A phase 1 study of bortezomib and romidepsin in patients with chronic lymphocytic leukemia/small lymphocytic lymphoma, indolent B-cell lymphoma, peripheral T-cell lymphoma, or cutaneous T-cell lymphoma.

A phase 1 study was conducted to determine the dose-limiting toxicities and maximum-tolerated dose (MTD) for bortezomib followed by romidepsin on days 1, 8, and 15 in patients with relapsed/refractory CLL/SLL or B- or T-cell lymphoma. Eighteen treated patients were evaluable for response. The MTD was 1.3 mg/m2 bortezomib and 10 mg/m2 romidepsin; median treatment duration was 3 cycles at this dose. The dose-limiting toxicities were grade 3 fatigue, vomiting, and chills. Two patients had partial responses, one lasting >2 years, 8 had stable disease, and 8 had progressive disease. The median duration of stable disease was 3.5 cycles. Correlative studies examining expression of NF-кB, XIAP, Bcl-xL, and Bim yielded variable results. The safety profile was consistent with that reported for single-agent bortezomib and romidepsin. This regimen has modest activity in heavily pretreated patients with relapsed/refractory CLL or B- or T-cell lymphoma. NCT00963274.

A Phase II Trial of AZD6244 (Selumetinib, ARRY-142886), an Oral MEK1/2 Inhibitor, in Relapsed/Refractory Multiple Myeloma.

PURPOSE: AZD6244 is a MEK1/2 inhibitor with significant preclinical activity in multiple myeloma cells. This phase II study used a two-stage Simon design to determine the AZD6244 response rate in patients with relapsed or refractory multiple myeloma. EXPERIMENTAL DESIGN: AZD6244 (75 mg) was administered orally, twice a day, continuously for 28-day cycles. Response was evaluated after three cycles. RESULTS: Thirty-six patients received therapy. The median age was 65 years (range: 43-81) and the median number of prior therapies was 5 (range: 2-11). The most common grade 3 and 4 toxicities included anemia, neutropenia, thrombocytopenia, diarrhea, and fatigue. Three deaths occurred possibly related to AZD6244 (2 due to sepsis, 1 due to acute kidney injury). After AZD6244 discontinuation, three additional deaths occurred due to disease progression. The response rate (CR + PR) was 5.6% with a mean duration of response of 4.95 months and median progression-free survival time of 3.52 months. One patient had a very good partial response (VGPR), 1 patient had a partial response, 17 patients had stable disease, 13 patients had progressive disease, and 4 patients could not be assessed for response. Pharmacodynamic studies revealed variable effects on bone marrow CD138(+) cell MEK1/2 and ERK1/2 phosphorylation. The best clinical response, a prolonged VGPR, occurred in a patient with an MMSET translocation. CONCLUSIONS: Single-agent AZD6244 was tolerable and had minimal activity in this heavily pretreated population.

Phase 1 trial of carfilzomib (PR-171) in combination with vorinostat (SAHA) in patients with relapsed or refractory B-cell lymphomas.

A phase 1 study with carfilzomib and vorinostat was conducted in 20 B-cell lymphoma patients. Vorinostat was given orally twice daily on days 1, 2, 3, 8, 9, 10, 15, 16, and 17 followed by carfilzomib (given as a 30-min infusion) on days 1, 2, 8, 9, 15, and 16. A treatment cycle was 28 days. Dose escalation initially followed a standard 3 + 3 design, but adapted a more conservative accrual rule following dose de-escalation. The maximum tolerated dose was 20 mg/m2 carfilzomib and 100 mg vorinostat (twice daily). The dose-limiting toxicities were grade 3 pneumonitis, hyponatremia, and febrile neutropenia. One patient had a partial response and two patients had stable disease. Correlative studies showed a decrease in NF-κB activation and an increase in Bim levels in some patients, but these changes did not correlate with clinical response.

Phase I trial of bortezomib (PS-341; NSC 681239) and "nonhybrid" (bolus) infusion schedule of alvocidib (flavopiridol; NSC 649890) in patients with recurrent or refractory indolent B-cell neoplasms.

PURPOSE: This phase I study was conducted to determine the dose-limiting toxicities (DLT) and maximum tolerated dose (MTD) for the combination of bortezomib and alvocidib in patients with B-cell malignancies (multiple myeloma, indolent lymphoma, Waldenstrom macroglobulinemia, and mantle cell lymphoma). EXPERIMENTAL DESIGN: Patients received bortezomib (intravenous push), followed by alvocidib (1-hour infusion), on days 1, 4, 8, and 11 of a 21-day treatment cycle. Patients experiencing responses or stable disease continued on treatment at the investigator's discretion. A standard 3+3 dose-escalation design was used to identify the MTD based on DLTs, and pharmacokinetic and pharmacodynamic studies were conducted. RESULTS: A total of 44 patients were enrolled, with 39 patients assessed for response. The MTD was established as 1.3 mg/m(2) for bortezomib and 40 mg/m(2) for alvocidib. The most common hematologic toxicities included leukopenia, lymphopenia, neutropenia, and thrombocytopenia. The most common nonhematologic toxicities included diarrhea, fatigue, and sensory neuropathy. Three complete remissions (8%) and 10 partial remissions (26%) were observed for a total response rate of 33%. Pharmacokinetic findings with the current dosing regimen were consistent with the comparable literature and the hybrid dosing regimen. Pharmacodynamic study results did not correlate with clinical responses. CONCLUSIONS: The combination of bortezomib and alvocidib is tolerable, and an MTD has been established for this schedule. The regimen appears to be efficacious in patients with relapsed/refractory multiple myeloma or indolent non-Hodgkin lymphoma. As the nonhybrid regimen is less cumbersome than the previous hybrid dosing schedule regimen, the current schedule is recommended for successor studies.

Fractal organization of the human T cell repertoire in health and after stem cell transplantation.

T cell repertoire diversity is generated in part by recombination of variable (V), diversity (D), and joining (J) segments in the T cell receptor ß (TCR) locus. T cell clonal frequency distribution determined by high-throughput sequencing of TCR ß in 10 stem cell transplantation (SCT) donors revealed a fractal, self-similar frequency distribution of unique TCR bearing clones with respect to V, D, and J segment usage in the T cell repertoire of these individuals. Further, ranking of T cell clones by frequency of gene segment usage in the observed sequences revealed an ordered distribution of dominant clones conforming to a power law, with a fractal dimension of 1.6 and 1.8 in TCR ß DJ and VDJ containing clones in healthy stem cell donors. This self-similar distribution was perturbed in the recipients after SCT, with patients demonstrating a lower level of complexity in their TCR repertoire at day 100 followed by a modest improvement by 1 year post-SCT. A large shift was observed in the frequency distribution of the dominant T cell clones compared to the donor, with fewer than one third of the VDJ-containing clones shared in the top 4 ranks. In conclusion, the normal T cell repertoire is highly ordered with a TCR gene segment usage that results in a fractal self-similar motif of pattern repetition across levels of organization. Fractal analysis of high-throughput TCR ß sequencing data provides a comprehensive measure of immune reconstitution after SCT.

Phase I trial of bortezomib (PS-341; NSC 681239) and alvocidib (flavopiridol; NSC 649890) in patients with recurrent or refractory B-cell neoplasms.

PURPOSE: A phase I study was conducted to determine the dose-limiting toxicities (DLT) and maximum tolerated dose (MTD) for the combination of bortezomib and alvocidib in patients with B-cell malignancies (multiple myeloma, indolent lymphoma, and mantle cell lymphoma). EXPERIMENTAL DESIGN: Patients received bortezomib by intravenous push on days 1, 4, 8, and 11. Patients also received alvocidib on days 1 and 8 by 30-minute bolus infusion followed by a 4-hour continuous infusion. Treatment was on a 21-day cycle, with indefinite continuation for patients experiencing responses or stable disease. Dose escalation employed a standard 3 + 3 design until the MTD was identified on the basis of DLTs. Pharmacokinetic studies and pharmacodynamic studies were conducted. RESULTS: Sixteen patients were treated. The MTD was established as 1.3 mg/m(2) for bortezomib and 30 mg/m(2) for alvocidib (both the 30-minute bolus and 4-hour infusions). Common hematologic toxicities included leukopenia, lymphopenia, neutropenia, and thrombocytopenia. Common nonhematologic toxicities included fatigue and febrile neutropenia. DLTs included fatigue, febrile neutropenia, and elevated aspartate aminotransferase (AST) levels. Two complete responses (CR; 12%) and five partial responses (PR; 31%) were observed at the MTD (overall response rate = 44%). Pharmacokinetic results were typical for alvocidib and pharmacodynamic studies yielded variable results. CONCLUSIONS: The combination of bortezomib and alvocidib is tolerable and an MTD has been established for the tested schedule. The regimen appears active in patients with relapsed and/or refractory multiple myeloma or non-Hodgkin's lymphoma, justifying phase II studies to determine the activity of this regimen more definitively.

Evaluation of molecular markers of mesenchymal phenotype in melanoma.

The epithelial to mesenchymal transition is a developmental process allowing epithelial cells to dedifferentiate into cells displaying mesenchymal phenotypes. The pathological role of epithelial to mesenchymal transition has been implicated in invasion and metastasis for numerous carcinomas, yet limited data exist addressing whether mesenchymal transition (MT) occurs in malignant melanoma cells. Our group developed an in-vitro three-dimensional culture system to address MT in melanoma cells upon transforming growth factor-ß/ tumor necrosis factor-α treatment. Loss of E-cadherin is one of the best indicators of MT in epithelial cells. Not surprisingly, E-cadherin was expressed in only three of 12 (25%) melanoma cell lines and all three mesenchymal proteins, N-cadherin, vimentin, and fibronectin, were expressed by seven (58%) melanoma cell lines. However, after cytokine treatment, two or more mesenchymal proteins were elevated in nine (75%) melanoma cell lines. Data support the transforming growth factor-ß production by melanoma cells which may induce/support MT. Evaluation of E-cadherin, N-cadherin, and Snail expression in melanoma tissue samples are consistent with an inverse coupling of E-cadherin and N-cadherin expression, however, there are also examples suggesting a more complex control of their expression. These results indicate that malignant melanoma cell lines are susceptible to MT after cytokine treatment and highlight the importance of understanding the effects of cytokines on melanoma to undergo MT.

The effect of intraoperative specimen inking on lumpectomy re-excision rates.

BACKGROUND: Lumpectomy re-excision to obtain negative margins is common. We compare the effect of two specimen orientation approaches on lumpectomy re-excision rates. METHODS: All women undergoing lumpectomy for breast cancer by a single surgeon between 03/2007 - 02/2009 were included. Lumpectomies underwent standard inking (SI) after surgery by a pathologist from 03/2007-02/2008 while intraoperative inking (II) with direct surgeon input was done from 03/2008-02/2009. Rates of margin positivity and re-excision were compared between these methods. RESULTS: 65 patients were evaluated, reflecting SI in 39 and II in 26 cases. Margin positivity rates of 46% [SI] vs. 23% [II] (p = 0.06) and re-excision rates of 38% [SI] vs. 19% [II] were observed. Residual disease at re-excision was found in 27% [SI] vs. 67% [II] of cases. CONCLUSIONS: Intraoperative inking in this practice offered a simple way to reduce re-excision rates after lumpectomy and affect an improvement in quality of patient care.

Treatment of ovarian cancer cell lines with 5-aza-2'-deoxycytidine upregulates the expression of cancer-testis antigens and class I major histocompatibility complex-encoded molecules.

PURPOSE: To test the hypothesis that decrease in DNA methylation will increase the expression of cancer-testis antigens (CTA) and class I major histocompatibility complex (MHC)-encoded molecules by ovarian cancer cells, and thus increase the ability of these cells to be recognized by antigen-reactive CD8(+) T cells. METHODS: Human ovarian cancer cell lines were cultured in the presence or absence of varying concentrations of the DNA demethylating agent 5-aza-2'-deoxycytidine (DAC) for 3-7 days. The expression levels of 12 CTA genes were measured using the polymerase chain reaction. The protein expression levels of class I MHC molecules and MAGE-A1 were measured by flow cytometry. T cell reactivity was determined using interferon-gamma ELISpot analysis. RESULTS: DAC treatment of ovarian cancer cell lines increased the expression of 11 of 12 CTA genes tested including MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, NY-ESO-1, TAG-1, TAG-2a, TAG-2b, and TAG-2c. In contrast, DAC treatment decreased the already low expression of the MAGE-A2 gene by ovarian cancer cells, a finding not previously observed in cancers of any histological type. DAC treatment increases the expression of class I MHC molecules by the cells. These effects were time-dependent over a 7-day interval, and were dose-dependent up to 1-3 microM for CTA and up to 10 microM for class I MHC molecules. Each cell line tested had a unique pattern of gene upregulation after exposure to DAC. The enhanced expression levels increased the recognition of 2 of 3 antigens recognized by antigen-reactive CD8(+) T cells. CONCLUSIONS: These results demonstrate the potential utility of combining DAC therapy with vaccine therapy in an attempt to induce the expression of antigens targeted by the vaccine, but they also demonstrate that care must be taken to target inducible antigens.

The use of gamma-irradiation and ultraviolet-irradiation in the preparation of human melanoma cells for use in autologous whole-cell vaccines.

BACKGROUND: Human cancer vaccines incorporating autologous tumor cells carry a risk of implantation and subsequent metastasis of viable tumor cells into the patient who is being treated. Despite the fact that the melanoma cell preparations used in a recent vaccine trial (Mel37) were gamma-irradiated (200 Gy), approximately 25% of the preparations failed quality control release criteria which required that the irradiated cells incorporate 3H-thymidine at no more than 5% the level seen in the non-irradiated cells. We have, therefore, investigated ultraviolet (UV)-irradiation as a possible adjunct to, or replacement for gamma-irradiation. METHODS: Melanoma cells were gamma- and/or UV-irradiated. 3H-thymidine uptake was used to assess proliferation of the treated and untreated cells. Caspase-3 activity and DNA fragmentation were measured as indicators of apoptosis. Immunohistochemistry and Western blot analysis was used to assess antigen expression. RESULTS: UV-irradiation, either alone or in combination with gamma-irradiation, proved to be extremely effective in controlling the proliferation of melanoma cells. In contrast to gamma-irradiation, UV-irradiation was also capable of inducing significant levels of apoptosis. UV-irradiation, but not gamma-irradiation, was associated with the loss of tyrosinase expression. Neither form of radiation affected the expression of gp100, MART-1/MelanA, or S100. CONCLUSION: These results indicate that UV-irradiation may increase the safety of autologous melanoma vaccines, although it may do so at the expense of altering the antigenic profile of the irradiated tumor cells.

Evaluation of the sentinel immunized node for immune monitoring of cancer vaccines.

BACKGROUND: We hypothesized that lymph nodes draining sites of cutaneous vaccination could be identified by sentinel node biopsy techniques, and that measuring T-cell response with lymphocytes obtained from these lymph nodes would provide a more sensitive measure of immunogenicity than would the same measurement made with peripheral blood lymphocytes (PBL). METHODS: ELISpot analysis was used to determine the magnitude of vaccine-specific T-cell response in the sentinel immunized nodes (SIN), random lymph nodes, and peripheral blood lymphocytes (PBL) obtained from patients enrolled in clinical trials of experimental melanoma vaccines. RESULTS: The SIN biopsy was successful in 97% of cases and morbidity was very low. The T-cell response to vaccination was detected with greater sensitivity in the SIN (57%) than in PBL (39%), and evaluation of T-cell responses in the SIN and the PBL together yielded T-cell responses in 63% of patients. When the T-cell responses from a SIN and a random lymph node were compared in four patients, immune responses were detected to one of the vaccine peptides in three of these four patients. In all of those cases, responses were present in the SIN but absent from the random lymph node. CONCLUSION: Measurements of T-cell responsiveness to cutaneous immunization are more frequently positive in the SIN than they are in the PBL, however evaluation of both the SIN and PBL permit a more sensitive measure of T-cell immunogenicity than use of either single source.

A multipeptide vaccine is safe and elicits T-cell responses in participants with advanced stage ovarian cancer.

Nine participants with epithelial ovarian, fallopian tube, or primary peritoneal carcinoma, who were human leukocyte antigen (HLA)-A1, HLA-A2, or HLA-A3, were eligible to enroll in a phase 1 study designed to assess the safety and immunogenicity of a peptide-based vaccine. Participants received 5 class I major histocompatibility complex-restricted synthetic peptides derived from multiple ovarian cancer-associated proteins plus a class II major histocompatibility complex-restricted synthetic helper peptide derived from tetanus toxoid protein. The vaccines were administered with granulocyte macrophage-colony stimulating factor in Montanide ISA-51 adjuvant over a 7-week period. All vaccine-related toxicities were grade 1 to 2, the most common being injection site reaction (grade 2, 100%), fatigue (grade 1, 78%), and headache (grade 1, 67%). Lymphocytes from the peripheral blood and a node draining a secondary vaccine site (sentinel immunized node) were harvested during the course of vaccination and T-cell responses to the peptides were evaluated using an enzyme-linked immunosorbent spot assay. CD8 T-cell responses were detected in 1 participant ex vivo and in 8 of 9 participants (89%) after in vitro stimulation. All 4 HLA-A2 and HLA-A3-restricted peptides were immunogenic. This includes 2 peptides, folate binding protein (FBP191-199) and Her-2/neu754-762, which had not previously been evaluated in vaccines in humans. Responding T cells required over 200 nM for half-maximal reactivity. These data support continued investigation of these peptides as immunogens for patients with ovarian cancer but, owing to low potency, also suggest a need for additional immunomodulation in combination with vaccines to increase the magnitude and to improve the quality of the T-cell responses.

The TAG family of cancer/testis antigens is widely expressed in a variety of malignancies and gives rise to HLA-A2-restricted epitopes.

The TAG-1, TAG-2a, TAG-2b, and TAG-2c cancer/testis genes, known to be expressed in an unusually high percentage of melanoma cell lines, are shown here to be expressed in a variety of tumor lines of diverse histologic type, including cancers of the brain, breast, colon, lung, ovary, pharynx, and tongue. The genes are also expressed in fresh, uncultured melanoma, and ovarian cancer cells. Epitope prediction algorithms were used to identify potential HLA-A1, HLA-A2, HLA-A3, HLA-B7, and HLA-B8 epitopes, and these potential epitopes were tested for their ability to stimulate a peptide-specific cytotoxic T lymphocyte response using lymphocytes from healthy donors. Two HLA-A2-restricted epitopes (SLGWLFLLL and LLLRLECNV) were identified using this approach. Cytotoxic T lymphocytes specific for each of these peptides were capable of recognizing tumor cells expressing both the corresponding class I major histocompatibility complex encoded molecule and the TAG genes. These results indicate that TAG-derived peptides may be good components of a therapeutic vaccine designed to target melanoma and a variety of epithelial cell-derived malignancies.

Immunological profiling of a panel of human ovarian cancer cell lines.

PURPOSE: The efficient identification of peptide antigens recognized by ovarian cancer-specific cytotoxic T lymphocytes (CTL) requires the use of well-characterized ovarian cancer cell lines. To develop such a panel of cell lines, 11 ovarian cancer cell lines were characterized for the expression of class I and class II major histocompatibility complex (MHC)-encoded molecules, 15 tumor antigens, and immunosuppressive cytokines [transforming growth factor beta (TGF-beta) and IL-10]. METHODS: Class I MHC gene expression was determined by polymerase chain reaction (PCR), and class I and class II MHC protein expression was determined by flow cytometry. Tumor antigen expression was determined by a combination of polymerase chain reaction (PCR) and flow cytometry. Cytokine expression was determined by ELISA. RESULTS: Each of the ovarian cancer cell lines expresses cytokeratins, although each cell line does not express the same cytokeratins. One of the lines expresses CD90, which is associated with a fibroblast lineage. Each of the cell lines expresses low to moderate amounts of class I MHC molecules, and several of them express low to moderate amounts of class II MHC molecules. Using a combination of PCR and flow cytometry, it was determined that each cell line expressed between six and thirteen of fifteen antigens tested. Little to no TGF-beta3 was produced by any of the cell lines, TGF-beta1 was produced by three of the cell lines, TGF-beta2 was produced by all of the cell lines, with four of the cell lines producing large amounts of the latent form of the molecule, and IL-10 was produced by one of the cell lines. CONCLUSIONS: Each of the 11 ovarian cancer lines is characterized by a unique expression pattern of epithelial/fibroblast markers, MHC molecules, tumor antigens, and immunosuppressive cytokines. Knowledge of these unique expression patterns will increase the usefulness of these cell lines in identifying the antigens recognized by ovarian cancer-specific CTL.

Use of selected reaction monitoring mass spectrometry for the detection of specific MHC class I peptide antigens on A3 supertype family members.

The development of peptide-based vaccines that are useful in the therapeutic treatment of melanoma and other cancers ultimately requires the identification of a sufficient number of antigenic peptides so that most individuals, regardless of their major histocompatibility complex (MHC)-encoded class I molecule phenotype, can develop a cytotoxic T lymphocyte (CTL) response against one or more peptide components of the vaccine. While it is relatively easy to identify antigenic peptides that are presented by the most prevalent MHC class I molecules in the population, it is problematic to identify antigenic peptides that are presented by MHC class I molecules that have less frequent expression in the population. One manner in which this problem can be overcome is by taking advantage of known MHC class I supertypes, which are groupings of MHC class I molecules that bind peptides sharing a common motif. We have developed a mass spectrometric approach which can be used to determine if an antigenic peptide is naturally processed and presented by any given MHC class I molecule. This approach has been applied to the A3 supertype, and the results demonstrate that some, but not all, A3 supertype family-associated peptides can associate with all A3 supertype family members. The approach also demonstrates the shared nature of several newly identified peptide antigens. The use of this technology negates the need to test peptides for their ability to stimulate CTL responses in those cases where the peptide is not naturally processed and bound to the target MHC class I molecule of interest, thus allowing resources to be focused on the most promising vaccine candidates.

Preventing the spontaneous modification of an HLA-A2-restricted peptide at an N-terminal glutamine or an internal cysteine residue enhances peptide antigenicity.

The p68-derived peptide, QIVDVCHDV, was identified by a reverse immunology approach as capable of reconstituting an epitope recognized by the melanoma-reactive cytotoxic T lymphocyte (CTL) line VMM5. The peptide has not been demonstrated definitively on the cell surface by mass spectrometry; thus, it is not yet considered appropriate for use in human melanoma vaccines. Interestingly, however, the antigenicity of this peptide was affected by spontaneous modifications at two distinct residues. Spontaneous modification of the QIVDVCHDV peptide can occur at the cysteine residue at position 6 or at the N-terminal glutamine residue, and both modifications dramatically affect CTL recognition. Avoidance of an acidic environment prevents the conversion of the N-terminal glutamine residue to pyroglutamic acid, a conversion that inhibits binding of the peptide to HLA-A2 and diminishes recognition by CTLs. Substitution of asparagine for the N-terminal glutamine and substitution of serine for the cysteine were shown to enhance the binding of the peptide to HLA-A2 and to enhance the recognition of the peptide by CTLs. These findings suggest general strategies for enhancing the antigenicity of other peptides containing similar amino acids in their sequence.

Identification of novel and widely expressed cancer/testis gene isoforms that elicit spontaneous cytotoxic T-lymphocyte reactivity to melanoma.

Multiple isoforms (TAG-1, TAG-2a, TAG-2b, and TAG-2c) of a novel cancer/testis antigen gene have been identified and are expressed in 84-88% of melanoma cell lines tested. The tumor antigen (TAG) genes are also expressed in K562, a myelogenous leukemia cell line, and they have homology to two chronic myelogenous leukemia-derived clones and a hepatocellular carcinoma clone in the human expressed sequence tags (EST) database, thus indicating that their expression is not restricted to melanomas. In contrast to the fact that many cancer/testis antigens are poorly immunogenic, the TAG-derived peptide, RLSNRLLLR, is recognized by HLA-A3-restricted, melanoma-specific CTLs that were obtained from a melanoma patient with spontaneous reactivity to the peptide. Unlike most cancer/testis antigen genes which are located on the X chromosome, the TAG genes are located on chromosome 5. The genes have the additional unusual features of being coded for in an open reading frame that is initiated by one of three nonstandard initiation codons, and the sequence coding the RLSNRLLLR peptide crosses an exon-exon boundary. The properties of the TAG antigens indicate that they are excellent vaccine candidates for the treatment of melanoma and perhaps other cancers.

Genomic organization, incidence, and localization of the SPAN-x family of cancer-testis antigens in melanoma tumors and cell lines.

PURPOSE: Members of the SPAN-X (sperm protein associated with the nucleus mapped to the X chromosome) family of cancer-testis antigens are promising targets for tumor immunotherapy because they are normally expressed exclusively during spermiogenesis on the adluminal side of the blood-testis barrier, an immune privileged compartment. EXPERIMENTAL DESIGN AND RESULTS: This study analyzed the human SPANX genomic organization, as well as SPAN-X mRNA and protein expression in somatic and cancer cells. The SPANX family consists of five genes, one of which is duplicated, all located in a gene cluster at Xq27.1. From the centromere, the arrangement of the five SPANX genes mapped on one contiguous sequence is SPANXB, -C, -A1, -A2, and -D. Reverse transcription-PCR analyses demonstrated expression of SPAN-X mRNA in melanoma and ovarian cell lines, and virtual Northern analysis established SPANX gene expression in numerous cancer cell lines. Immunoblot analysis using polyclonal antisera raised against recombinant SPAN-X confirmed the translation of SPAN-X proteins in melanoma and ovarian tumor cell lines. The immunoreactive proteins migrated between M(r) 15,000 and M(r) 20,000 similar to those observed in spermatozoa. Immunoperoxidase labeling of melanoma cells and tissue sections demonstrated SPAN-X protein localization in the nucleus, cytoplasm, or both. Ultrastructurally, in melanoma cells with nuclear SPAN-X, the protein was associated with the nuclear envelope, a localization similar to that observed in human spermatids and spermatozoa. Significantly, the incidence of SPAN-X-positive immunostaining was greatest in the more aggressive skin tumors, particularly in distant, nonlymphatic metastatic melanomas. CONCLUSIONS: The data herein suggest that the SPAN-X protein may be a useful target in cancer immunotherapy.

Identification of a shared epitope recognized by melanoma-specific, HLA-A3-restricted cytotoxic T lymphocytes.

We previously established a melanoma-reactive cytotoxic T lymphocyte (CTL) line that recognizes multiple epitopes in the context of HLA-A3. To increase the number of peptides available for use in a vaccine for the treatment of melanoma, we identified one of these epitopes, SQNFPGSQK, through a combination of epitope reconstitution experiments and mass spectrometry. The SQNFPGSQK peptide was also found to be associated with HLA-A3 on an additional melanoma tumor line, thus indicating that the peptide is not unique to the melanoma tumor line from which it was isolated and thus, unlikely to arise through a mutational event. Although the protein origin of SQNFPGSQK has yet to be established, the shared nature of this epitope and the fact that it elicits a natural immune response indicates that it warrants further study to determine its usefulness as a vaccine component for the treatment of melanoma. The peptide may also be useful as a research tool for evaluating spontaneous anti-tumor immune responses in patients with melanoma.

A fractal clonotype distribution in the CD8+ memory T cell repertoire could optimize potential for immune responses.

The nature of CD8(+) T cell memory is still incompletely understood. We have previously reported that the response to an HLA-A2-restricted influenza-derived peptide results in a complex T cell repertoire. In this study we extend this analysis and describe the repertoire with more rigor. In one individual we defined 141 distinct T cell clonotypes on the basis of the unique DNA sequence of the third complementarity-determining region of the TCR beta-chain. The frequency distribution of the clonotypes is not what is expected of a normal distribution but is characterized by a large low-frequency tail. The existence of a complex population indicates a mechanism for maintaining a large number of Ag-specific clonotypes at a low frequency in the memory pool. Ranking the clonotypes allowed us to describe the population in terms of a power law-like distribution with a parameter of decay of approximately 1.6. If the repertoire is divided into subsets, such as clonotypes that use BJ2.7 or those whose third complementarity-determining region encodes the amino acid sequence IRSS, the clonotype frequencies could also be described by a power law-like distribution. This indicates a self similarity to the repertoire in which smaller pieces are slightly altered copies of the larger piece. The power law-like description is stable with time and was observed in a second individual. The distribution of clonotypes in the repertoire could be mapped onto a polygonal spiral using a recursive algorithm. Self similarity, power laws, and recursive mapping algorithms are associated with fractal systems. Thus, Ag-specific memory CD8 T cell repertoires can be considered as fractal, which could indicate optimized flexibility and robustness.