Engineered T cells directed at tumors with defined allelic loss.

We describe an approach to cancer therapy based on exploitation of common losses of genetic material in tumor cells (loss of heterozygosity) (Basilion et al., 1999; Beroukhim et al., 2010). This therapeutic concept addresses the fundamental problem of discrimination between tumor and normal cells and can be applied in principle to the large majority of tumors. It utilizes modular activator/blocker elements that integrate signals related to the presence and absence of ligands displayed on the cell surface (Fedorov et al., 2013). We show that the targeting system works robustly in vitro and in a mouse cancer model where absence of the HLA-A*02 allele releases a brake on engineered T cells activated by the CD19 surface antigen. This therapeutic approach potentially opens a route toward a large, new source of cancer targets.

Immune deficiency augments the prevalence of p53 loss of heterozygosity in spontaneous tumors but not bi-directional loss of heterozygosity in bone marrow progenitors.

p53 loss of heterozygosity (LOH) is a frequent event in tumors of somatic and Li-Fraumeni syndrome patients harboring p53 mutation. Here, we focused on resolving a possible crosstalk between the immune-system and p53 LOH. Previously, we reported that p53 heterozygous bone-marrow mesenchymal progenitor cells undergo p53 LOH in-vivo. Surprisingly, the loss of either the wild-type p53 allele or mutant p53 allele was detected with a three-to-one ratio in favor of losing the mutant allele. In this study, we examined whether the immune-system can affect the LOH directionality in bone marrow progenitors. We found that mesenchymal progenitor cells derived from immune-deficient mice exhibited the same preference of losing the mutant p53 allele as immune-competent matched cells, nevertheless, these animals showed a significantly shorter tumor-free survival, indicating the possible involvement of immune surveillance in this model. Surprisingly, spontaneous tumors of p53 heterozygous immune-deficient mice exhibited a significantly higher incidence of p53 LOH compared to that observed in tumors derived of p53 heterozygous immune-competent mice. These findings indicate that the immune-system may affect the p53 LOH prevalence in spontaneous tumors. Thus suggesting that the immune-system may recognize and clear cells that underwent p53 LOH, whereas in immune-compromised mice, those cells will form tumors with shorter latency. In individuals with a competent immune-system, p53 LOH independent pathways may induce malignant transformation which requires a longer tumor latency. Moreover, this data may imply that the current immunotherapy treatment aimed at abrogating the inhibition of cellular immune checkpoints may be beneficial for LFS patients.

Loss of heterozygosity at 6p21 underlying [corrected] HLA class I downregulation in Chinese primary esophageal squamous cell carcinomas.

Loss or downregulation of human leukocyte antigen (HLA) class I molecules is a widespread mechanism used by tumor cells to avoid tumor recognition by cytotoxic T lymphocytes favoring tumor immune escape. Multiple molecular mechanisms are responsible for these altered HLA class I tumor phenotypes, such as the loss of heterozygosity (LOH) at chromosome region 6p21.3. In this study, we used immunohistological techniques with a highly selective panel of anti-HLA monoclonal antibodies to analyze the expression of HLA class I molecules in 84 formalin-fixed, paraffin-embedded section and 49 frozen-fresh tissues of primary esophageal squamous cell carcinomas (pESCC) from Chinese patients. To elucidate the underlying mechanism of HLA class I loss or downregulation, we also analyzed LOH of previously selected microsatellite markers located in chromosomes 6 and 15 by polymerase chain reaction. DNA was obtained from frozen-fresh tumor tissues and surrounding stroma to define the LOH associated with chromosomes 6p21 and 15q21. Our results showed that HLA-A, HLA-B/C, HLA class I heavy chain, beta2-microglobuline, and HLA class I complex were lost or downregulated in pESCC (P<0.0001), and were moderately associated with the microsatellite alterations in HLA class I gene regions, correlated with patients' age, tumor's location, and stage, and indicated that LOH at 6p21.3 is a frequent mechanism that leads to HLA class I abnormalities in pESCC.

Frequent HLA class I loss is an early event in cervical carcinogenesis.

Loss at chromosome 6p21.3, the human leukocyte antigen (HLA) region, is the main cause of HLA downregulation, occurring in the majority of invasive cervical carcinomas. To identify the stage of tumor development at which HLA class I aberrations occur, we selected 12 patients with cervical carcinoma and adjacent cervical intraepithelial neoplasia (CIN). We investigated HLA class I and beta2-microglobulin expression by immunohistochemistry in tumor and adjacent CIN. Loss of heterozygosity (LOH) was studied using microsatellite markers covering the HLA region. Fluorescent in situ hybridization with HLA class I probes was performed to investigate the mechanism of HLA loss. Immunohistochemistry showed absent or weak HLA class I expression in 11/12 cases. In 10 of these 11 cases, downregulation occurred in both tumor and CIN. Only in one case did the concomitant CIN lesion show normal expression. In 9/12 cases, LOH was present for at least one marker in both tumor and CIN, 1 case showed only LOH in the CIN lesion, and 1 case showed retention of heterozygosity for all markers in both tumor and CIN. We conclude that HLA class I aberrations occur early and frequently in cervical carcinogenesis. This might allow premalignant CIN lesions to escape immune surveillance and progress to invasive cancer.

Novel germ-line deletion of SNF5/INI1/SMARCB1 gene in neonate presenting with congenital malignant rhabdoid tumor of kidney and brain primitive neuroectodermal tumor.

We describe a neonate who had a rare tumor combination of a malignant rhabdoid tumor of the kidney (MRTK) and a brain primitive neuroectodermal tumor (PNET). Genetic alterations of the SNF5/INI1/SMARCB1 gene were investigated by PCR-single-strand conformation polymorphism (SSCP), loss of heterozygosity (LOH), sequence, and karyotyping analyses, and the gene expression level was determined by real-time quantitative RT-PCR analysis. PCR band signals of each exon of the hSNF5/INI1 were weak or nearly undetectable in both MRTK and PNET, whereas those of the corresponding normal kidney were clearly detected. Aberrantly migrating SSCP bands led to identification of a nucleotide change in intron 8. Although this was regarded as a polymorphism, only the changed nucleotide was observed in the normal kidney of the patient. Allelic states in the parents were heterozygous for the polymorphism in the father and homozygous for the normal sequence in the mother. Thus, it was evident that a substantial genetic part of the maternal normal allele including SNF5/INI1 was deleted as a de novo germ-line mutation. In both tumors, LOH at microsatellite loci on the long arm of chromosome 22 was evident, and expression of SNF5/INI1 mRNA was drastically decreased compared to that in control tissues (0.7-3.9 vs. 123.6-153.5). Deletion of a substantial genetic part demonstrated in our patient is the novel appearance of a germ-line deletion of the SNF5/INI1 gene. Additional large somatic deletions resulted in total inactivation of the gene in both tumors. Our patient provides evidence for an important role of SNF5/INI1germ-line mutation in predisposing patients to multiple rhabdoid tumors.

Loss of a single allele of SHIP exacerbates the immunopathology of Pten heterozygous mice.

Phosphatidylinositol 3-kinase (PI3K) has emerged as a critical component of multiple immune system intracellular signalling pathways. The levels and relative ratios of PI3K products, phosphatidylinositol (3,4) bisphosphate (PI(3,4)P(2)) and phosphatidylinositol (3,4,5) trisphosphate (PIP(3)), are regulated by inositol phosphatases such as Pten and SHIP. Interestingly, mice heterozygous for Pten, a 3'-inositol phosphatase, develop a progressive lymphoproliferative syndrome with autoimmune features. Given the importance of PIP(3) species in regulating immune responses, we hypothesized that heterozygosity for the 5'-inositol phosphatase SHIP might exacerbate the autoimmune phenotype of Pten(+/-) mice. In keeping with this, mice heterozygous for both Pten and SHIP developed lymphoproliferation, hypergammaglobulinaemia, autoantibody titres and renal pathology that were more severe than that of Pten(+/-) mice. These results suggest that the relative levels of phosphatidylinositol phosphatases are likely critical to immune system homeostasis and they also highlight the potential for gene dosage effects in regulating susceptibility and/or severity of autoimmunity.

Criteria to define HLA haplotype loss in human solid tumors.

Short tandem repeat (STR) markers are currently used to define loss of heterozygosity (LOH) of genes and chromosomes in tumors. Chromosome 6 and chromosome 15 STR markers are applied to define loss of HLA and related genes (e.g. TAP and beta2m). The number of STR identified in the HLA region is still increasing. In this study, seven representative STR markers covering the 6p/6q arms of chromosome 6 including the HLA region and two for chromosome 15 flanking the beta2m gene, were selected as minimally required for reliable LOH studies. A multiplex polymerase chain reaction (PCR) strategy is proposed when small number of cells are available in microdissected tumor samples.