Expression of cancer testis antigens CT10 (MAGE-C2) and GAGE in gastrointestinal stromal tumors.

INTRODUCTION: Expression of cancer testis antigens (CTAs) has been associated with prognosis in gastrointestinal stromal tumors (GIST) and other malignancies. CTAs are currently being investigated for cancer immunotherapy. MATERIALS AND METHODS: We analyzed two CTAs, CT10/MAGE-C2 and GAGE, in 51 GIST by immunohistochemistry and correlated it with established histopathological criteria for malignancy. RESULTS: GAGE expression was found in 6/51 (12%) patients, whereas 5/51 (10%) patients expressed CT10/MAGE-C2. 7/51(14%) patients expressed at least one of both CTAs, in 4/51 (8%) patients both CTAs were positive. High-grade GIST are more likely to express GAGE (p = 0.002) and CT10/MAGE-C2 (p = 0.007) compared to less aggressive tumors. All patients with GAGE or CT10/MAGE-C2 expression had moderate- or high-risk of recurrence according to the established risk criteria. The presence of GAGE correlates with mitotic rate (p = 0.001) and tumor size (p = 0.02), but not with tumor location (p = 0.60). CT10/MAGE-C2 also significantly correlates with mitotic rate (p = 0.004) and tumor size (p = 0.002), whereas no correlation could be found with tumor location (p = 0.36). DISCUSSION: CT10/MAGE-C2 and GAGE should be explored together with other previously described CTAs as targets for immunotherapy of GIST in cases, which are refractory to conventional therapy.

NY-ESO-1 antibody as a novel tumour marker of gastric cancer.

BACKGROUND: NY-ESO-1 antibodies are specifically observed in patients with NY-ESO-1-expressing tumours. We analysed whether the NY-ESO-1 humoral immune response is a useful tumour marker of gastric cancer. METHODS: Sera from 363 gastric cancer patients were screened by enzyme-linked immunosorbent assay (ELISA) to detect NY-ESO-1 antibodies. Serial serum samples were obtained from 25 NY-ESO-1 antibody-positive patients, including 16 patients with curative resection and 9 patients who received chemotherapy alone. RESULTS: NY-ESO-1 antibodies were detected in 3.4% of stage I, 4.4% of stage II, 25.3% of stage III, and 20.0% of stage IV patients. The frequency of antibody positivity increased with disease progression. When the NY-ESO-1 antibody was used in combination with carcinoembryonic antigen and CA19-9 to detect gastric cancer, information gains of 11.2% in stages III and IV, and 5.8% in all patients were observed. The NY-ESO-1 immune response levels of the patients without recurrence fell below the cutoff level after surgery. Two of the patients with recurrence displayed incomplete decreases. The nine patients who received chemotherapy alone continued to display NY-ESO-1 immune responses. CONCLUSION: When combined with conventional tumour markers, the NY-ESO-1 humoral immune response could be a useful tumour marker for detecting advanced gastric cancer and inferring the post-treatment tumour load in seropositive patients.

Cancer-testis antigen expression and immunogenicity in AL amyloidosis.

Light-chain amyloidosis (AL) is a plasma cell dyscrasia closely related to multiple myeloma. In multiple myeloma, the cancer-testis antigens (CTAs) CT7 (MAGE-C1), CT10 (MAGE-C2) and MAGE-A CTAs are expressed in up to 80% of cases. In this study, we investigated the expression and immunogenicity of several CTAs in patients with AL amyloidosis in a total of 38 bone marrow specimens by employing standard immunohistochemistry techniques on paraffin-embedded archival tissues. Plasma samples from 35 patients (27 with matched bone marrow samples) were also analyzed by ELISA for sero reactivity to a group of full-length CTA proteins. CT7 was present in 25/38 (66%) while CT10 was demonstrated in 3/38 and GAGE in 1/38 AL amyloid cases. The expression pattern was mostly focal. There were no significant differences with regard to organ involvement, response to treatment, or prognosis in CTA positive compared to negative cases. None of the specimens showed spontaneous humoral immunity to CT7, but sero reactivity was observed in individual patients to other CTAs. This study identifies CT7 as the prevalent CTA in plasma cells of patients with AL amyloidosis. Further analyses determining the biology of CTAs in AL amyloidosis and their value as potential targets for immunotherapy are warranted.

Negative argininosuccinate synthetase expression in melanoma tumours may predict clinical benefit from arginine-depleting therapy with pegylated arginine deiminase.

BACKGROUND: Arginine-depleting therapy with pegylated arginine deiminase (ADI-PEG20) was reported to have activity in advanced melanoma in early phase I-II trial, and clinical trials are currently underway in other cancers. However, the optimal patient population who benefit from this treatment is unknown. METHODS: Advanced melanoma patients with accessible tumours had biopsy performed before the start of treatment with ADI-PEG20 and at the time of progression or relapse when amenable to determine whether argininosuccinate synthetase (ASS) expression in tumour was predictive of response to ADI-PEG20. RESULTS: Twenty-seven of thirty-eight patients treated had melanoma tumours assessable for ASS staining before treatment. Clinical benefit rate (CBR) and longer time to progression were associated with negative expression of tumour ASS. Only 1 of 10 patients with ASS-positive tumours (ASS+) had stable disease, whereas 4 of 17 (24%) had partial response and 5 had stable disease, when ASS expression was negative (ASS-), giving CBR rates of 52.9 vs 10%, P=0.041. Two responding patients with negative ASS expression before therapy had rebiopsy after tumour progression and the ASS expression became positive. The survival of ASS- patients receiving at least four doses at 320 IU m(-2) was significantly better than the ASS+ group at 26.5 vs 8.5 months, P=0.024. CONCLUSION: ADI-PEG20 is safe and the drug is only efficacious in melanoma patients whose tumour has negative ASS expression. Argininosuccinate synthetase tumour positivity is associated with drug resistance and tumour progression.

Arginine deiminase PEG20 inhibits growth of small cell lung cancers lacking expression of argininosuccinate synthetase.

BACKGROUND: Some cancers have been shown to lack expression of argininosuccinate synthetase (ASS), an enzyme required for the synthesis of arginine and a possible biomarker of sensitivity to arginine deprivation. Arginine deiminase (ADI) is a microbial enzyme capable of efficiently depleting peripheral blood arginine. METHODS: Argininosuccinate synthetase expression was assessed in human small cell lung cancer (SCLC) by immunohistochemistry (IHC), with expression also assessed in a panel of 10 human SCLC by qRT-PCR and western blot. Proliferation assays and analyses of apoptosis and autophagy assessed the effect of pegylated ADI (ADI-PEG20) in vitro. The in vivo efficacy of ADI-PEG20 was determined in mice bearing SCLC xenografts. RESULTS: Approximately 45% of SCLC tumours and 50% of cell lines assessed were negative for ASS. Argininosuccinate synthetase-deficient SCLC cells demonstrated sensitivity to ADI-PEG20, which was associated with the induction of autophagy and caspase-independent cell death. Arginine deiminase-PEG20 treatment of ASS-negative SCLC xenografts caused significant, dose-dependent inhibition of tumour growth of both small and established tumours. CONCLUSION: These results suggest a role for ADI-PEG20 in the treatment of SCLC, and a clinical trial exploring this therapeutic approach in patients with ASS-negative SCLC by IHC has now been initiated.

Cancer-testis antigen expression in triple-negative breast cancer.

BACKGROUND: cancer-testis (CT) antigens, frequently expressed in human germline cells but not in somatic tissues, may become aberrantly reexpressed in different cancer types. The aim of this study was to investigate the expression of CT antigens in breast cancer. PATIENTS AND METHODS: a total of 100 selected invasive breast cancers, including 50 estrogen receptor (ER) positive/HER2 negative and 50 triple negative (TN), were examined for NY-ESO-1 and MAGE-A expression by immunohistochemistry. RESULTS: a significantly higher expression of MAGE-A and NY-ESO-1 was detected in TN breast cancers compared with ER-positive tumors (P = 0.04). MAGE-A expression was detected in 13 (26%) TN cancers compared with 5 (10%) ER-positive tumors (P = 0.07). NY-ESO-1 expression was confirmed in nine (18%) TN tumor samples compared with two (4%) ER-positive tumors. CONCLUSIONS: MAGE-A and NY-ESO-1 CT antigens are expressed in a substantial proportion of TN breast cancers. Because of the limited therapeutic options for this group of patients, CT antigen-based vaccines might prove to be useful for patients with this phenotype of breast cancer.

Melanoma or not? Cancer testis antigens may help.

BACKGROUND: Cancer testis antigens (CTAs) are expressed in a variety of malignant tumours. No CTA expression is found in normal adult tissues, except in male germ cells and occasionally placenta. To date, more than 20 CTAs or antigen families have been identified. OBJECTIVES: Owing to their tumour-associated expression pattern, CTAs may be useful for making a distinction between benign and malignant neoplasms. The present study was done to analyse the value of CTAs for the discrimination of cutaneous melanoma and naevi. PATIENTS AND METHODS: Primary melanomas (38) and 19 naevi were analysed for their expression of CTAs by immunohistochemistry using the following monoclonal antibodies (mAb) to the following antigens (mAb/antigen): MA454/MAGE-A1, 57B/MAGE-A4, ES121/NY-ESO-1. In a subset of melanomas (n = 26), the CTA panel was extended to three additional CTAs (mAb/antigen): CT7-33/MAGE-C1, M3H67/MAGE-A3 and GAGE/GAGE. RESULTS: All 19 naevi were negative for the mAbs MA454, M3H67, 57B, ES121, CT7-33 and GAGE. In melanoma, the immunoreactivity was as follows: MA454: 8/38 (21%), 57B 11/38 (29%), ES121 9/38 (24%). However, 19/38 (50%) were positive for at least one CTA. When 26 melanomas were tested for the expression of six different CTAs 20/26 (77%) were positive for at least one CTA. CONCLUSIONS: CTAs may be useful in the determination of suspected malignancy in cutaneous melanomas. The low incidence of particular CTAs can be overcome by increasing the number of CTAs analysed.

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.

Growth suppression of intracranial xenografted glioblastomas overexpressing mutant epidermal growth factor receptors by systemic administration of monoclonal antibody (mAb) 806, a novel monoclonal antibody directed to the receptor.

A mutant epidermal growth factor receptor (variously called DeltaEGFR, de2-7 EGFR, or EGFRvIII) containing a deletion of 267 amino acids of the extracellular domain is frequently highly expressed in human malignant gliomas and has been reported for cancers of the lung, breast, and prostate. We tested the efficacy of a novel monoclonal anti-DeltaEGFR antibody, mAb 806, on the growth of intracranial xenografted gliomas in nude mice. Systemic treatment with mAb 806 significantly reduced the volume of tumors and increased the survival of mice bearing xenografts of U87 MG.DeltaEGFR, LN-Z308.DeltaEGFR, or A1207.DeltaEGFR gliomas, each of which expresses high levels of DeltaEGFR. In contrast, mAb 806 treatment was ineffective with mice bearing the parental U87 MG tumors, which expressed low levels of endogenous wild-type EGFR, or U87 MG.DK tumors, which expressed high levels of kinase-deficient DeltaEGFR. A slight increase of survival of mice xenografted with a wild-type EGFR-overexpressing U87 MG glioma (U87 MG.wtEGFR) was effected by mAb 806 concordant with its weak cross-reactivity with such cells. Treatment of U87 MG.DeltaEGFR tumors in mice with mAb 806 caused decreases in both tumor growth and angiogenesis, as well as increased apoptosis. Mechanistically, in vivo mAb 806 treatment resulted in reduced phosphorylation of the constitutively active DeltaEGFR and caused down-regulated expression of the apoptotic protector, Bcl-XL. These data provide preclinical evidence that mAb 806 treatment may be a useful biotherapeutic agent for those aggressive gliomas that express DeltaEGFR.

Expression of melanocytic differentiation markers in malignant melanomas of the oral and sinonasal mucosa.

Malignant melanomas of the oral and sinonasal mucosa are rare tumors. Amelanotic variants can, on occasion, be difficult to recognize by routine light microscopy. Immunohistochemical studies may be needed for a final diagnosis. A number of new monoclonal antibodies to melanocytic differentiation antigens have been studied recently on primary cutaneous and metastatic melanoma. However, little is known about these antibodies for the diagnosis of mucosal melanomas. In this study the authors analyzed 79 oral and sinonasal mucosal melanomas of 65 patients. A total of 35 tumors originated from the oral mucosa (21 primary tumors, eight local recurrences, and six metastases) and 44 melanomas were from the sinonasal tract (27 primary tumors, nine local recurrences, and eight metastases). Immunohistochemical studies were performed on paraffin-embedded tissues, using the following antibodies: anti-S-100 protein, T311 (anti-tyrosinase), A103 (anti-Mart-1/Melan-A), D5 (antimicrophthalmia-associated transcription factor), and HMB-45 (anti-gp100). Of 35 oral mucosal tumors, 34 (97%) were positive with anti-S-100 protein, 33 (94%) with T311, 30 (85%) with A103, 26 (74%) with D5, and 25 (71%) with HMB-45. All five desmoplastic melanomas of the oral mucosa were positive for S-100 protein, four for tyrosinase, and one each for HMB-45 and A103. No desmoplastic melanoma was positive with D5. All 44 sinonasal melanomas were positive for tyrosinase and Mart-1/Melan-A (100%). Forty-three (98%) were positive with HMB-45, 42 (95%) with anti-S-100 protein, and 40 (91%) with D5. These results reveal that T311 is the most sensitive marker for sinonasal melanomas and closely approaches the sensitivity of anti-S-100 protein for oral mucosal melanomas. For desmoplastic mucosal tumors, anti-S-100 protein remains the most sensitive marker.

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.

Effect of TNF gene depletion on liver regeneration after partial hepatectomy in mice.

BACKGROUND: Tumor necrosis factor-alpha (TNF) is thought to act as a stimulator for initiating hepatocyte proliferation after partial hepatectomy (PH). At the same time, TNF induces a series of inflammatory responses that may be detrimental for the liver and other remote organs. The purpose of this study was to investigate the effect of TNF on the pathophysiologic state after PH. METHODS: Wild-type (TNF+/+) and TNF-deficient (TNF-/-) mice underwent 70% PH. Hepatocyte proliferation was assessed by bromodeoxyuridine labeling and mitotic index. Liver function was evaluated by alanine aminotransferase (ALT) and total bilirubin levels in serum after PH. Myeloperoxidase activity in the liver and lung was measured as a marker for neutrophil activation. RESULTS: No differences were observed in liver regeneration or hepatocyte proliferation between TNF+/+ and TNF-/- mice. The survival of TNF-/- mice on day 1 after PH was significantly higher than that of TNF+/+ mice, but both groups had similar survival thereafter. The ALT level was significantly higher in TNF+/+ mice 6 hours after PH and myeloperoxidase activities in both liver and lung were markedly elevated in TNF+/+ mice compared with TNF-/- mice. CONCLUSIONS: These findings demonstrate that TNF gene-depleted mice do not demonstrate delayed liver regeneration but do suppress neutrophil activation after PH compared with results in wild-type (TNF +/+) mice.

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.

Xanthogranulomas with inconspicuous foam cells and giant cells mimicking malignant melanoma: a clinical, histologic, and immunohistochemical study of three cases.

Histiocytic proliferations can mimic melanocytic tumors and vice versa. The authors describe the clinical, histologic, and immunohistochemical findings of three predominantly mononuclear xanthogranulomas that were misdiagnosed as malignant melanoma by experienced pathologists. All lesions occurred in male patients ranging in age from 14 to 75 years. The tumors presented as dermal nodules, two of which were surrounded by an epidermal collarette and were ulcerated focally. The tumors were composed of a mixed population of large epithelioid and plump spindle cells with pink or pale cytoplasm arranged in nests and short fascicles. Occasional mononuclear cells had cytoplasmic vacuolar changes, but none had well-developed foamy cytoplasm. Rare, multinucleated giant cells were present, but they were not of the Touton type. Mitotic figures were found in all lesions. Immunohistochemically, most tumor cells (80%-90%) were strongly positive for CD68 and a minority of cells (10%-15%), located typically at the periphery of the tumor, was positive for factor XIIIa. Two tumors contained rare cells positive for S-100 protein (5% of tumor cells or less). All tumors were completely negative for tyrosinase (T311), gp100 (HMB-45), and Melan-A (A103). Giant and foam cell-poor variants of juvenile xanthogranuloma have been reported previously, mainly in young children. Their occurrence in adolescents and adults is underrecognized. Knowledge of this variant is important to avoid misdiagnosing a benign tumor as malignant melanoma.

Immunohistochemical distinction of epithelioid histiocytic proliferations from epithelioid melanocytic nevi.

Histiocytic tumors can be confused with melanocytic nevi and malignant melanoma and vice versa. To explore the use of immunohistochemistry for this diagnostic problem, we examined the expression of S-100 protein, gp100 (the antigen recognized by HMB-45), tyrosinase (T311), Melan-A (A103), Factor XIIIa (FXIIIa), and CD68 in 10 juvenile xanthogranulomas (JXGs), five epithelioid histiocytomas (EHs), and 15 melanocytic nevi composed of large epithelioid cells. All epithelioid melanocytic nevi were immunoreactive for Melan-A, tyrosinase, and S-100 protein in most melanocytes. Four nevi were completely negative with HMB-45. Nine nevi had only a minor HMB-45-positive component in the superficial dermis. Two nevi were diffusely HMB-45-positive. Melanocytes in all nevi were completely negative for FXIIIa. Thirteen nevi were completely negative for CD68. Two nevi contained rare cells with weak staining for CD68. All 15 histiocytic proliferations were completely negative for Melan-A, tyrosinase, and gp100. They lacked expression of S-100 protein or had at most 10% immunopositive cells. In JXGs, most cells were strongly reactive for CD68, although only a few were positive for FXIIIa. In EHs, 40% to 60% of cells were immunoreactive for FXIIIa, and only 20% to 30% were positive for CD68. Our results demonstrate that Melan-A and tyrosinase are sensitive and specific markers to distinguish epithelioid melanocytic nevi from epithelioid histiocytic tumors.

The rabbit antibody repertoire as a novel source for the generation of therapeutic human antibodies.

The rabbit antibody repertoire, which in the form of polyclonal antibodies has been used in diagnostic applications for decades, would be an attractive source for the generation of therapeutic human antibodies. The humanization of rabbit antibodies, however, has not been reported. Here we use phage display technology to select and humanize antibodies from rabbits that were immunized with human A33 antigen which is a target antigen for the immunotherapy of colon cancer. We first selected rabbit antibodies that bind to a cell surface epitope of human A33 antigen with an affinity in the 1 nm range. For rabbit antibody humanization, we then used a selection strategy that combines grafting of the complementarity determining regions with framework fine tuning. The resulting humanized antibodies were found to retain both high specificity and affinity for human A33 antigen.

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.