Fanconi anemia (FA) is a rare autosomal recessive cancer susceptibility disorder characterized by cellular hypersensitivity to mitomycin C (MMC). Six FA genes have been cloned, but the gene or genes corresponding to FA subtypes B and D1 remain unidentified. Here we show that cell lines derived from FA-B and FA-D1 patients have biallelic mutations in BRCA2 and express truncated BRCA2 proteins. Functional complementation of FA-D1 fibroblasts with wild-type BRCA2 complementary DNA restores MMC resistance. Our results link the six cloned FA genes with BRCA1 and BRCA2 in a common pathway. Germ-line mutation of genes in this pathway may result in cancer risks similar to those observed in families with BRCA1 or BRCA2 mutations.

Melanoma lines MEL.A and MEL.B were derived from metastases removed from patient LB33 in 1988 and 1993, respectively. The MEL.A cells express several antigens recognized by autologous cytolytic T lymphocytes (CTL) on HLA class I molecules. The MEL.B cells have lost expression of all class I molecules except for HLA-A24. By stimulating autologous lymphocytes with MEL.B, we obtained an HLA-A24-restricted CTL clone that lysed these cells. A novel gene, PRAME, encodes the antigen. It is expressed in a large proportion of tumors and also in some normal tissues, albeit at a lower level. Surprisingly, the CTL failed to lyse MEL.A, even though these cells expressed the gene PRAME. The CTL expresses an NK inhibitory receptor that inhibits its lytic activity upon interaction with HLA-Cw7 molecules, which are present on MEL.A cells and not on MEL.B. Such CTL, active against tumor cells showing partial HLA loss, may constitute an intermediate line of anti-tumor defense between the CTL, which recognize highly specific tumor antigens, and the NK cells, which recognize HLA loss variants.

Using molecular dynamics simulations with a many-body force field, we studied the deformation of single crystal Ni and NiCu random alloy nanowires subjected to uniform strain rates but kept at 300 K. For all strain rates, the Ni nanowire is elastic up to 7.5% strain with a yield stress of 5.5 GPa, far above that of bulk Ni. At high strain rates, we find that for both systems the crystalline phase transforms continuously to an amorphous phase, exhibiting a dramatic change in atomic short-range order and a near vanishing of the tetragonal shear elastic constant perpendicular to the tensile direction. This amorphization which occurs directly from the homogeneous, elastically deformed system with no chemical or structural inhomogeneities exhibits a new mode of amorphization.[ S0031-9007(99) We used molecular dynamics (MD) simulations to strain fcc crystalline nanowires at a uniform rate along the ͗001͘ crystallographic direction [2][3][4]. Here, to realize an infinite nanowire we applied 1D periodic boundary condition in the c direction (an initial length of ten fcc unit cells, ഠ4 nm, while the a and b directions are five fcc cells long, ഠ2 nm). We studied the deformation behavior at constant strain rates (0.05% to 5% ps 21 ) and constant temperature (300 K). The tensile strain component´3 3 was applied uniformly (in increments of 0.5%) to obtain the specified strain rate. The average stress components s ij in the specimen were computed at and following each applied strain increment. At the strain rates reported here, the stress distribution relaxes between strain increments to a homogeneous stationary equilibrium state of uniaxial tensile stress. We observe a crystal to glass transformation at 300 K above a critical strain rate.Such large strain rates are observed experimentally only in shock wave and high velocity impact studies, where it has been difficult to control temperature or to obtain details about dynamic structural changes. Experiments at such high strain rates lead to shear localization arising from adiabatic heat dissipation and local thermal softening of the material, which results in highly nonequilibrium systems that are difficult to study experimentally. The MD simulations follow the effects of loading and loading rate independently from those arising from heat dissipation and concomitant temperature increases.To illustrate the effect of strain rate on the detailed deformation, Fig. 1 displays snapshots of MD simulations on an fcc NiCu random alloy nanowire (at 300 K) deformed to 100% strain at strain rates from 0.5% and 5% ps 21 in the z direction. For ᠨ 0.5% ps 21 cooperative shear events within the crystal produce coherent shear bands, which are often coherent "twins." Multiple coherent shearing events finally lead to necking before failure. For ᠨ 5% ps 21 , the behavior is fundamentally different. No coherent shear bands or twins form as the system is strained. Instead, the specimen transforms homogeneously to an amorphous state at strains of only 0.15. This homogeneously disordered material un...

We have studied the patterns of antigens recognized by autologous cytolytic T lymphocytes (CTL) on two melanoma cell lines derived from metastases that were removed from patient LB33 at several years distance. Cell line LB33-MEL.A was obtained after surgery in 1988. A large number of CTL clones directed against LB33-MEL.A was obtained with blood lymphocytes collected from the patient in 1990. In vitro selection of melanoma cells that were resistant to these CTL clones indicated that at least five different antigens were recognized on LB33-MEL.A by autologous CTL. Four of these antigens were found to be presented by HLA-A28, B13, B44 and Cw6, respectively. The patient remained disease-free until 1993 when a metastasis was detected and was used to obtain cell line LB33-MEL.B. This cell line proved resistant to lysis by all the CTL clones directed against the LB33-MEL.A cells and showed no expression of HLA class I molecules except for HLA-A24. Using LB33-MEL.B cells to stimulate blood lymphocytes collected from the patient in 1994 we derived CTL clones that lysed these cells. All these CTL clones recognized a new antigen presented by HLA-A24. These results suggest that in patient LB33 the melanoma cells may have lost the expression of several HLA molecules under the selective pressure of an anti-tumor CTL response.

Tightly regulated at the level of transcription, expression of MHC class II molecules varies significantly among gastrointestinal cancers. High levels of MHC class II expression are often associated with a better prognosis, which is indicative of the involvement of CD4 þ lymphocytes in tumor suppression, but the molecular mechanism by which MHC class II expression is regulated remains unclear. In the present study, we investigated the expression of one inducible MHC class II molecule, HLA-DR, and its coactivators in a panel of colorectal and gastric cancer cell lines. Interferon-c induced expression of HLA-DR in 14 of 20 cell lines tested; the remaining six cell lines did not express HLA-DR. Analysis of the expression of transcription factors and coactivators associated with HLA-DR revealed that the loss of CIITA expression was closely associated with the absence of HLA-DR induction. Moreover, DNA methylation of the 5 0 CpG island of CIITA-PIV was detected in all cancer cells that lacked CIITA. The methylation and resultant silencing of CIITA-PIV depended on the activities of two DNA methyltransferases, DNMT1 and DNMT3B, and their genetic inactivation restored CIITA-PIV expression. It thus appears that CIITA methylation is a key mechanism that enables some gastrointestinal cancer cells to escape immune surveillance.

Summary. PRAME (Preferentially expressed antigen of melanoma) has been previously identified as a melanoma antigen recognized by cytotoxic T cells (CTLs) and found to be expressed in a variety of cancer cells including leukaemic cells. We have screened 98 Japanese patients with leukaemia and lymphoma for expression of the PRAME gene using semiquantitative reverse transcription polymerase chain reaction (RT-PCR). Forty-one patients (42%) showed high levels of PRAME expression. Eight of these patients were then monitored using real-time PCR for a period of 10±37 months. Significant reductions in the PRAME expression were observed in all patients after chemotherapy. An increased expression was detected in the two patients who relapsed, one of which was before cytological diagnosis. These changes were correlated with those of other known genetic markers, such as the bcr-abl gene. Therefore, quantitative monitoring of the PRAME gene using real-time PCR method may be useful for detecting minimal residual disease and to predict subsequent relapse, especially in patients without known genetic markers. In addition, a PRAME-positive leukaemia cell line and fresh leukaemic cells were found to be susceptible to lysis by PRAME-specific CTLs established from a patient with melanoma, suggesting that the PRAME peptide can also be a target leukaemia antigen for T cells.

We reported previously a HLA-A24-restricted antigenic peptide, survivin-2B80-88 (AYACNTSTL), recognized by CD8 + CTL. This peptide was derived from survivin protein, an inhibitor of apoptosis proteins, expressed in a variety of tumors, such as adenocarcinoma, squamous cell carcinoma, and malignant melanoma. In this report, we provide further evidence that survivin-2B80-88 peptide might serve as a potent immunogenic cancer vaccine for various cancer patients. Overexpression of survivin was detected in surgically resected primary tumor specimens of most breast and colorectal cancers and some gastric cancers as assessed by immunohistochemical study. HLA-A24/survivin-2B80-88 tetramer analysis revealed that there existed an increased number of CTL precursors in peripheral blood mononuclear cells (PBMC) of HLA-A24 + cancer patients, and in vitro stimulation of PBMCs from six breast cancer patients with survivin-2B80-88 peptide could lead to increases of the CTL precursor frequency. Furthermore, CTLs specific for this peptide were successfully induced from PBMCs in all 7 (100%) patients with breast cancers, 6 of 7 (83%) patients with colorectal cancers, and 4 of 7 (57%) patients with gastric cancers. These data indicate that survivin expressed in tumor tissues is antigenic in cancer patients, and survivin-2B80-88-specific CTLs are present in PBMCs of various cancer patients. Our study raises the possibility that this peptide may be applicable as a general cancer vaccine to a large proportion of HLA-A24 + cancer patients.

To investigate the immunogenic property of peptides derived from the synovial sarcoma-specific SYT-SSX fusion gene, we synthesized four peptides according to the binding motif for HLA-A24. The peptides, SS391 (PYGYDQIMPK) and SS393 (GYDQIMPKK), were derived from the breakpoint of SYT-SSX, and SS449a (AWTHRLRER) and SS449b (AWTHRLRERK) were from the SSX region. These peptides were tested for their reactivity with CTL precursors (CTLps) in 16 synovial sarcoma patients using HLA-A24/SYT-SSX peptide tetramers and also for induction of specific CTLs from four HLA-A24+ synovial sarcoma patients. Tetramer analysis indicated that the increased CTLp frequency to the SYT-SSX was associated with pulmonary metastasis in synovial sarcoma patients (p < 0.03). CTLs were induced from PBLs of two synovial sarcoma patients using the peptide mixture of SS391 and SS393, which lysed HLA-A24+ synovial sarcoma cells expressing SYT-SSX as well as the peptide-pulsed target cells in an HLA class I-restricted manner. These findings suggest that aberrantly expressed SYT-SSX gene products have primed SYT-SSX-specific CTLps in vivo and increased their frequency in synovial sarcoma patients. The identification of SYT-SSX peptides may offer an opportunity to design peptide-based immunotherapeutic approaches for HLA-A24+ patients with synovial sarcoma.