Reovirus is under development as a therapeutic for numerous types of cancer. In contrast to other oncolytic viruses, the safety and efficacy of reovirus have not been improved through genetic manipulation. Here, we tested the oncolytic capacity of recombinant strains (rs) of prototype reovirus laboratory strains T1L and T3D (rsT1L and rsT3D, respectively) in a panel of non-small cell lung cancer (NSCLC) cell lines. We found that rsT1L was markedly more cytolytic than rsT3D in the large cell carcinoma cell lines tested, whereas killing of adenocarcinoma cell lines was comparable between rsT1L and rsT3D. Importantly, non-recombinant T1L and T3D phenocopied the kinetics and magnitude of cell death induced by recombinant strains. We identified gene segments L2, L3, and M1 as viral determinants of strain-specific differences cell killing of the large cell carcinoma cell lines. Together, these results indicate that recombinant reoviruses recapitulate the cell killing properties of non-recombinant, tissue culture-passaged strains. These studies provide a baseline for the use of reverse genetics with the specific objective of engineering more effective reovirus oncolytics. This work raises the possibility that type 1 reoviruses may have the capacity to serve as more effective oncolytics than type 3 reoviruses in some tumor types.

AIM: Identifying the genetic expression profile of CD133
METHOD: Circulating CD133
RESULTS: Data analysis of stem cells related genes in CD133
CONCLUSION: KRT15 can be used to differentiate between circulating CD133

Different therapeutic methods have been developed for the B-cell and T-cell subtypes of acute lymphoblastic leukemia (ALL). The identification of molecular biomarkers that can accurately discriminate between B-cell and T-cell ALLs will facilitate the quick determination of therapeutic plans, as well as reveal the intrinsic mechanisms underlining the two different ALL subtypes. This study computationally screened the high-throughput transcriptome dataset for multiple candidate biomarkers and verified their discrimination abilities in an independent sample set using quantitative real-time polymerase chain reaction (PCR) technology. Both technologies suggest that the two genes CD3D and PKRCQ together provided a good model for classification of B-cell and T-cell ALLs, whereas the individual genes did not show consistent discrimination between the two ALL subtypes. Supplementary material is available at http://healthinformaticslab.org/supp/.

PURPOSE: The primary aim was to derive evidence for or against the clinical importance of several biologic processes in patients treated with neoadjuvant chemotherapy (NAC) by assessing expression of selected genes with prior implications in prognosis or treatment resistance. The secondary aim was to determine the prognostic impact in residual disease of the genes' expression.
EXPERIMENTAL DESIGN: Expression levels of 24 genes were quantified by NanoString nCounter on formalin-fixed paraffin-embedded residual tumors from 126 patients treated with NAC and 56 paired presurgical biopsies. The paired t test was used for testing changes in gene expression, and Cox regression and penalized elastic-net Cox Regression for estimating HRs.
RESULTS: After NAC, 12 genes were significantly up- and 8 downregulated. Fourteen genes were significantly associated with time to recurrence in univariable analysis in residual disease. In a multivariable model, ACACB, CD3D, MKI67, and TOP2A added prognostic value independent of clinical ER(-), PgR(-), and HER2(-) status. In ER(+)/HER2(-) patients, ACACB, PAWR, and ERBB2 predicted outcome, whereas CD3D and PAWR were prognostic in ER(-)/HER2(-) patients. By use of elastic-net analysis, a 6-gene signature (ACACB, CD3D, DECORIN, ESR1, MKI67, PLAU) was identified adding prognostic value independent of ER, PgR, and HER2.
CONCLUSIONS: Most of the tested genes were significantly enriched or depleted in response to NAC. Expression levels of genes representing proliferation, stromal activation, metabolism, apoptosis, stemcellness, immunologic response, and Ras-ERK activation predicted outcome in residual disease. The multivariable gene models identified could, if validated, be used to identify patients needing additional post-neoadjuvant treatment to improve prognosis. Clin Cancer Res; 22(10); 2405-16. ©2016 AACR.

PURPOSE: APOBEC3 DNA cytosine deaminase family members normally defend against viruses and transposons. However, deregulated APOBEC3 activity causes mutations in cancer. Because of broad expression profiles and varying mixtures of normal and cancer cells in tumors, including immune cell infiltration, it is difficult to determine where different APOBEC3s are expressed. Here, we ask whether correlations exist between APOBEC3 expression and T-cell infiltration in high-grade serous ovarian cancer (HGSOC), and assess whether these correlations have prognostic value.
EXPERIMENTAL DESIGN: Transcripts for APOBEC3G, APOBEC3B, and the T-cell markers, CD3D, CD4, CD8A, GZMB, PRF1, and RNF128 were quantified by RT-qPCR for a cohort of 354 HGSOC patients. Expression values were correlated with each other and clinical parameters. Two additional cohorts were used to extend HGSOC clinical results. Immunoimaging was used to colocalize APOBEC3G and the T-cell marker CD3. TCGA data extended expression analyses to additional cancer types.
RESULTS: A surprising positive correlation was found for expression of APOBEC3G and several T cell genes in HGSOC. Immunohistochemistry and immunofluorescent imaging showed protein colocalization in tumor-infiltrating T lymphocytes. High APOBEC3G expression correlated with improved outcomes in multiple HGSOC cohorts. TCGA data analyses revealed that expression of APOBEC3D and APOBEC3H also correlates with CD3D across multiple cancer types.
CONCLUSIONS: Our results identify APOBEC3G as a new candidate biomarker for tumor-infiltrating T lymphocytes and favorable prognoses for HGSOC. Our data also highlight the complexity of the tumor environment with respect to differential APOBEC family gene expression in both tumor and surrounding normal cell types. Clin Cancer Res; 22(18); 4746-55. ©2016 AACR.

DNA methylation has a substantial impact on gene expression, affecting the prognosis of breast cancer (BC) patients dependent on molecular subtypes. In this study, we investigated the prognostic relevance of the expression of genes reported as aberrantly methylated, and the link between gene expression and DNA methylation in BC subtypes. The prognostic value of the expression of 144 aberrantly methylated genes was evaluated in ER+/HER2-, HER2+, and ER-/HER2- molecular BC subtypes, in a meta-analysis of two large transcriptomic cohorts of BC patients (n = 1,938 and n = 1,640). The correlation between gene expression and DNA methylation in distinct gene regions was also investigated in an independent dataset of 104 BCs. Survival and Pearson correlation analyses were computed for each gene separately. The expression of 48 genes was significantly associated with BC prognosis (p < 0.05), and 32 of these prognostic genes exhibited a direct expression-methylation correlation. The expression of several immune-related genes, including CD3D and HLA-A, was associated with both relapse-free survival (HR = 0.42, p = 3.5E-06; HR = 0.35, p = 1.7E-08) and overall survival (HR = 0.50, p = 5.5E-04; HR = 0.68, p = 4.5E-02) in ER-/HER2- BCs. On the overall, the distribution of both positive and negative expression-methylation correlation in distinct gene regions have different effects on gene expression and prognosis in BC subtypes. This large-scale meta-analysis allowed the identification of several genes consistently associated with prognosis, whose DNA methylation could represent a promising biomarker for prognostication and clinical stratification of patients with distinct BC subtypes.

In the canonical pathway, infection of cells by the wild-type mammalian orthoreovirus Type 3 Dearing (T3D) is dependent on the interaction of the viral spike protein σ1 with the high-affinity cellular receptor junction adhesion molecule-A (JAM-A). We previously demonstrated that the human glioblastoma cell line U-118 MG does not express JAM-A and resists reovirus T3D infection in standard cell culture conditions (SCCC). Heterologous JAM-A expression sensitises U-118 MG cells to reovirus T3D. Here we studied reovirus infection in U-118 MG cells grown in spheroid cultures with the premise that cells in such cultures resemble cells in tumours more than those grown under standard adherent cell culture conditions on a plastic surface. Although the U-118 MG cells in spheroids do not express JAM-A, they are susceptible to reovirus T3D infection. We show that this can be attributed to factors secreted by cells in the spheroids. The concentration of active extracellular proteases cathepsin B and L in the medium of spheroid cultures was increased 19- and 24-fold, respectively, as compared with SCCC. These enzymes can convert the reovirus particles into a form that can infect the U-118 MG cells independent of JAM-A. Taken together, these data demonstrate that infection of tumour cells by wild-type reovirus T3D is not strictly dependent on the expression of JAM-A on the cell surface.

BACKGROUND: T-cells play an important role in the immune response and are activated in response to the presentation of antigens bound to major histocompatibility complex (MHC) molecules participating with the T-cell receptor (TCR). T-cell receptor complexes also contain four CD3 (cluster of differentiation 3) subunits. The TCR-CD3 complex is vital for T-cell development and plays an important role in intervening cell recognition events. Since microRNAs (miRNAs) are highly stable in blood serum, some of which may target CD3 molecules, they could serve as good biomarkers for early cancer detection. The aim of this study was to see whether there is a relationship between cancers and the amount of miRNAs -targeted CD3 molecules.
METHODS: Bioinformatics tools were used in order to predict the miRNA targets for these genes. Subsequently, these highly conserved miRNAs were evaluated to see if they are implicated in various kinds of cancers. Consequently, human disease databases were used. According to the latest research, this study attempted to investigate the possible down- or upregulation of miRNAs cancer patients.
RESULTS: We identified miRNAs which target genes producing CD3 subunit molecules. The most conserved miRNAs were identified for the CD3G gene, while CD247 and CD3EAP genes had the least number and there were no conserved miRNA associated with the CD3D gene. Some of these miRNAs were found to be responsible for different cancers, following a certain pattern.
CONCLUSIONS: It is highly likely that miRNAs affect the CD3 molecules, impairing the immune system, recognizing and destroying cancer tumor; hence, they can be used as suitable biomarkers in distinguishing cancer in the very early stages of its development.

Glioblastoma (GB) is a devastating disease for which new treatment modalities are needed. Efficacious therapy requires the removal of stem-cell like cells, these cells drive tumor progression because of their ability to self-renew and differentiate. In glioblastoma, the GB stem-like cells (GSC) form a small population of tumor cells and possess high resistance to chemo and radiation therapies. To assess the sensitivity of GSC to reovirus-mediated cytolysis, a panel of GSC cultures was exposed to wild-type reovirus Type 3 Dearing (T3D) and its junction adhesion molecule-A (JAM-A)-independent mutant, jin-1. Several parameters were evaluated, including the fraction of cells expressing the JAM-A reovirus receptor, the fraction of cells synthesizing reovirus proteins, the number of infectious reovirus particles required to reduce cell viability, the amount of infectious progeny reovirus produced and the capacity of the reoviruses to infect the GSC in 3-dimensional (3D) tumor cell spheroids. Our data demonstrate a marked heterogeneity in the susceptibility of the cultures to reovirus-induced cytolysis. While in monolayer cultures the jin-1 reovirus was generally more cytolytic than the wild-type reovirus T3D, in the 3D GSC spheroids, these viruses were equally effective. Despite the variation in reovirus sensitivity between the different GSC cultures, our data support the use of reovirus as an oncolytic agent. It remains to be established whether the variation in the reovirus sensitivity correlates with a patient's response to reovirus therapy. Moreover, our data show that the expression of the JAM-A receptor is not a major determinant of reovirus sensitivity in 3D GSC cultures.

BACKGROUND: Epstain-Barr virus (EBV) can transform human B lymphocytes making them immortalized and inducing tumorigenic ability in vitro, but the molecular mechanisms remain unclear. The aim of the present study is to detect and analyze differentially expressed genes in two types of host cells, normal human lymphocytes and coupled EBV-transformed lymphoblasts in vitro using gene chips, and to screen the key regulatory genes of lymphocyte transformation induced by EB virus.
METHODS: Fresh peripheral blood samples from seven healthy donors were collected. EBV was used to transform lymphocytes in vitro. Total RNA was extracted from 7 cases of the normal lymphocytes and transformed lymphoblasts respectively, marked with dihydroxyfluorane after reverse transcription, then hybridized with 4 × 44 K Agilent human whole genome microarray. LIMMA, String, Cytoscape and other softwares were used to screen and analyze differentially expressed genes. Real-time PCR was applied to verify the result of gene expression microarrays.
RESULTS: There were 1745 differentially expressed genes that had been screened, including 917 up-regulated genes and 828 down-regulated genes. According to the results of Generank, String and Cytoscape analyses, 38 genes may be key controlled genes related to EBV-transformed lymphocytes, including 22 up-regulated genes(PLK1, E2F1, AURKB, CDK2, PLCG2, CD80, PIK3R3, CDC20, CDC6, AURKA, CENPA, BUB1B, NUP37, MAD2L1, BIRC5, CDC25A, CCNB1, RPA3, HJURP, KIF2C, CDK1, CDCA8) and 16 down-regulated genes(FYN, CD3D, CD4, CD3G, ZAP70, FOS, HCK, CD247, PRKCQ, ITK, LCP2, CXCL1, CD8A, ITGB5, VAV3, CXCR4), which primarily control biological processes such as cell cycle, mitosis, cytokine-cytokine pathway, immunity response and so on.
CONCLUSIONS: Human lymphocyte transformation induced by EB virus is a complicated process, involving multiple-genes and -pathways in virus-host interactions. Global gene expression profile analysis showed that EBV may transform human B lymphocytes by promoting cell cycle and mitosis, inhibiting cell apoptosis, hindering host immune function and secretion of cytokines.

INTRODUCTION: Infiltration of breast tumors by tumor-infiltrating lymphocytes (TIL) has been associated with sensitivity to anthracycline-based chemotherapy. However, it is unclear whether this is true within the estrogen receptor-alpha (ER)-negative subset of breast tumors that frequently manifest high TIL levels.
METHODS: The association of TIL with short-term and long-term clinical response to anthracycline-based therapy was assessed in two independent ER-negative breast cancer cohorts in which patients were categorized as TIL-high or TIL-low. We defined an eight-gene lymphocyte mRNA expression signature (including CD19, CD3D, CD48, GZMB, LCK, MS4A1, PRF1, and SELL) and used unsupervised hierarchical clustering to examine the association between TIL and short-term response to neoadjuvant chemotherapy in a previously published cohort of ER-negative tumors (n = 113). We also examined the association between TIL and long-term chemotherapeutic efficacy in a second cohort of ER-negative tumors (n = 255) with longer than 6 years of median follow-up by using tissue microarrays and immunohistochemistry (IHC) for detection of CD3, CD8, CD4, CD20, and TIA-1.
RESULTS: In patients with ER-negative tumors treated with neoadjuvant anthracycline-based chemotherapy, pathologic complete responses (pCRs) were achieved by 23 (74%) of 31 TIL-high patients and 25 (31%) of 80 TIL-low patients (odds ratio (OR), 6.33; 95% confidence interval (CI), 2.49 to 16.08; P < 0.0001). Multivariate logistic regression with standard clinicopathologic features demonstrated that only tumor size (P = 0.037) and TIL status (P = 0.001) were independent predictors of anthracycline response. In the second cohort, adjuvant anthracycline-based therapy was associated with increased disease-free survival (DFS) only in patients with high levels of intraepithelial CD3+ TIL (P = 0.0023). In contrast, outcomes after CMF treatment (cyclophosphamide, methotrexate, and fluorouracil) showed no association with CD3 status. In both cohorts, cytotoxic T-cells were the primary TIL subtype associated with anthracycline sensitivity. Finally, TIL significantly predicted anthracycline sensitivity for both the Her2-positive and triple-negative tumor phenotypes.
CONCLUSIONS: ER-negative breast cancers with high levels of TIL have heightened sensitivity to anthracycline-based chemotherapy, as assessed by the immediate response to neoadjuvant therapy and long-term outcome following adjuvant therapy. Investigations of TIL-based predictive tests to identify patients likely to benefit from anthracycline-based treatments are warranted.

PURPOSE Preclinical data suggest a contribution of the immune system to chemotherapy response. In this study, we investigated the prespecified hypothesis that the presence of a lymphocytic infiltrate in cancer tissue predicts the response to neoadjuvant chemotherapy. METHODS We investigated intratumoral and stromal lymphocytes in a total of 1,058 pretherapeutic breast cancer core biopsies from two neoadjuvant anthracycline/taxane-based studies (GeparDuo, n = 218, training cohort; and GeparTrio, n = 840, validation cohort). Molecular parameters of lymphocyte recruitment and activation were evaluated by kinetic polymerase chain reaction in 134 formalin-fixed, paraffin-embedded tumor samples. Results In a multivariate regression analysis including all known predictive clinicopathologic factors, the percentage of intratumoral lymphocytes was a significant independent parameter for pathologic complete response (pCR) in both cohorts (training cohort: P = .012; validation cohort: P = .001). Lymphocyte-predominant breast cancer responded, with pCR rates of 42% (training cohort) and 40% (validation cohort). In contrast, those tumors without any infiltrating lymphocytes had pCR rates of 3% (training cohort) and 7% (validation cohort). The expression of inflammatory marker genes and proteins was linked to the histopathologic infiltrate, and logistic regression showed a significant association of the T-cell-related markers CD3D and CXCL9 with pCR. CONCLUSION The presence of tumor-associated lymphocytes in breast cancer is a new independent predictor of response to anthracycline/taxane neoadjuvant chemotherapy and provides useful information for oncologists to identify a subgroup of patients with a high benefit from this type of chemotherapy.

The Reoviridae are a family of viruses with a non-enveloped icosahedral capsid and a segmented double-stranded RNA genome. Prototypes of the mammalian Orthoreoviruses have been isolated from human respiratory and enteric tracts and are not associated with human disease. One of these, human reovirus type 3 Dearing (T3D), usually serves as a model for the family. In the last decade the mammalian Orthoreoviruses, especially T3D, have been evaluated as oncolytic agents in experimental cancer therapy. This is based on the observation that reoviruses induce cell death and apoptosis in tumor cells, but not in healthy non-transformed cells. Several clinical trials have been initiated in Canada, the USA, and the UK, to study the feasibility and safety of this approach. Due to the segmented structure of their double-stranded RNA genomes genetic modification of Reoviridae has been notoriously difficult. Several techniques have been described recently that facilitate the genetic modification of reovirus genomes. The basis for reverse genetics of reovirus is the discovery in 1990 that reovirus RNA is infectious. Subsequently, it took ten years before a foreign gene was introduced into the reovirus genome. Here we review the methods for reovirus modification and their use for generating new reovirus-derived oncolytic agents.

Although remission rates for metastatic melanoma are generally very poor, some patients can survive for prolonged periods following metastasis. We used gene expression profiling, mitotic index (MI), and quantification of tumor infiltrating leukocytes (TILs) and CD3+ cells in metastatic lesions to search for a molecular basis for this observation and to develop improved methods for predicting patient survival. We identified a group of 266 genes associated with postrecurrence survival. Genes positively associated with survival were predominantly immune response related (e.g., ICOS, CD3d, ZAP70, TRAT1, TARP, GZMK, LCK, CD2, CXCL13, CCL19, CCR7, VCAM1) while genes negatively associated with survival were cell proliferation related (e.g., PDE4D, CDK2, GREF1, NUSAP1, SPC24). Furthermore, any of the 4 parameters (prevalidated gene expression signature, TILs, CD3, and in particular MI) improved the ability of Tumor, Node, Metastasis (TNM) staging to predict postrecurrence survival; MI was the most significant contributor (HR = 2.13, P = 0.0008). An immune response gene expression signature and presence of TILs and CD3+ cells signify immune surveillance as a mechanism for prolonged survival in these patients and indicate improved patient subcategorization beyond current TNM staging.

Human Orthoreovirus Type 3 Dearing is not pathogenic to humans and has been evaluated clinically as an oncolytic agent. Its transduction efficiency and the tumor cell selectivity may be enhanced by incorporating ligands for alternative receptors. However, the genetic modification of reoviruses has been difficult, and genetic targeting of reoviruses has not been reported so far. Here we describe a technique for generating genetically targeted reoviruses. The propagation of wild-type reoviruses on cells expressing a modified sigma 1-encoding segment embedded in a conventional RNA polymerase II transcript leads to substitution of the wild-type genome segment by the modified version. This technique was used for generating reoviruses that are genetically targeted to an artificial receptor expressed on U118MG cells. These cells lack the junction adhesion molecule-1 and therefore resist infection by wild-type reoviruses. The targeted reoviruses were engineered to carry the ligand for this receptor at the C terminus of the sigma 1 spike protein. This demonstrates that the C terminus of the sigma 1 protein is a suitable locale for the insertion of oligopeptide ligands and that targeting of reoviruses is feasible. The genetically targeted viruses can be propagated using the modified U118MG cells as helper cells. This technique may be applicable for the improvement of human reoviruses as oncolytic agents.

In order to identify genes associated with primary chemotherapy-resistance, gene expression profiles (GEP) in tumour tissue from 37 patients with de novo diffuse large B-cell lymphoma (DLBCL), stage II-IV, either in continuous complete remission (n = 24) or with progressive disease during primary treatment (n = 13), were examined using spotted 55K oligonucleotide arrays. Immunohistochemistry was used for confirmation at the protein level. The top 86 genes that best discriminated between the two cohorts were chosen for further analysis. Only seven of 86 genes were overexpressed in the refractory cohort, e.g. RABGGTB and POLE, both potential targets for drug intervention. Seventy-nine of 86 genes were overexpressed in the cured cohort and mainly coded for proteins expressed in the tumour microenvironment, many of them involved in proteolytic activity and remodelling of extra cellular matrix. Furthermore, major histocompatibility complex class I molecules, CD3D and ICAM1 were overexpressed, indicating an enhanced immunological reaction. Immunohistochemistry confirmed the GEP results. The frequency of tumour infiltrating lymphocytes, macrophages, and reactive cells expressing ICAM-1, lysozyme, cathepsin D, urokinase plasminogen activator receptor, signal transducer and activator of transcription 1, and galectin-3 was higher in the cured cohort. These findings indicate that a reactive microenvironment has an impact on the outcome of chemotherapy in DLBCL.

Reovirus T3D preferentially kills tumor cells expressing Ras oncogenes and has shown great promise as an anticancer agent in various preclinical tumor models. Here, we investigated whether reovirus can infect and kill tumor cell cultures and tissue fragments isolated from resected human colorectal tumors, and whether this was affected by the presence of endogenous oncogenic KRAS. Tissue fragments and single-cell populations isolated from human colorectal tumor biopsies were infected with reovirus virions or with intermediate subviral particles (ISVPs). Reovirus virions were capable of infecting neither single-cell tumor cell populations nor small fragments of intact viable tumor tissue. However, infection of tumor cells with ISVPs resulted in transient viral protein synthesis, irrespective of the presence of oncogenic KRAS, but this did not lead to the production of infectious virus particles, and tumor cell viability was largely unaffected. ISVPs failed to infect intact tissue fragments. Thermolysin treatment of tumor tissue liberated single cells from the tissue and allowed infection with ISVPs, but this did not result in the production of infectious virus particles. Immunohistochemistry on tissue microarrays showed that junction adhesion molecule 1, the major cellular reovirus receptor, was improperly localized in the cytoplasm of colorectal tumor cells and was expressed at very low levels in liver metastases. This may contribute to the observed resistance of tumor cells to reovirus T3D virions. We conclude that infection of human colorectal tumor cells by reovirus T3D requires processing of virions to ISVPs, but that oncolysis is prevented by a tumor cell response that aborts viral protein synthesis and the generation of infectious viral particles, irrespective of KRAS mutation status.

Nonchromaffin paragangliomas of the head and neck region, also known as glomus tumors, are usually benign neoplasms consisting of clusters of chief cells surrounded by sustentacular cells arranged in so-called 'Zellballen.' Most of the patients have a familial background. In a previous study, examining all chromosome arms, we found loss of heterozygosity (LOH) predominantly at the chromosome 11q22-q23 region, where the disease causing gene PGL1 has been located by linkage analysis. However, all tumors showed only partial loss of allele signal intensities, and it was not clear whether this represented allelic imbalance or cellular heterogeneity. In the current study, we have performed LOH analysis for the 11q22-q23 region on DNA-aneuploid tumor cells, enriched by flow sorting, and on purified chief cell fractions obtained by single-cell microdissection. Complete LOH was found for two markers (D11S560 and CD3D) in the flow-sorted aneuploid fractions, whereas no LOH was found in the diploid fractions of three tumors. The microdissected chief cells from two of these tumors also showed complete LOH for both markers, indicating that the chief cells are clonal proliferated tumor cells. These results indicate that the PGL1 gene is likely to be a tumor suppressor gene, which is inactivated according to the two-hit model of Knudson. Furthermore, it shows that chief cells are a major if not the sole neoplastic component of paragangliomas.

Hereditary nonchromaffin paragangliomas (PGL; glomus tumors; MIM 168000) are mostly benign, slow-growing tumors of the head and neck region, inherited from carrier fathers in an autosomal dominant fashion subject to genomic imprinting. Genetic linkage analysis in two large, unrelated Dutch families assigned PGL loci to two regions of chromosome 11, at 11q23 (PGL1) and 11q13.1 (PGL2). We ascertained a total of 11 North American PGL families and confirmed maternal imprinting (inactivation). In three of six families, linkage analysis provided evidence of linkage to the PGL1 locus at 11q23. Recombinants narrowed the critical region to an approximately 4.5-Mb interval flanked by markers D11S1647 and D11S622. Partial allelic loss of strictly maternal origin was detected in 5 of 19 tumors. The greatest degree of imbalance was detected at 11q23, distal to D11S1327 and proximal to CD3D. Age at onset of symptoms was significantly different between fathers and children (Wilcoxon rank-sum test, P < .002). Affected children had an earlier age at onset of symptoms in 39 of 57 father-child pairs (chi2 = 7.74, P < .006). However, a more conservative comparison of the number of pairs in which a child had > or = 5 years earlier age at onset (n = 33) vis-a-vis that of complementary pairs (n = 24) revealed no significant difference (chi2 = 1.42, P > .2). Whether these data represent genetic anticipation or ascertainment bias can be addressed only by analysis of a larger number of father-child pairs.

Paragangliomas of the head and neck region, also known as glomus tumours, are mostly benign tumours of neuro-ectodermal origin. We mapped the familial form by linkage analysis in 6 families to chromosome region 11q22-q23, between the markers STMY and CD3D which currently span a 16-cM interval. Here, we performed detailed haplotype analysis of this region in a single Dutch multibranch 7-generation family. A region of 2 cM between the markers D11S938/D11S4122 and D11S1885 was shared between all patients of whom disease haplotypes could be reconstructed. In support of this localization, a recombination observed in a small French family with 2 affected nieces places the PGL gene proximal to marker D11S908, genetically coincident with D11S1885.

We examined the pattern of allelic loss in 76 adenocarcinomas of the lung using 14 highly informative microsatellite markers on the long arm of chromosome 11. Loss of heterozygosity was found in 48 of 76 tumors (63%). Three distinct regions of deletion were identified. The first region, the most centromeric, lies between markers D11S940 and CD3D: the second, delimited by markers D11S924 and D11S925, is estimated to be 4 Mb in length, and has never been previously described; a third, more telomeric region, the length of which is also estimated to be in the range of 4 Mb, is bracketed by loci D11S1345 and D11S1328. These findings suggest the presence of at least three tumor suppressor genes on the long arm of chromosome 11, and confirm the relevance of 11q22-24, a region frequently deleted in carcinomas of the breast, ovary, uterine, cervix, colon, and malignant melanoma in the pathogenesis of solid tumors. The characterization of minimal regions of loss could provide the basis for the identification and cloning of the critical genes.

A long-term T-cell line, termed SP+, was developed from a human T-cell leukemia virus type I (HTLV-I)-infected patient with adult T-cell leukemia that is dependent on exogenous IL-2 for growth. The SP+ expresses a full complimentation of HTLV-I-specific viral proteins, and contains replication competent viral particles. Restriction enzyme digestion followed by Southern blot analysis demonstrated the presence of a single integrated proviral copy and limiting dilution analysis confirmed the clonality of the cell line. Interestingly, phenotypically, the SP+ cell line is CD2+, CD3+ and coexpresses CD4 and CD8, yet lacks TCR alpha beta and TCR tau delta expression. Further ontogenetic characterization of the SP+ cell line demonstrated the lack of thymic T-cell precursor markers, including absence of cell surface expression of CD1, intracellular thymic terminal deoxynucleotidyl transferase (TdT) enzyme, as well as message expression for V(D)J recombinase activating gene-1 (RAG-1). Furthermore, the SP+ cell did express the message for the CD3 delta chain. Taken together, these data suggest that the SP+ cell line resulted from HTLV-I infection of a mature CD4+/CDB+ lymphocyte. This cell line can be potentially useful as a model, both for regulation of cellular functions by HTLV-I and for immunologic functions of mature dual CD4/CD8 positive T-cells.

Expression of an extended panel of cytokine genes was investigated by reverse polymerase chain reaction (PCR) in 10 freshly excised melanoma metastases infiltrated by lymphocytes (TIL). cDNA encoding for CD3-delta and tyrosinase could be amplified in all samples, confirming the presence of T lymphocytes and melanoma cells. Cytokine genes possibly transcribed by both cell types, such as GM-CSF, IL-6 and IL-10 could be amplified from 5, 2 and 2 samples respectively. In contrast, IL-1 beta and TNF-alpha mRNA were never detectable, IL-1 alpha, IL-3 and IL-7 mRNA could be observed only in one case each. Transcripts encoding for TGF-beta 1 were observed in 8 samples, while TGF-beta 2 and 3 mRNA were detectable in only 2 specimens. mRNA encoding for cytokine genes typically transcribed by antigen-stimulated T lymphocytes, such as IL-2, IL-4 and IFN-gamma were rarely or never detectable (none, none and 1 of the samples respectively). In one case, where no cytokine gene transcription was detectable at the time of surgery, we addressed the question of the antigenicity of the tumor and of the functional competence of TIL. A primary tumor cell line was generated and cultured TIL were induced to transcribe IL-2 and IFN-gamma genes by incubation with the autologous irradiated tumor cell line, but not with autologous EBV-transformed cells. In these conditions, tumor-specific cytotoxic T lymphocytes (CTL) could be generated only after 3 weekly re-stimulations. In contrast, if autologous irradiated EBV-transformed cells were added to the cultures, specific CTL could be detected after one single tumor stimulation. Thus, signs of active responsiveness in terms of lymphokine gene mRNA are seldom detectable in melanoma metastases. Tumor-specific responses, however, including IL-2 and IFN-gamma gene expression and generation of CTL can be produced in vitro from specimens in which no cytokine gene mRNA is detectable ex vivo.

The gene expression of myeloperoxidase (MPO), CD3 epsilon, and CD3 delta molecules, the gene rearrangement of T-cell receptor (TCR) delta, gamma, and beta and immunoglobulin heavy (IgH) chain, and the expression of cell-surface antigens were investigated in seven cases of CD7+ CD5- CD2- and four cases of CD7+ CD5+ CD2- acute lymphoblastic leukemia or lymphoblastic lymphoma (ALL/LBL) blasts, which were negative for cytochemical myeloperoxidase (cyMPO). More mature T-lineage blasts were also investigated in a comparative manner. In conclusion, the CD7+ CD5- CD2- blasts included four categories: undifferentiated blasts without lineage commitment, T-lineage blasts, T-/myeloid lineage blasts, and cyMPO-negative myeloblasts. The CD7+ CD5+ CD2- blasts included two categories; T-lineage and T-/myeloid lineage blasts. The 11 cases were of the germ-line gene (G) for TCR beta and IgH. Four cases were G for TCR delta and TCR gamma. The others were of the monoclonally rearranged gene (R) for TCR delta and G for TCR gamma or R for both TCR delta and TCR gamma. The expression or in vitro induction of CD13 and/or CD33 antigens correlated with the immaturity of these neoplastic T cells, since it was observed in all 11 CD7+ CD5- CD2- and CD7+ CD5+ CD2-, and some CD7+ CD5+ CD2+ (CD3- CD4- CD8-) cases, but not in CD3 +/- CD4+ CD8+ or CD3+ CD4+ CD8- cases. CD3 epsilon mRNA, but not CD3 delta mRNA, was detected in two CD7+ CD5- CD2- cases, while mRNA of neither of the two CD3 molecules was detected in the other tested CD7+ CD5- CD2- cases. In contrast, mRNA of both CD3 epsilon and CD3 delta were detected in all CD7+ CD5+ CD2- cases, indicating that CD7+ CD5- CD2- blasts at least belong to T-lineage. The blasts of two CD7+ CD5- CD2- cases with entire germ-line genes and without mRNA of the three molecules (MPO, CD3 epsilon, and CD3 delta) were regarded as being at an undifferentiated stage prior to their commitment to either T- or myeloid-lineage. The co-expression of the genes of MPO and CD3 epsilon in a CD7+ CD5- CD2- case MPO, CD3 epsilon, and CD3 delta in a CD7+ CD5+ CD2- case suggested the presence of some overlapping phase for T- and myeloid-lineage commitment during immature stages of differentiation. This helps understand the conversion of some T-ALL/LBL cases to acute myeloblastic leukemia (AML).(ABSTRACT TRUNCATED AT 400 WORDS)

A novel immature human T-ALL cell line, UHKT-42, was established from a 12 year old male patient with acute undifferentiated leukemia. The cell line expressed surface CD7, CD5 and cytoplasmic CD3 antigens. All other T-lymphocytic antigens were undetectable on the surface or in the cytoplasm of cultured cells. Expression of the T-cell receptor (TCR) beta, TCR delta, CD3 delta and CD3 epsilon genes was detected by Northern blotting in total cellular RNA extracts, however, the expression of TCR alpha and TCR gamma was undetectable. After stimulation by TPA for 3 days, only the appearance of CD25 (Tac antigen) was detected by immunofluorescence and flow cytometry. Secretion of interleukin-2 (IL-2) into the culture media was also detected after stimulation by PHA or TPA, but not in unstimulated cells. These results suggest that UHKT-42 cells are early precursors of T cells, with TCR beta/delta expression.

Paragangliomas of the head and neck are slow-growing tumors that rarely show malignant progression. Familial transmission has been described, consistent with an autosomal dominant gene that is maternally imprinted. Clinical manifestations of hereditary paraganglioma are determined by the sex of the transmitting parent. All affected individuals have inherited the disease gene from their father, expression of the phenotype is not observed in the offspring of an affected female or female gene carrier until subsequent transmittance of the gene through a male gene carrier. Recently, we assigned the gene responsible for paragangliomas (PGL) to chromosome 11q23-qter by linkage in a single large Dutch kindred. We now report confirmation of this localization in five unrelated Dutch families with hereditary paragangliomas. On the basis of segregation of haplotypes in the available family material, we localize the PGL locus between markers STMY and CD3D on chromosome 11q22.3-q23.

Cytogenetic studies in patients with acute leukemia showed structural abnormalities on chromosome 11 at band q23 in five cases. Four of these had acute lymphoblastic leukemia (ALL) associated with t(4;11)(q21;q23) and one case had acute nonlymphoblastic leukemia (ANLL) (M5) associated with t(11;17)(q23;q21). We examined the CD3D and c-ets-1 genes in the t(11;17)(q23;q21) patient to ascertain any association between them and the chromosome change. In situ hybridization results showed that unlike in other studied cases with rearrangements of 11q23, the CD3D gene in the t(11;17)(q23;21) is transposed to the der(17) chromosome, providing evidence for a different breakpoint in the 11q23 region.

Twelve cases of sinonasal 'T'-cell lymphoma were studied immunohistologically with a panel of antibodies that included two antibodies to CD3, Leu4 and rabbit anti-human CD3 epsilon. Leu4 was positive in five cases, whereas rabbit anti-human CD3 epsilon was positive in 11. This discordant reaction of the antibodies, suggestive of cytoplasmic CD3 epsilon +, CD3 delta-, and CD3 gamma-, has recently been demonstrated in activated adult natural killer (NK) cells also. Leu4-negative cases had the phenotype CD2+, CD5-, CD7+/-, T-cell receptor (TCR)-, and NK cell markers+, and had non-rearranged TCR genes. These findings suggest that a large group of sinonasal 'T'-cell lymphomas is derived from NK cells.

Twenty-four patients whose cells contained a variety of 11q23 rearrangements, including translocations, insertions, and an inversion, were studied using fluorescence in situ hybridization with cosmid, phage, and plasmid probes mapped to 11q22-24. In 17 patients, the breakpoints of the common 11q23 translocations involving chromosomes 4, 6, 9, and 19 as well as some uncommon translocations involving 3q23, 17q25, 10p11, and an insertion 10;11 were all located in the breakpoint cluster region of the MLL gene, regardless of age, phenotype of disease, or involvement of a third chromosome. The breakpoints in 11q23 in the other 7 patients with a t(7;11)(p15;q23), inv(11)(p11q23), t(4;11)(q23;q23), der(5)t(5;11)(q13;q23), ins(10;11)(p11;q23q24), t(11;14)(q23;q11), or t(11;18;11) (p15;q21;q23) were located either centromeric to CD3D or telomeric to THY1. Thus, although most 11q23 rearrangements, involve the same breakpoint cluster region of MLL, there is heterogeneity in the breakpoint in some of the rare rearrangements.

We present four patients with adult T-cell leukemia (ATL) derived from a novel T-cell subset (CD4-, CD8- [double-negative, DN], T-cell receptor [TCR] alpha beta+). In the ATL cells of these patients, neither gene nor surface expression of CD4 and CD8 antigens was detected. Clinical and laboratory data showed no difference between DN-ATL and CD4+ATL patients. In contrast to typical CD4+ATL cells, DN-ATL cells were shown to express the protein and messenger RNA (mRNA) for S100 beta in immunocytochemical assay and the reverse-transcription polymerase chain reaction assay. The mean fluorescence intensity of the TCR/CD3 complex was extremely low in all four DN-ATL patients as well as in typical CD4+ ATL. All four patients had TCR beta and gamma chain gene rearrangements, with deletion of TCR delta chain gene and mRNA expression for TCR alpha, beta, and CD3 delta but not for TCR gamma and delta chain genes. Thus, CD4- CD8- TCR alpha beta T cells are also a target for human T-cell lymphotrophic virus type I-induced leukemogenesis. In addition, expression of the TCR alpha beta/CD3 complex on the DN-ATL cells was further diminished by the addition of anti-CD3 or anti-TCR alpha beta monoclonal antibody. These results suggest that the decreased expression of the TCR alpha beta/CD3 complex by ATL cells plays a key role in the development of ATL, irrespective of CD4 expression.