumor and neighboring normal tissues, respectively. Finally, the exon I/promoter usage for aromatase expression in eight cell lines (skin fibroblast, MCF-7, MDA-MB-231, T-47D, SK-BR-3, JAR, OVCAR-3, and human adipose stromal cells) was examined by primer-directed RT-PCR analyses. These studies provide a basis for further evaluation of the control mechanism of aromatase expression and estrogen biosynthesis in breast tumors.

Endothelial area as a prognostic indicator for invasive breast carcinoma.

Vascular enumeration using antibodies to Factor VIII has been reported to be an independent prognostic indicator of invasive breast carcinoma.

Spontaneous regression of hepatocellular carcinoma

A surgically unresectable, biopsy‐proven hepatocellular carcinoma (HCC) developed in a 63‐year‐old man with cirrhosis. He survived 24 months without treatment. During that time the tumor burden decreased as demonstrated both radiologically and by the normalization of alpha‐fetoprotein levels. The patient died of complications secondary to repeated esophageal variceal hemorrhage. Necropsy demonstrated prominent, ulcerated esophageal varices and liver cirrhosis without evidence of neoplasia either grossly or on a subsequent thorough microscopic examination. This case represents the first confirmation of HCC spontaneous regression in which a primary histologic diagnosis was confirmed by immunohistochemical and flow cytometric DNA analysis, and where tumor regression was proven by a thorough necropsy examination.

Effect of yttrium‐90‐labeled anti‐carcinoembryonic antigen monoclonal antibody on the morphology and phenotype of human tumors grown as peritoneal carcinomatosis in athymic mice

Grossly visible peritoneal carcinomatosis resembling that seen in man was produced in athymic mice 7 days after intraperitoneal injection of 8 × 105 cells of the carcinoembrionic antigen (CEA)‐producing human colon carcinoma cell line LS174T. The mice received intraperitoneal injections of 40 to 160 μCi of yttrium‐90 (90Y)‐labeled anti‐CEA monoclonal antibody (MAb). When the mice were killed 12 days after injection, a significant inhibition of tumor growth, ranging from 40% to 95%, was observed in the treated animals when compared to the growth of tumors in the untreated animals (P < 0.001). No mortality secondary to the therapy was seen. The bone marrow was depleted significantly at the higher doses of labeled MAb, but total recovery was observed 4 weeks after treatment. Histologically, the treated tumors showed extensive radiation effects early in the posttherapy period and massive necrosis at later times. Minute foci of viable tumor remained in the periphery. New tumor outgrowths with histologic features similar to those in the untreated controls began to appear 3 weeks after therapy. The CEA expression of the treated tumors was similar to that of the untreated controls during the early posttreatment period, diminishing progressively as the tumors became necrotic. Newly grown tumor nodules in the treated animals lacked significant CEA expression both initially and at later times. Our studies suggest that therapy with 90Y‐anti‐CEA MAb therapy results in selection of tumor clones lacking CEA, and that a single large dose of 90Y‐MAb should be more effective than multiple fractions of smaller doses.

Effects of recombinant human gamma-interferon on carcinoembryonic antigen expression of human colon cancer cells.

The effects of human recombinant gamma-interferon (gamma-IFN) on the levels of carcinoembryonic antigen (CEA) expression were investigated in vitro in three human colon adenocarcinoma cell lines (WiDr, HT29, and SW403). Subconfluent cultures were exposed continuously to IFN at concentrations of 1-1,000 antiviral units/ml for up to 6 consecutive days. IFN resulted in a significant increase in CEA levels when assayed by cellular enzyme-linked immunosorbent assay (ELISA), with higher concentrations and longer exposure times resulting in greater CEA enhancement. A three to five-fold enhancement of CEA was observed after 5-6 days of continuous exposures at concentrations of 100-1,000 antiviral units/ml. CEA levels returned to baseline over a 4-day period after discontinuation of IFN. Levels of IFN that resulted in CEA enhancement also resulted in cell growth inhibition, with a direct correlation observed. Flow cytometric studies, which evaluated changes in CEA membrane expression of only the viable cells remaining after IFN exposure, gave similar results to cellular ELISA. Quantitative CEA ELISA, which quantitated changes in total cellular CEA content, demonstrated greater increase in CEA than predicted by cellular ELISA. Continuous IFN exposures for 5-6 days at 1,000 U/ml led to a 96-, 26-, and 5-fold increase in total CEA for the WiDr, HT29, and SW403 cell lines, respectively. WiDr cells exposed to daily 6-h IFN pulses demonstrated intermediate increases in CEA compared with cells exposed continuously to IFN.(ABSTRACT TRUNCATED AT 250 WORDS)

Sensitivity and specificity of Gold types 1 to 5 anti-carcinoembryonic antigen monoclonal antibodies: Immunohistologic characterization in colorectal cancer and normal tissues

Carcinoembryonic antigen (CEA) is one of the better-studied oncodevelopmental antigens to which numerous monoclonal antibodies (MoAbs) have been generated. Many of these MoAbs have been recently grouped (Gold classification) according to their epitope recognition. The present study was designed to immunocharacterize various MoAbs, each representative of the five Gold groups, on colorectal cancers and normal tissues using semiquantitative immunohistochemistry. Sensitivity, based on the number of colorectal cancer cases (n = 100) with positive reaction (> 5% cells), was greater with Gold groups 1 and 2 (93% each) than with groups 3, 4, and 5 (78%, 83%, and 87%, respectively). The intensity of the strain also correlated with the Gold groups, with 24%, 20%, and 18% of cancer cases displaying weak or negative staining (0 or 1+) when reacted with MoAbs from groups 3, 4, and 5, respectively, versus 6% and 12% with Gold 1 and 2 antibodies. Cross-reactivity of the anti-CEA antibodies with CEA-related molecules was found to be significant with Gold 5 antibody, which stained most of the normal lung, liver, stomach, and intestinal tissues tested. Strong staining also was seen in granulocytes when they were reacted with Gold 4 and 5 antibodies. The other antibodies showed much less and variable cross-reactivity with normal tissues, with a not statistically significant advantage for Gold 1 antibodies. In addition to lower sensitivity in CEA detection, Gold 3 to 5 MoAbs were less specific due to cross-reaction with one or more of the CEA-related macromolecules expressed by normal tissues. Based on these results and given the broad clinical applications of anti-CEA MoAbs, it is essential to characterize each MoAb to be used for clinical purposes in order to avoid interpretation errors of potential relevance resulting from poor sensitivity/specificity. The use of antibodies that recognize the epitope of group 1 or 2 is recommended to maximize sensitivity and specificity for CEA detection.

γ-Interferon Enhancement of Carcinoembryonic Antigen Expression in Human Colon Carcinoma Xenografts

Athymic nu/nu mice bearing a subcutaneous human colon cancer xenograft (WiDr, low CEA expression) were treated with gamma-interferon (gamma IFN) at varying doses, frequencies, and periods of duration. CEA content (micrograms/g) and uptake of radiolabeled anti-CEA monoclonal antibody (MAB) (percent injected dose per gram, % ID/g) were measured at 48 h following administration of the MAB, The optimal enhancement of tumor CEA content and tumor localization of [111In] anti-CEA monoclonal antibody (MAB) was seen at gamma IFN doses of 100,000 U i.p. every 8 h for 4 days (4.7 micrograms/g; 29% ID/g) compared to control animals (0.9 micrograms/g; 10% ID/g). The effects of gamma IFN on CEA content and MAB localization were less pronounced when administered (a) at lower doses: 5,000 to 50,000 U i.p. every 8 h, (b) at varying frequencies: 300,000 U/day delivered in divided doses every 4 or 24 h, or (c) for varying periods: 2 or 6 days of therapy. In each case, the biologic effects on tumor CEA content and uptake of [111In]MAB correlated closely with the serum gamma IFN level. Therefore, we conclude that enhancement of in vivo CEA expression by gamma IFN may have clinical relevance for tumor imaging and therapy using radiolabeled monoclonal antibodies.

GAMMA-INTERFERON ADMINISTRATION AFTER 90YTTRIUM RADIOLABELED ANTIBODY THERAPY: SURVIVAL AND HEMATOPOIETIC TOXICITY STUDIES

PURPOSE Hematopoietic toxicity is the dose-limiting factor for radioimmunotherapeutic regimens. Cytokines have been shown to decrease hematopoietic toxicity in animals exposed to whole-body irradiation. The purpose of this study was to investigate the effects of murine gamma-interferon (gamma-IFN) on survival and hematopoietic toxicity in mice treated with high dose 90yttrium labeled anticarcinoembryonic antigen (CEA) monoclonal antibody. METHODS AND MATERIALS Balb/c nu/nu mice were injected intravenously with 250 Ci 90Y-T84.66 (a murine anti-CEA monoclonal antibody). Thirty thousand units of gamma-IFN was administered i.v. 24 h later. Control mice received either 250 Ci 90Y-T84.66 alone or 30,000 units gamma-IFN alone. Survival, antibody biodistribution, and bone marrow histologic studies were then performed. RESULTS Only 7% of the animals treated with 90Y-T84.66 survived up to 40 days posttreatment, when the study was terminated. In contrast, 52% of the mice treated with both 90Y-T84.66 and gamma-IFN survived 40 days posttherapy. No toxic deaths were seen in the control group administered gamma-interferon alone. Histologic examination of the bone marrow of animals receiving 90Y-T84.66 and gamma-IFN showed cellular depletion of 40-70% of the hematopoietic cells by 48 h. Cell depletion was 50-70% and 20% by 72 h and 8 days posttherapy, respectively. The marrow of the 90Y-T84.66-treated control group was depleted to a level of 50-80% at 48 h, and remained depleted at 90% at 72 h and 8 days posttherapy. No marrow cell reduction was seen in the gamma-IFN-only treated group. Biodistribution studies showed no alterations in antibody biodistribution or kinetics that could account for the changes in bone marrow toxicity after gamma-IFN. CONCLUSION These results demonstrate that gamma-IFN can reduce the hematologic toxicity resulting from high dose radioimmunotherapy. Histologic studies of bone marrow suggest that gamma-IFN acts primarily to accelerate myelorestoration of the bone marrow. Further studies exploring the use of gamma-IFN as an adjunct to radioimmunotherapy are therefore warranted.