Monophasic and biphasic synovial sarcomas abundantly express cancer/testis antigen NY-ESO-1 but not MAGE-A1 or CT7.

Synovial sarcomas are high-grade malignant mesenchymal tumors with biphasic (BSS) and monophasic (MSS) variants that carry a pathognomonic cytogenetic alteration, t(X;18), involving the SYT gene on chromosome 18 and one of several SSX genes on chromosome X, usually SSX1 or SSX2. Cancer/testis (CT) antigens are expressed in a variety of malignant neoplasms but, in normal tissues, are restricted to male germ cells. Previous analysis revealed a high incidence and homogeneous expression of MAGE CT antigen in synovial sarcomas. The present study was performed to analyze the expression of 3 CT antigens, NY-ESO-1, MAGE-A1 and CT7, by immunohistochemistry with 3 monoclonal antibodies (MAbs), ES121 (anti-NY-ESO-1), MA454 (anti-MAGE-A1) and CT7-33 (anti-CT7), in 25 synovial sarcomas (12 MSS, 13 BSS) typed for the t(X;18)-derived fusion transcript by RT-PCR (19 SYT-SSX1, 6 SYT-SSX2). NY-ESO-1 immunoreactivity was found in 20/25 (80%) cases, and antigen expression was homogeneous in 14/20 NY-ESO-1-positive cases. Both morphologic variants and both translocation types were NY-ESO-1-positive, whereas 5 SYT-SSX1 tumors (1 MSS, 4 BSS) were NY-ESO-1-negative. MAb MA454 was immunoreactive with 4/25 cases (2 MSS, 2 BSS; 3 SYT-SSX1, 1 SYT-SSX2), and MAb CT7-33 was immunoreactive with only 2/25 cases (both BSS, SYT-SSX1). Expression of MAGE-A1 and CT7 was heterogeneous in all positive cases. Our study shows that NY-ESO-1 is highly expressed in a homogeneous pattern in synovial sarcomas of both morphologic variants and both translocation types, making these tumors an attractive target for NY-ESO-1 antigen-based immunotherapy.

Immunohistochemical analysis of NY-ESO-1 antigen expression in normal and malignant human tissues.

NY-ESO-1, a member of the CT (cancer/testis) family of antigens, is expressed in normal testis and in a range of human tumor types. Knowledge of NY-ESO-1 expression has depended on RT-PCR detection of mRNA and there is a need for detecting NY-ESO-1 at the protein level. In the present study, a method for the immunochemical detection of NY-ESO-1 in paraffin-embedded tissues has been developed and used to define the expression pattern of NY-ESO-1 in normal tissues and in a panel of human tumors. No normal tissue other than testis showed NY-ESO-1 reactivity, and expression in testis was restricted to germ cells particularly spermatogonia. In human tumors, the frequency of NY-ESO-1 antigen expression corresponds with past analysis of NY-ESO-1 mRNA expression e.g., 20-30% of lung cancers, bladder cancers and melanoma, and no expression in colon and renal cancer. Co-typing of NY-ESO-1 antigen and mRNA expression in a large panel of lung cancers showed a good correlation. There is great variability in NY-ESO-1 expression in individual tumors, ranging from an infrequent homogeneous pattern of staining to highly heterogeneous antigen expression.

Immunohistochemical and reverse transcription-polymerase chain reaction expression analysis of tyrosinase and microphthalmia-associated transcription factor in angiomyolipomas.

Angiomyolipomas (AMLs) show a characteristic immunoreactivity with melanocyte differentiation markers such as monoclonal antibody (mAb) HMB45, which detects melanocyte differentiation antigen gp100 and mAb A103 reacting with Melan-A/MART-1. Monoclonal antibody T311 to tyrosinase (a key enzyme of melanogenesis) and mAb D5 to the microphthalmia (Mitf) antigen are two newly available markers of melanocytic differentiation. The authors tested 15 AMLs with T311 and D5 by immunohistochemistry and a subset of 3 cases by reverse transcription-polymerase chain reaction for their expression of tyrosinase and Mitf mRNA. T311 showed poor sensitivity in AMLs because only focal staining was seen in 1 out of 15 cases, although tyrosinase mRNA was found in all tested cases. Mitf mRNA was present in 3 of 3 tested cases, and D5 was positive in 15 of 15 AMLs. However, D5 immunostaining often was focal and not as homogeneous as A103, which was analyzed in a previous study. D5 staining also could be seen in other cell types such as normal renal tubular cells, macrophages, and renal cell carcinoma. The current results show that in contrast with HMB45 and A103, T311 has little or no value in the diagnosis of AMLs. D5 may be useful in a panel of antibodies in the diagnosis of AMLs.

Analysis of microphthalmia transcription factor expression in normal tissues and tumors, and comparison of its expression with S-100 protein, gp100, and tyrosinase in desmoplastic malignant melanoma.

Microphthalmia transcription factor (Mitf) is a nuclear protein involved in the development of melanocytes and the regulation of melanin synthesis. Recent studies have suggested that Mitf may be a more sensitive and specific melanocyte marker than S-100 protein and gp100. However, there is insufficient knowledge on the specificity of Mitf, and a systematic examination of its use for the recognition of desmoplastic melanoma has not yet been performed. In this study, we compared the expression of Mitf with S-100 protein, gp100, and tyrosinase in 20 desmoplastic melanomas by using the antibodies D5 (anti-Mitf), anti-S100P, HMB-45 (anti-gp100), and T311 (anti-tyrosinase). All 20 melanomas were positive for S-100 protein, 7 were positive for Mitf, 6 for gp100, and 11 for tyrosinase. To examine the specificity of Mitf, a panel of normal tissue and 386 samples of miscellaneous tumors, including dermal and subcutaneous spindle cell lesions relevant for the differential diagnosis of desmoplastic melanoma, were examined by immunohistochemistry. Furthermore, normal tissue samples were tested for Mitf mRNA by reverse transcriptase polymerase chain reaction (rt-PCR). Immunoreactivity for Mitf was seen not only in melanocytes of normal skin, but also in macrophages, lymphocytes, fibroblasts, Schwann cells, and smooth muscle cells at various sites, and tumors derived thereof. Our results indicate that the antibody D5 lacks sufficient sensitivity and specificity for widespread diagnostic use. Especially in re-excisions, when immunohistochemistry is often needed to distinguish an inflamed scar tissue from tumor, the presence of immunopositive inflammatory cells and fibroblasts limits the diagnostic use of D5.

Monoclonal antibody MA454 reveals a heterogeneous expression pattern of MAGE-1 antigen in formalin-fixed paraffin embedded lung tumours.

Cancer/testis (CT) antigens such as those encoded by the MAGE-gene family are expressed in a wide variety of malignant neoplasms. In normal tissues, expression is generally restricted to testis. Current knowledge of the expression pattern of CT antigens is mainly based on mRNA analysis. Little is known about actual protein expression. We previously developed MA454, a monoclonal antibody (mAb) to MAGE-1 recombinant protein. By employing antigen retrieval techniques, we show that MA454 is reactive on formalin-fixed paraffin-embedded tissues. Immunohistochemical (IHC) analysis of a normal tissue panel revealed staining solely in germ cells of testes. A series of 59 lung tumours was co-typed for MAGE-1 expression by RT-PCR and by immunohistochemistry with MA454. MA454 was positive in 19/59 cases (32%). MAGE-1 mRNA was found in 17 of the 54 cases (32%) available for RT-PCR. Of the 19 MA454-reactive tumours, 15 showed a highly heterogeneous pattern of expression. The other 4 MA454 positive cases revealed immunoreactivity in >25% of tumour areas. Of the 53 cases typed for both, mRNA and protein expression, 48 co-typed whereas 5 cases were discrepant, a likely consequence of heterogeneous MAGE-1 expression. The predominantly focal expression of MAGE-1 suggests that this antigen might not be sufficient as a sole target for immunotherapeutic approaches.

T311--an anti-tyrosinase monoclonal antibody for the detection of melanocytic lesions in paraffin embedded tissues.

Tyrosinase is a key enzyme in melanin biosynthesis and represents a marker of melanocytic differentiation. We previously generated T311, a murine monoclonal antibody to the tyrosinase recombinant protein. This study was performed to evaluate T311 as a diagnostic immunohistochemical reagent for use on formalin-fixed paraffin-embedded pathological material. We analyzed the specificity of the antibody on a panel of normal and neoplastic tissues, and we assessed its sensitivity in a large number of metastatic and primary malignant melanomas, nevi, three angiomyolipomas, and two vitiligo specimens. T311 revealed intense reactivity on paraffin-embedded material. Immunoreactivity was limited to cells of melanocytic differentiation and no immunostaining was present in unrelated normal tissues and tumors. Eighty-four percent of metastatic malignant melanomas were immunoreactive with T311 and showed predominantly a homogeneous expression pattern. However, in primary melanomas of the desmoplastic/spindle cell type, T311 revealed a poor immunoreactivity. Nevi showed intense staining at the junctional zone, while the dermal component revealed decreasing reactivity towards deeper areas. Only one angiomyolipoma was focally immunoreactive with T311. Vitiligo specimens were immunonegative. We conclude that T311 is a specific and sensitive marker for the detection of melanocytic lesions in formalin-fixed paraffin-embedded tissues and a useful serological reagent for diagnostic pathology.

Expression of MAGE-antigens in normal tissues and cancer.

The human MAGE gene family encodes products that can be recognized by autologous cytotoxic T cells. Because MAGE genes are silent in most normal tissues except testis but are activated in a variety of neoplastic lesions, MAGE antigens represent ideal targets for immunotherapy. Current knowledge of MAGE gene expression is based primarily on mRNA typing and relatively little is known about MAGE protein expression. Monoclonal antibody (MAb) 57B, originally thought to be specific for MAGE-3, but now known to be reactive with other MAGE components, was used in the present study to analyze MAGE expression in a panel of normal and malignant tissues. In tests with a wide range of normal tissues, only spermatogenic cells of testis were reactive with 57B. In tumor tissues, significant immunoreactivity was observed in malignant melanomas and carcinomas of the lung, head and neck as well as urinary bladder. No 57B reactivity was seen with colorectal, prostatic or renal cell carcinomas. Lipo- and myosarcomas, as well as malignant fibrous histiocytoma (MFH), were negative, but synovial sarcomas showed intense immunoreactivity. A subset of seminomas was also strongly reactive with 57B. Tumor specimens showed great variability in the number of tumor cells showing 57B reactivity, with some tumors showing only small isolated clusters of positive cells to other tumors with uniform staining throughout the tumor.

Expression of MAGE antigens and analysis of the inflammatory T-cell infiltrate in human seminoma.

The MAGE gene family encodes antigens that are recognized by cytotoxic T-cells. The expression of MAGE antigens has been linked to tumor stage, and MAGE peptides are under investigation as possible vaccines. Seminomas are tumors that are typically accompanied by a heavy inflammatory infiltrate, but have not been studied with regard to their MAGE antigen expression and its correlation with the inflammatory infiltrate. We investigated, therefore, MAGE protein expression, the amount of cytotoxic T-cells, clonality of the lymphocytic infiltrate, apoptotic activity and occurrence of necrosis. Specimens of 27 patients with classical seminoma were examined by immunohistochemistry for CD4, CD8, CD56, CD45R0, beta2-microglobulin and HLA-DR. MAGE expression was detected with the monoclonal antibody 57B, reactive with MAGE-1, -3, -4, -6 and -12. Clonality of the inflammatory infiltrate was examined by multiplex polymerase chain reaction (PCR) analysis of the T-cell receptor rearrangement. Apoptotic cells were detected by DNA nick-end labeling of fragmented DNA, and the apoptotic index was determined semi-quantitatively. Expression of 57B was found in 19 (70%) of 27 seminomas. In all cases, more than 70% of T-cells expressed CD45R0. In four cases, a predominant infiltration of CD8-positive cytotoxic T-cells (CD4/CD8 ratio < 1) was present. However, 15 seminomas showed a CD4/CD8 ratio > 1. In all cases, infiltration of CD56-positive natural killer cells was only focal. HLA-DR expression was not detectable in tumor tissue; beta2-microglobulin was only focal in three cases. Analysis of the T-cell clonality revealed a polyclonal population. The apoptotic index was not significantly different in 57B-positive seminomas (4.15%) compared with 57B negative seminomas (3.80%). Also, no correlation between the 57B expression and the occurrence of necrosis was found. MAGE antigens are homogeneously expressed in most seminomas, but their presence does not appear to represent a dominant epitope responsible for the lymphocytic infiltrate.

Expression of melanocyte-associated markers gp-100 and Melan-A/MART-1 in angiomyolipomas. An immunohistochemical and rt-PCR analysis.

Angiomyolipomas are tumours of uncertain histogenesis, most often occurring in association with the kidney. A characteristic finding is their reactivity with HMB-45, a monoclonal antibody to the melanocyte-associated antigen gp-100. We tested 18 angiomyolipomas for their reactivity with A103, a monoclonal antibody to Melan-A (MART-1), another melanocyte-associated marker, and compared it with HMB-45. All cases were positive with both antibodies, yet most cases showed a more homogeneous staining pattern with A103. Normal kidney was immunohistochemically negative for both antibodies. We also performed RT-PCR assays for gp-100 and Melan-A in 4 of the 18 angiomyolipoma samples and in three normal kidney samples. All 4 angiomyolipoma specimens revealed mRNA for both melanocyte differentiation markers. gp-100 mRNA was found in the samples of normal kidney, but Melan-A mRNA was not. Our study shows that angiomyolipomas express the melanocyte-associated antigens Melan-A and gp-100 at the protein and at the mRNA level, suggesting a true expression of these antigens rather than cross-reacting epitopes. Based on the mRNA expression pattern, immunohistochemical analysis is the preferred method for the detection of gp-100, while Melan-A can be used at the protein and mRNA levels. Our study demonstrates that A103 is a useful marker for the diagnosis of angiomyolipomas.

Expression of melan-A (MART1) in benign melanocytic nevi and primary cutaneous malignant melanoma.

The Melan-A (MART1) gene encodes an antigen recognized by cytotoxic T cells. Although its expression in metastatic melanoma has been documented in the literature by several investigators, little is known about its distribution in primary melanomas and benign melanocytic nevi. In this study, we evaluated Melan-A expression immunohistochemically on sections from paraffin-embedded material of 50 benign nevi and 40 primary cutaneous melanomas using the monoclonal antibody A103. To evaluate a potential role of A103 in the differential diagnosis of melanocytic from nonmelanocytic tumors, we also analyzed a number of benign and malignant peripheral nerve sheath tumors, fibrohistiocytic tumors, and leiomyosarcomas. Immunoreactivity with A103 was present in all "nonneurotized" nevi and in all nondesmoplastic primary melanomas, both in the intraepidermal and the dermal component. Only two nevi that underwent prominent neurotization showed no staining with A103. Although all melanomas with epithelioid cells tended to be strongly positive with A103, only 4 of 13 spindle cell and desmoplastic melanomas (all positive with anti-S-100 and negative with HMB-45) were immunoreactive with A103 (two focally, two diffusely). None of the nonmelanocytic lesions expressed Melan-A. Our results confirm that Melan-A protein is broadly expressed in the majority of benign and malignant melanocytic lesions and suggest that A103 can be helpful diagnostically, not only for metastatic tumors, but also for primary skin lesions. Its use in distinguishing between melanocytic and peripheral nerve sheath tumors, however, is limited because of the low or absent expression of Melan-A in nevi that underwent neurotization and spindle cell and desmoplastic melanomas.

A103: An anti-melan-a monoclonal antibody for the detection of malignant melanoma in paraffin-embedded tissues.

Melan-A is a previously defined, melanocyte differentiation antigen, and an anti-Melan-A murine monoclonal antibody, A103, was recently developed by our group. In this study, we evaluated A103 immunoreactivity on formalin-fixed, paraffin-embedded tissues, exploring the potential of A103 in the diagnosis of metastatic melanoma. Seventy-five metastatic melanomas, 10 primary melanomas, and 10 benign melanocytic nevi were tested. The reactivity of A103 was compared with HMB-4, an anti-gp100 antibody. Results showed that all nevi were A103 positive, and most primary melanomas were A103 and HMB45 positive. Of 75 metastatic melanomas, 61 (81%) were A103 positive, and 56 (75%) were HMB45 positive. Of 19 HMB45-negative lesions, 8 were A103 positive; of 14 A103-negative lesions, 3 were HMB45 positive. Eleven metastatic lesions, as well as 2 of 10 primary melanomas, were dual negative. These negative cases consisted mainly of the spindle cell and desmoplastic variants. Of the positive cases, A103 showed homogeneous staining in a significantly higher proportion of cases than HMB45 (72% versus 52%). In addition, focal staining with less than 5% reactive tumor cells was seen more frequently in HMB45 (12 of 56) than in A103 (5 of 61). These results indicated that A103 can be used as a first-line antibody in the diagnosis of metastatic melanoma. Our results also showed that A103 reacted with angiomyolipoma, which is known to be HMB45 positive. Of normal tissues, unexpected A103 reactivity was observed in the adrenal cortex, granulosa and theca cells of the ovary, and Leydig cells of the testis. This A103 immunoreactivity in benign and neoplastic tissues of nonmelanocytic origin, the basis of which is unclear, could also be of potential diagnostic value.

Rapid and specific targeting of monoclonal antibody A33 to a colon cancer xenograft in nude mice.

Monoclonal antibody (mAb) A33 detects a glycoprotein homogeneously expressed by > 95% of human colon cancers and by normal colon cells. The A33 antigen is not secreted or shed and after mAb A33 binds to antigen on the cell membrane, a fraction of membrane-bound mAb A33 is internalized into endosomes. Phase I 131I-mAb A33 biodistribution studies have shown consistent, specific tumor-targeting, and phase I radioimmunotherapy trials with 131I- or 125I-mAb A33 have demonstrated antitumor effects. Here we describe a nude mouse model that was established using a human colon cancer cell line, SW1222, which grows as a relatively hypovascular, invasive heterotransplant when injected i.m. Peak uptake of 131I-labeled or 111In-chelated mAb A33 was observed at 48-96 h, with a mean of 34% (SE +/- 5.0) and 46.7% (SE +/- 1.7) injected dose per gram of tumor tissue, respectively. 111In-mAb A33 was retained in tumor tissue longer than halide radioimmunoconjugates. The specificity of antibody localization was assessed using a control antibody (tumor uptake and pharmacokinetics), a control tumor, corrections for vascular antibody blood-pooling in tumor tissue, and blocking of radiolabeled mAb A33 localization by pretreating mice with excess unlabeled mAb A33. These experiments demonstrate that mAb A33 localization in tumor was specific, and they emphasize the unexpected rapidity with which the antibody localizes. Our conclusions were confirmed by immunohistochemical techniques which allowed direct visualization of localization and distribution of the humanized version of mAb A33 in tumor tissue. Furthermore, antibody doses approximating tumor-saturating doses demonstrated that a homogeneous distribution of antibody in tumor is possible. This model will be valuable for studies focusing on general physiologic aspects of antibody-to-tumor cell localization and critical as a guide to the evaluation of various A33 antibody constructs and combinations with other therapies for the treatment of colon cancer.

Immunoreactivity for A103, an antibody to melan-A (Mart-1), in adrenocortical and other steroid tumors.

The Melan-A (MART-1) gene encodes an antigen recognized by cytotoxic T cells. It has been said to be restricted in its expression to melanocytes. However, here we report the presence of immunoreactivity for A103, an antibody to Melan-A, in five adrenocortical adenomas, 16 primary and 13 metastatic adrenocortical carcinomas, four Leydig cell tumors of the testis, and three Sertoli-Leydig cell tumors of the ovary. To evaluate the potential diagnostic role of this antibody, we studied immunoreactivity for A103 in 111 carcinomas, 40 germ cell tumors, and 33 miscellaneous nonmelanocytic epithelioid tumors. All of them were negative for A103. Our findings suggest that once melanoma is excluded, A103 can aid in the recognition of steroid hormone-producing tumors and may be particularly useful in the diagnosis of adrenocortical carcinoma. The presence of immunoreactivity for A103 practically excludes any other carcinoma that may enter into the differential diagnosis of adrenocortical tumors.

Serological analysis of Melan-A(MART-1), a melanocyte-specific protein homogeneously expressed in human melanomas.

Recent progress in the structural identification of human melanoma antigens recognized by autologous cytotoxic T cells has led to the recognition of a new melanocyte differentiation antigen, Melan-A(MART-1). To determine the properties of the Melan-A gene product, Melan-A recombinant protein was produced in Escherichia coli and used to generate mouse monoclonal antibodies (mAbs). Two prototype mAbs, A103 and A355, were selected for detailed study. Immunoblotting results with A103 showed a 20-22-kDa doublet In Melan-A mRNA positive melanoma cell lines and no reactivity with Melan-A mRNA-negative cell lines. A355, in addition to the 20-22-kDa doublet, recognized several other protein species in Melan-A mRNA-positive cell lines. Immunocytochemical assays on cultured melanoma cells showed specific and uniform cytoplasmic staining in Melan-A mRNA-positive cell lines. Immunohistochemical analysis of normal human tissues with both mAbs showed staining of adult melanocytes and no reactivity with the other normal tissues tested. Analysis of 21 melanoma specimens showed homogenous staining of tumor cell cytoplasm in 16 of 17 Melan-A mRNA-positive cases and no reactivity with the three Melan-A mRNA-negative cases.

Immunophenotyping of melanomas for tyrosinase: implications for vaccine development.

Tyrosinase (EC 1.14.18.1), the key enzyme in melanin synthesis, has been shown to be one of the targets for cytotoxic T-cell recognition in melanoma patients. To develop serological reagents useful for immunophenotyping melanoma for tyrosinase, human tyrosinase cDNA was expressed in an Escherichia coli expression vector. The purified recombinant tyrosinase was used to generate mouse monoclonal and rabbit polyclonal antibodies. The prototype monoclonal antibody, T311, recognized a cluster of protein moieties ranging from 70 to 80 kDa in tyrosinase mRNA-positive melanoma cell lines and melanoma specimens as well as in L cells transfected with tyrosinase cDNA. Untransfected L cells and L cells transfected with tyrosinase-related protein 1, TRP-1(gp75), were nonreactive. Immunohistochemical analysis of melanomas with T311 showed tyrosinase in melanotic and amelanotic variants, and tyrosinase expression correlated with the presence of tyrosinase mRNA. Melanocytes in skin stained with T311, whereas other normal tissues tested were negative. The expression pattern of three melanosome-associated proteins--tyrosinase, TRP-1(gp75), and gp100--in melanoma was also compared. Tyrosinase and gp100 are expressed in a higher percentage of melanomas than TRP-1(gp75), and the expression of these three antigens was discordant. Tyrosinase expression within individual tumor specimen is usually homogenous, distinctly different from the commonly observed heterogeneous pattern of gp100 expression.

HLA expression in hepatocellular carcinoma cell lines.

The present study undertook to investigate the biological significance of human leucocyte antigen expression in hepatocellular carcinoma and to elucidate the role of potential modulating agents on human leucocyte antigen expression. These studies used several hepatic tumour-derived cell lines as in vitro model systems. The cell lines included PLC/PRF/5 (Alexander cell line), Hep3B, HepG2, TONG PHC, HA22T/VGH, HA59T/VGH and Mahlavu. The cell lines K562 and Raji were used as negative and positive controls, respectively. K562, a B lymphoid-derived cell line, was shown to express negligible amounts of human leucocyte antigens, while Raji, an erythromyeloid-derived cell line, expressed both class I and class II human leucocyte antigens as well as their respective invariant chains, beta 2-microglobulin and Ii. Using an ELISA, experiments performed on these cell lines confirmed the natural expression of class I and class II antigens by the HA22T/VGH and HA59T/VGH cell lines, whereas PLC/PRF/5 displayed class II surface antigens only. The effects of modulating agents such as interferon-gamma sodium butyrate and clofazimine on human leucocyte antigen expression were investigated using the HA22T/VGH, HA59T/VGH and TONG PHC cell lines. These agents increased class II and class II human leucocyte antigen expression on HA22T/VGH and TONG PHC cells, but had no effect on the HA59T/VGH cell line. The results suggest a potential use for these agents as modulators of human leucocyte antigen expression by human heptocellular cell lines.

Antitumor effects of doxorubicin in combination with anti-epidermal growth factor receptor monoclonal antibodies.

BACKGROUND: A variety of human tumors frequently express high levels of epidermal growth factor (EGF) receptor and its ligand, transforming growth factor alpha (TGF-alpha), which in some tumors is associated with poor prognosis. Monoclonal antibodies (MAbs) that block the binding of TGF-alpha or EGF to the receptor can inhibit proliferation of tumor cells that express the receptor. Studies suggest that these MAbs may enhance the antitumor effects of chemotherapy. PURPOSE: Our purpose was to study, in vitro and in vivo, the antitumor effects of doxorubicin in combination with anti-EGF receptor MAbs against tumor cells expressing high levels of EGF receptor. Our goal was to achieve maximum initial cytoreduction with high-dose doxorubicin in association with prolonged blockade of EGF receptor with MAbs. METHODS: Anti-EGF receptor MAbs 528 (isotype IgG2a) and 225 (isotype IgG1) were used in combination with doxorubicin against cells from human A431 squamous cell carcinoma and human MDA-468 breast adenocarcinoma. Both A431 and MDA-468 cells express high levels of EGF receptors and TGF-alpha. Cultured cells were treated with doxorubicin (range, 0-10 nM) in the presence or absence of MAb 528 or 225 (range, 0-30 nM). At 48 hours, doxorubicin-containing medium was removed, and treatment with antibody was continued for 5 days, when cell proliferation assays were performed. The activity of the agents and the combinations against well-established xenografts in BALB/c nude mice was also studied. In nude mice, doxorubicin was given at doses of 50-100 micrograms/20 g body weight on 2 successive days, and MAbs 528 and 225 were given at a dose range of 0-2 mg intraperitoneally twice a week. RESULTS: MAbs 528 and 225 both enhanced the antitumor effects of doxorubicin against A431 and MDA-468 tumor cells, producing additive growth suppression in cell cultures. MAb 528 increased the antitumor effects of doxorubicin by 32%-42%, and similar results were obtained with MAb 225. In BALB/c athymic mice, the treatment of well-established xenografts with either doxorubicin or anti-EGF receptor MAb alone temporarily inhibited growth, but the combination of both agents substantially enhanced antitumor activity over that of doxorubicin alone in A431 and MDA-468 cell xenografts. The combination treatment of mice bearing A431 xenografts resulted in tumor eradication of 40%-100% in the surviving mice in several independent experiments. The enhanced antitumor activity was dose dependent. CONCLUSIONS: Our results suggest that anti-EGF receptor MAbs substantially enhance the effects of doxorubicin against well-established xenografts of tumor cells expressing high levels of EGF receptors. IMPLICATIONS: Clinical trials with anti-EGF receptor MAbs are being conducted, and trials with anti-EGF receptor MAbs combined with doxorubicin are planned.