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Sarcomas are rare but aggressive malignant tumors associated with high mortality, for which the efficacy of standard therapies remains limited. In order to develop immunotherapeutic approaches for the treatment of sarcoma, we studied the relevance of cancer/testis antigens (CTAs), a group of antigens whose expression is developmentally regulated and that is specifically found in some tumor types, as sarcoma vaccine targets. CTA expression was assessed by PCR and/or immunohistochemistry (IHC) in sarcoma tumor samples that included different histological subtypes and sarcoma cell lines. Expression of HLA class I was assessed by IHC in tumor samples and by FACS analysis in cell lines. More than 70% of the tumor samples expressed at least one CTA. The majority of tumors and cell lines expressed normal levels of HLA class I. HLA class I expression in cell lines was enhanced upon treatment with IFN-gamma. CTA expression was enhanced or induced by treatment with the demethylating agent 5-aza-2'-deoxycytidine, resulting in recognition by specific CTLs. Interestingly, a spontaneous humoral and CD8+ T cellular response to the CTA NY-ESO-1 was detected in a synovial sarcoma patient. Together, these findings strongly support the implementation of CTA-based immunotherapy of sarcoma as a means to improve the efficacy of the standard therapy.

The efficacy of current standard therapies for the treatment of sarcoma remains limited. With the aim of identifying target antigens relevant to the development of vaccine-based immunotherapy of sarcoma, we have addressed the relevance of tumor-specific antigens encoded by genes belonging to the SSX family as vaccine targets in sarcoma tumors. Expression of SSX-1 to -5 was analyzed in a collection of sarcoma tumors of diverse histological subtypes and in sarcoma cell lines. We found expression of at least one SSX-encoded antigen in 42% of sarcoma tumors, including 5 of 7 different histological subtypes, and in 50% of sarcoma cell lines. SSX-1 was the most frequently expressed family member, followed by SSX-4, -2 and -5. Expression of SSX-3 was detected in only one sample. Importantly, most SSX positive samples co-expressed more than one family member. In addition, assessment of CD8+ T cell recognition of HLA-A2+ SSX-2+ sarcoma cells showed that the latter were efficiently recognized and lysed by SSX-2-specific CTLs. The results of this study indicate that SSX antigens are relevant targets for the development of vaccine-based immunotherapy of sarcoma and encourage the start of vaccination trials using SSX-derived immunogens in sarcoma patients.

The cancer-testis antigen NY-ESO-1 is one of the most promising candidates for generic vaccination of cancer patients. Here we analyzed the CD8(+) T cell response to a NY-ESO-1 peptide vaccine composed of the two previously defined peptides 157-165 and 157-167, administered with GM-CSF as a systemic adjuvant. The NY-ESO-1 peptide vaccine elicited a CD8(+) T cell response directed against multiple distinct epitopes in the 157-167 region, as revealed by using A2/peptide multimers incorporating overlapping A2 binding peptides in this region. However, only a minor fraction of the elicited CD8(+) T cells, namely those recognizing the peptide 157-165 with sufficiently high functional avidity, recognized the naturally processed target on NY-ESO-1(+) tumor cells. In contrast, the majority of peptide 157-165-specific CD8(+) T cells exhibited lower functional avidity and no tumor reactivity. In addition, vaccine-elicited CD8(+) T cells specific for other overlapping epitopes in the 157-167 region failed to significantly recognize NY-ESO-1-expressing tumor targets. Thus, because of the complexity of the CD8(+) T cell repertoire that can be elicited by vaccination with synthetic peptides, a precise definition of the targeted epitope, and hence, of the corresponding peptide to be used as immunogen, is required to ensure a precise tumor targeting.

Antisera have been raised to human leukemic blast cells from individual patients in mice rendered tolerant with cyclophosphamide to remission leukocytes from the same individual. 10 antisera were raised against acute myelogenous leukemia (AML) cells and 5 antisera were raised against acute lymphoblastic leukemia (ALL) cells. Antisera to AML cells were absorbed with ALL cells, and antisera to ALL cells were absorbed with AML cells. Unabsorbed and absorbed antisera as well as antisera raised in nontolerant mice were tested for cytotoxicity against various cells of a panel containing myeloblasts from 35 patients with AML, lymphoblasts from 7 patients with ALL, myeloblasts from 7 patients with chronic myelogenous leukemia (CML) in blast crisis, peripheral blood leukocytes from 12 patients with acute leukemia in remission and 30 nonleukemic patients, and nucleated bone marrow cells from 10 nonleukemic patients. Unabsorbed antisera to AML or ALL cells raised in tolerant mice were highly cytotoxic to leukemic blasts cells but significantly less cytotoxic to remission and control cells. Antisera to AML cells absorbed with ALL cells retained measurable cytotoxicity against AML cells but were not cytotoxic to ALL cells or control cells. Similarly, antisera to ALL cells absorbed with AML cells retained significant cytotoxicity only to ALL cells. Control antisera raised in nontolerant mice were cytotoxic to all cells tested. Although species specific, histocompatibility, differentiation, maturation, and cell cycle-associated antigens may be responsible in part for the cytotoxic activity of the unabsorbed antisera, the absorbed antisera are probably detecting antigens specific for their leukemic cell type.

A method is described for raising antisera to human leukemia cells of an individual patient in mice rendered tolerant with cyclophosphamide to platelets obtained from the same patient. The resulting antisera are able to distinguish serologically between leukemic blast cells and remission cells of patients with acute leukemia and may be recognizing leukemia-associated antigens. The antisera are similar in activity to antisera raised following tolerance-induction with remission leukocytes, but larger volumes of anti-leukemia antiserum can be raised using the more easily obtainable platelets. This technique provides further evidence that human platelets share many of the antigens present on human leukocytes.

Blast cells were obtained from 17 patients with acute undifferentiated leukemia and 13 patients with chronic myelogenous leukemia in blast crisis. The blasts were tested with anti-i serum in cytotoxicity tests and with antisera to myeloblastic leukemia-associated antigens in immunofluorescence tests. The terminal deoxynucleotidyl transferase (TDT) content of the blasts was also measured. Lymphoblasts react strongly with anti-i, do not react with anti-myeloblast serum, and have high levels of TDT; myeloblasts react weakly with anti-i, do not react with anti-myeloblast serum, and have very low levels of TDT. Of the 17 patients with acute undifferentiated leukemia, there were six with blasts which reacted like lymphoblasts, six with blasts which reacted like myeloblasts, and five with blasts bearing different combinations of these lymphoblastic and myeloblastic markers. Eight of the 11 patients with lymphoblastic or mixed lymphoblastic-myeloblastic markers, but only one of the six with myeloblastic markers, achieved complete or partial remission in response to therapy. Thus, in acute undifferentiated leukemia, classification of blasts with these markers may be of prognostic value. Of the 13 patients with chronic myelogenous leukemia in blast crises, the markers were concordant (for myeloblasts) in only two cases. Three of the 13 patients had TDT-positive blasts, but the reactions of these cells with anti-i and with anti-myeloblast serum differed from those seen with lymphoblasts from patients with acute lymphoblastic leukemia. Although the cell involved in "lymphoid" blast crisis of chronic myelogenous leukemia is similar in many respects to that involved in acute lymphoblastic leukemia, these cells are not identical.

Peripheral blood myeloblasts from five patients with acute myeloblastic leukemia and peripheral remission leukocytes from two of these patients were radiolabeled by the lactoperoxidase-catalyzed surface radioiodination technique and incubated in a nutrient medium at 37 degrees. Radioactive materials shed from viable cells into the supernatant at 24 hr were purified by gel filtration and by DEAE-cellulose chromatography. The radiolabeled leukemic cells shed relatively few molecular species into the culture medium. The DEAE-cellulose eluate usually contained one major peak in which radioactivity and protein levels were coincident; the molecular weight of this compound was 350,000 to 400,000, and it contained carbohydrate as well as protein. Glycoprotein shed from leukemic cells was specifically reactive in a coprecipitation assay with defined antimyeloblast alloantisera obtained from leukemic patients receiving immunotherapy. No reaction was seen with antisera directed against HLA or B-cell antigens. Material shed from remission cells did not coprecipitate with antileukemic antisera. The isolation of radioactively labeled antigen derived from myeloblasts may ultimately allow the monitoring of human antigen levels in leukemic blood by radioimmunoassay.

We have studied immunologic reactivity to leukemia-associated antigens in patients with chronic myelocytic leukemia (CML) treated with chemotherapy and adjunctive immunotherapy. All patients were immunologically competent as measured by skin test reactivity to dinitrochlorobenzene. Immunotherapy consisted of allogeneic irradiated leukemic myeloblasts injected intradermally, with BCG vaccine (Research Foundation, Chicago, Ill.) given by multiple puncture at the same site. 10(9) cells plus BCG were given weekly for 4 wk, and 10(8) cells plus BCG were given at monthly intervals thereafter. Eight patients judged clinically to be in the stable phase of their disease developed circulating antibody against the immunizing blast cells demonstrable by cytotoxicity and immunofluorescence assays. The antibody also showed reactivity against a panel of myeloblasts (12 paients) but not against the corresponding remission lymphocytes (five patients) or normal lymphocytes (20 donors). In two cases the antibody showed reactivity against the patient's own leukemic blasts. Seven of these eight patients have maintained a steady clinical course ranging from 20 to 40 mo, while one entered the blastic phase and died. Six patients were judged to be in the aggressive phase of CML because of progressive leukocytosis and splenomegaly or increasing myeloblastosis; five died an average of 16 mo after diagnosis. Humoral antibodies were not detected in these patients after repeated courses of BCG and allogeneic leukemic cells. We conclude that specific active immunotherapy of patients with CML can abet the production of humoral antibody against blast cell antigens and that this response may be impaired during the aggressive phase of the disease.

Antigenic compounds from the surface of leukemic myeloblasts are shed in vitro on short-term culture. Blast cells radiolabeled by lactoperoxidase iodination release soluble compounds that react immunologically with alloantisera to leukemia-associated antigens. Partially characterized soluble antigens were used to raise heteroantisera in monkeys that are selectively reactive with leukemic myeloblasts and unreactive with nonleukemic cells. Monkey heteroantisera were used to further characterize soluble leukemia antigens. Sera from patients with acute myeloblastic leukemia inhibit the reactivity of the heteroantisera, suggesting that soluble leukemic antigen is released in vivo as well.

We tested serial bone-marrow samples from 47 adults with acute myeloblastic leukemia in remission for reactivity with heteroantiserums to leukemia-associated antigens, to determine whether imminent relapse could be detected in patients with acute leukemia. Of 26 patients who relapsed by standard morphologic criteria, 21 had increased immunoreactivity of bone marrow for one to six months (mean, 3.7 months) before relapse. High concordance was observed between a positive test and relapse during the period of study (chi-square = 27.53, P less than 0.001). The median time to relapse after a positive test was four months, as compared with the median remission duration of 19 months for the whole group (P less than 0.02, Peto's log-rank analysis). Serologic detection of leukemia-associated antigens in marrow may be a reliable indicator of imminent relapse in acute myeloblastic leukemia.