HAGE (DDX43) is a biomarker for poor prognosis and a predictor of chemotherapy response in breast cancer.

BACKGROUND: HAGE protein is a known immunogenic cancer-specific antigen. METHODS: The biological, prognostic and predictive values of HAGE expression was studied using immunohistochemistry in three cohorts of patients with BC (n=2147): early primary (EP-BC; n=1676); primary oestrogen receptor-negative (PER-BC; n=275) treated with adjuvant anthracycline-combination therapies (Adjuvant-ACT); and primary locally advanced disease (PLA-BC) who received neo-adjuvant anthracycline-combination therapies (Neo-adjuvant-ACT; n=196). The relationship between HAGE expression and the tumour-infiltrating lymphocytes (TILs) in matched prechemotherapy and postchemotherapy samples were investigated. RESULTS: Eight percent of patients with EP-BC exhibited high HAGE expression (HAGE+) and was associated with aggressive clinico-pathological features (Ps<0.01). Furthermore, HAGE+expression was associated with poor prognosis in both univariate and multivariate analysis (Ps<0.001). Patients with HAGE+did not benefit from hormonal therapy in high-risk ER-positive disease. HAGE+and TILs were found to be independent predictors for pathological complete response to neoadjuvant-ACT; P<0.001. A statistically significant loss of HAGE expression following neoadjuvant-ACT was found (P=0.000001), and progression-free survival was worse in those patients who had HAGE+residual disease (P=0.0003). CONCLUSIONS: This is the first report to show HAGE to be a potential prognostic marker and a predictor of response to ACT in patients with BC.

Virulence loss and amastigote transformation failure determine host cell responses to Leishmania mexicana.

The effect of alterations in virulence and transformation by long-term in vitro culture of Leishmania mexicana promastigotes on infectivity and immune responses was investigated. Fresh parasite cultures harvested from Balb/c mice were passaged 20 times in vitro. Infectivity was decreased and was completely avirulent after 20 passages. The qPCR results showed a down-regulation of GP63, LPG2, CPC, CPB2, CPB2.8, CHT1, LACK and LDCEN3 genes after passage seven concomitant with a reduced and absence of infectivity by passages seven and 20, respectively. Parasites at passages one and 20 are referred to as virulent and avirulent, respectively. The growth of avirulent and virulent parasite was affected by conditioned media derived from macrophages or monocytes infected with parasites for 2 h. Giemsa staining showed the failure of avirulent but not virulent parasites to transform to the amastigote stage in infected host cells with both virulent and avirulent modulating the expression of CCL-22, Tgad51, Cox2, IL-1, IL-10, TGF-ß, TNF-α, Rab7, Rab9 and A2 genes; virulent but not avirulent L. mexicana significantly up-regulated Th2-associated cytokines, but down-regulated Rab7 and Rab9 gene expression. In conclusion, a model for L. mexicana is reported, which is of potential value in studying host-parasite interaction.

Mutation-specific reporter for optimization and enrichment of prime editing.

Prime editing is a versatile genome-editing technique that shows great promise for the generation and repair of patient mutations. However, some genomic sites are difficult to edit and optimal design of prime-editing tools remains elusive. Here we present a fluorescent prime editing and enrichment reporter (fluoPEER), which can be tailored to any genomic target site. This system rapidly and faithfully ranks the efficiency of prime edit guide RNAs (pegRNAs) combined with any prime editor variant. We apply fluoPEER to instruct correction of pathogenic variants in patient cells and find that plasmid editing enriches for genomic editing up to 3-fold compared to conventional enrichment strategies. DNA repair and cell cycle-related genes are enriched in the transcriptome of edited cells. Stalling cells in the G1/S boundary increases prime editing efficiency up to 30%. Together, our results show that fluoPEER can be employed for rapid and efficient correction of patient cells, selection of gene-edited cells, and elucidation of cellular mechanisms needed for successful prime editing.

Interleukin 12 (IL-12) induces tyrosine phosphorylation of JAK2 and TYK2: differential use of Janus family tyrosine kinases by IL-2 and IL-12.

Interleukin (IL-12) has many effects on the function of natural killer and T cells, and is important in the control of cell-mediated immunity. IL-2 and IL-12 display many similar activities, yet each also induces a distinct set of responses. A human IL-12 receptor subunit has recently been cloned and, like the IL-2R beta and IL-2R gamma, is a member of the hematopoietic receptor superfamily; however, the molecular mechanisms of IL-12 action are unknown. In this report we show that IL-12 and IL-2 induce tyrosine phosphorylation of distinct members of the Janus (JAK) family of protein tyrosine kinases in human T lymphocytes. IL-12, but not IL-2, stimulates the tyrosine phosphorylation of TYK2 and JAK2, whereas JAK1 and JAK3, which are phosphorylated in response to IL-2, are not phosphorylated after IL-12 treatment. The use of distinct but related JAK family tyrosine kinases by IL-12 and IL-2 may provide a biochemical basis for their different biological activities.

A validated gene expression profile for detecting clinical outcome in breast cancer using artificial neural networks.

Gene expression microarrays allow for the high throughput analysis of huge numbers of gene transcripts and this technology has been widely applied to the molecular and biological classification of cancer patients and in predicting clinical outcome. A potential handicap of such data intensive molecular technologies is the translation to clinical application in routine practice. In using an artificial neural network bioinformatic approach, we have reduced a 70 gene signature to just 9 genes capable of accurately predicting distant metastases in the original dataset. Upon validation in a follow-up cohort, this signature was an independent predictor of metastases free and overall survival in the presence of the 70 gene signature and other factors. Interestingly, the ANN signature and CA9 expression also split the groups defined by the 70 gene signature into prognostically distinct groups. Subsequently, the presence of protein for the principal prognosticator gene was categorically assessed in breast cancer tissue of an experimental and independent validation patient cohort, using immunohistochemistry. Importantly our principal prognosticator, CA9, showed that it is capable of selecting an aggressive subgroup of patients who are known to have poor prognosis.

CTL responses to Leishmania mexicana gp63-cDNA vaccine in a murine model.

Immunity to Leishmania is believed to be strongly dependent upon the activation of Th1 immune responses, although the exact role of cytotoxic T lymphocytes (CTLs) has not yet been determined. The aims of this study were to establish a suitable cytotoxicity assay to measure CTL activity and to compare immunity induced by Leishmania mexicana gp63 cDNA via i.m. injection and gene gun immunization in the BALB/c mouse model. The CTL activity was evaluated by short-term (51)Cr-release cytotoxicity assays against CT26 tumour cells transfected with L. mexicana gp63 cDNA and dendritic cells (DCs) loaded with soluble Leishmania antigen (SLA) as targets. The results clearly demonstrated that higher protection to L. mexicana infection was induced by gene gun DNA-immunization vs. i.m. injection. Cytotoxic T lymphocyte activity of splenocytes was observed in mice immunized either with L. mexicana gp63 cDNA or SLA and long-lived CTL activity was observed in immunized and/or re-challenged mice but not naïve mice infected with the parasite.

Cancer/testis antigens for therapeutic use.

Since van der Bruggen and colleagues first identified specific human tumour-associated antigens of the MAGE family, numerous potential immunotherapeutic targets have been discovered, often belonging to the so-called cancer/ testis gene family. Several members of this group have been described as immunogenic and have been utilised in clinical trials. In a search for interesting targets within this family, our laboratory has focussed its works for a number of years on two novel cancer/testis antigens called T21 and HAGE. In this article, we will focus our discussion on their levels of expression in a wide variety of both normal and cancer tissues, their possible role in tumour cell development and proliferation, and their immunogenic potential.

Identification of immunogenic MHC class II Tyrosinase-derived peptides using HLA-DR1 and HLA-DR4 transgenic mice.

The immunogenicity of "novel" MART-1 and Tyrosinase class-II peptides was assessed in transgenic mice. Tyrosinase(141-161) peptide was found to be immunogenic and endogenously processed in the HLA-DRbeta1*0101 and HLA-DRbeta1*0401 transgenic mice with peptide specific production of IFNgamma or IL-5 respectively. The MART-1(29-43) peptide was only found immunogenic in HLA-DRbeta1*0101 mice.

Nm23 protein expression in ductal in situ and invasive human breast carcinoma.

BACKGROUND: Mortality associated with human breast carcinoma is almost entirely due to subsequent metastatic disease, but the molecular basis of this metastasis is not understood. Elucidation of the genetic control of metastatic propensity of a tumor is important in determining prognosis and choice of therapy. Expression of nm23, a putative metastasis suppressor gene, has been detected in human breast cancers, but studies have not consistently shown high levels of the Nm23 messenger RNA or protein to be associated with better histological differentiation. This inconsistency suggests that Nm23 protein may act independently as a metastasis suppressor. PURPOSE: The purpose of this retrospective study was to investigate the relationship of Nm23 protein expression with 1) histology in ductal breast carcinoma in situ and 2) the variables considered to be the major prognostic indicators in invasive breast carcinoma. METHODS: We obtained formalin-fixed biopsy specimens of breast tissue excised from 128 patients with breast lesions detected by mammography. Of these patients, 35 had been diagnosed with benign breast disease, 26 with ductal carcinoma in situ (DCIS), and 67 with invasive carcinoma. Tissue sections were embedded in paraffin blocks, and immunohistochemical staining was used to determine Nm23 expression. Specimens were rated positive if all lesional epithelium was stained and negative if any lesional epithelium was unstained. Statistical analysis was performed by multiple regression analysis because of nonorthogonality of the data. RESULTS: All 35 examples of benign breast disease showed uniform epithelial cell staining. The seven cases of comedo DCIS were negative for Nm23 protein; all 18 noncomedo types were positive. Nm23 negativity was significantly associated with worsening invasive ductal carcinoma grade and advancing lymph node stage but not with tumor diameter or vascular invasion. Despite the putative antimetastatic role of the nm23 gene, no statistically significant association was found between Nm23 protein expression and vascular invasion. CONCLUSIONS: The precise role of the nm23 gene remains to be established, but our simplified immunohistochemical rating system shows an association between Nm23 protein expression and the two most significant prognostic factors relating to histologic grade and stage. Nm23 negativity distinguished comedo ductal carcinoma in situ from the other histological types, a finding consistent with the fact that comedo histology is known to have a higher likelihood of becoming invasive and of having higher cell proliferation rates and higher expression of growth factor (c-erb B2) receptor.

Rat natural killer cell activity against lymphoid and nonlymphoid tumor cells and normal rat cell lines.

Natural cytotoxicity in the rat was assessed against solid tumors and normal and embryonic rat monolayer culture lines, and the results were compared with rat natural killer activity towards syngeneic, allogeneic, and xenogeneic lymphoma targets. The targets tested showed a very wide range of susceptibility to lysis by peripheral blood and spleen effector cells in both 4- and 18-hr cytotoxicity assays. Cytotoxicity toward the mouse lymphoma, YAC-1, and rat sarcoma LT1 targets was relatively high in a 4-hr assay, compared with that toward the other cell lines used in the study. At 18 hr, increased killing was seen against YAC-1, LT1, SP3 (rat pheochromoblastoma), F2304 (rat embryonic cells), and normal rat kidney cells, while a significant increase in susceptibility with time was not observed with other rat targets: W/Fu-T (sarcoma); ERTh/V-G (sarcoma); R35 (mammary tumor); and W/Fu-P21 (embryo fibroblasts) targets. The cytotoxicity against all of the targets tested was not age restricted and was potentiated by rat interferon. Natural cytotoxic reactivity was seen with effector cells from the blood, spleen, and peritoneal cavity of both W/Fu (euthymic) and Rowett nude (congenitally athymic) rats. Cytotoxic effector cells from the blood were present in the low-density fractions recovered from discontinuous Percoll density gradients and, as shown previously for lymphoma target cells, a strong correlation was observed between the killing of embryonic, normal, and solid tumor targets and the presence of large granular lymphocytes. These results indicate that the naturally cytotoxic rat effector cells for normal fibroblast and bone marrow targets, lymphomas-leukemias, embryonic cell lines, and solid tumor targets are all included in the large granular lymphocyte subpopulation.

Preclinical evaluation of "whole" cell vaccines for prophylaxis and therapy using a disabled infectious single cycle-herpes simplex virus vector to transduce cytokine genes.

The development of genetically modified "whole" tumor cell vaccines for cancer therapy relies on the efficient transduction and expression of genes by vectors. In the present study, we have used a disabled infectious single cycle-herpes simplex virus 2 (DISC-HSV-2) vector constructed to express cytokine or marker genes upon infection. DISC-HSV-2 is able to infect a wide range of tumor cells and efficiently express the beta-galactosidase reporter gene, granulocyte-macrophage colony-stimulating factor (GM-CSF), or IL-2 genes. Gene expression occurred rapidly after infection of tumor cells, and the level of production of the gene product (beta-galactosidase, GM-CSF, or IL-2) was shown to be both time-and dose-dependent. Vaccination with irradiated DISC-mGM-CSF or DISC-hIL-2-infected murine tumor cells resulted in greatly enhanced immunity to tumor challenge with live parental tumor cells compared with control vaccines. When used therapeutically to treat existing tumors, vaccination with irradiated DISC-mGM-CSF-infected tumor cells significantly reduced the incidence and growth rates of tumors when administered locally adjacent to the tumor site, providing up to 90% protection. The prophylactic and therapeutic efficacy of DISC-mGM-CSF-infected cells was shown initially using a murine renal cell carcinoma model (RENCA), and the results were confirmed in two additional murine tumor models: the M3 melanoma and 302R sarcoma. Therapy with DISC-infected RENCA "whole" cell vaccines failed to reduce the incidence or growth of tumor in congenitally T-cell deficient (Nu+/Nu+) mice or mice depleted of CD4+ and/or CD8+ T-lymphocytes, confirming that both T-helper and T-cytotoxic effector arms of the immune response are required to promote tumor rejection. These preclinical results suggest that this "novel" DISC-HSV vector may prove to be efficacious in developing genetically modified whole-cell vaccines for clinical use.

Cytoplasmic PML promotes TGF-ß-associated epithelial-mesenchymal transition and invasion in prostate cancer.

Epithelial-mesenchymal transition (EMT) is a key event that is involved in the invasion and dissemination of cancer cells. Although typically considered as having tumour-suppressive properties, transforming growth factor (TGF)-ß signalling is altered during cancer and has been associated with the invasion of cancer cells and metastasis. In this study, we report a previously unknown role for the cytoplasmic promyelocytic leukaemia (cPML) tumour suppressor in TGF-ß signalling-induced regulation of prostate cancer-associated EMT and invasion. We demonstrate that cPML promotes a mesenchymal phenotype and increases the invasiveness of prostate cancer cells. This event is associated with activation of TGF-ß canonical signalling pathway through the induction of Sma and Mad related family 2 and 3 (SMAD2 and SMAD3) phosphorylation. Furthermore, the cytoplasmic localization of promyelocytic leukaemia (PML) is mediated by its nuclear export in a chromosomal maintenance 1 (CRM1)-dependent manner. This was clinically tested in prostate cancer tissue and shown that cytoplasmic PML and CRM1 co-expression correlates with reduced disease-specific survival. In summary, we provide evidence of dysfunctional TGF-ß signalling occurring at an early stage in prostate cancer. We show that this disease pathway is mediated by cPML and CRM1 and results in a more aggressive cancer cell phenotype. We propose that the targeting of this pathway could be therapeutically exploited for clinical benefit.

Transfection of tissue transglutaminase into a highly malignant hamster fibrosarcoma leads to a reduced incidence of primary tumour growth.

Reduced expression of the tissue transglutaminase in both murine and human tumours has been consistently associated with tumour growth and progression. To investigate the functional effects of transglutaminase expression we have transfected a constitutive human tissue transglutaminase expression construct into a highly malignant hamster fibrosarcoma cell line Met B. Met B clones expressing the exogenous tissue transglutaminase exhibited a reduced incidence of primary tumour formation and an increased adherence to tissue culture plastic and fibronectin coated surfaces when compared to transfected and non transfected control cells. Transglutaminase transfected clones exhibited no significant differences in their growth rates measured in vitro, cell morphology or levels of spontaneous apoptosis measured by the determination of detergent insoluble apoptotic envelopes. The data demonstrates a suppressive effect of tissue transglutaminase on tumour growth and confirms its importance in the phenotypic changes associated with the cancer process.

Apoptosis: a potential role for cytosolic transglutaminase and its importance in tumour progression.

The relationship between transglutaminase activity, apoptosis and the propensity of a tumour to metastasise, was investigated in a series of metastatic variants of an HSV-2 induced hamster fibrosarcoma and two metastatic variants of the B16 mouse melanoma. The data suggest an inverse relationship between metastatic potential and cytosolic transglutaminase activity. A direct relationship was found between measured cytosolic activity and the levels of the endogenous product of transglutaminase, the protein crosslink epsilon(gamma-glutamyl)lysine. Increasing metastatic potential and decreasing cytosolic transglutaminase activity was accompanied by a corresponding decrease in the number of detergent-insoluble apoptotic envelopes isolated from variant cell lines. These apoptotic envelopes were found to be highly crosslinked structures, containing more than 85% of the cells content of epsilon(gamma-glutamyl)lysine. These data are in keeping with the idea that a major role for the cytosolic transglutaminase is in the formation of the highly crosslinked apoptotic envelope during programmed cell death and that perturbation of this function may be an important determinant in the development of the metastatic phenotype.

The existence of an inactive form of transglutaminase within metastasising tumours.

Separation by anion exchange chromatography of detergent extracts from a poorly metastatic HSV-2-induced hamster fibrosarcoma, its highly metastatic variant and a highly metastatic rat fibrosarcoma indicated the presence of an inactive form of transglutaminase antigen, when eluent fractions were assayed for transglutaminase activity and antigen. This inactive antigenic transglutaminase was clearly separable from the particulate and cytosolic forms of the transglutaminase enzyme. Unlike tumours, its presence could not be demonstrated in extracts from normal rat liver. Measurement of activity levels during tumour growth indicated that the progression of the two highly metastatic tumours was accompanied by a decrease in cytosolic transglutaminase activity, whilst the activity of this enzyme form remained constant in the poorly metastatic tumour. Measurement of antigen levels indicated an inverse relationship between the level of inactive transglutaminase and the level of cytosolic transglutaminase activity, suggesting that the two forms are inter-related. Gel filtration indicated the molecular weight of the inactive form to be greater than both the particulate and cytosolic forms, and it was estimated to be 120,000. Partial proteolysis of the semi-purified inactive form, by either trypsin or thrombin, led to its activation and to the appearance of a transglutaminase similar in molecular weight and ionic mobility, both by anion-exchange chromatography and electrophoresis, to the cytosolic transglutaminase.

Gene therapy with autologous, interleukin 2-secreting tumor cells in patients with malignant melanoma.

We vaccinated metastatic melanoma patients with irradiated, autologous melanoma cells genetically engineered to secrete interleukin 2 (IL-2) to investigate whether an anti-tumor immune response would be induced. Melanoma cell cultures were established from surgical specimens and were engineered to secrete IL-2 by infection with recombinant retrovirus. Twelve patients were vaccinated subcutaneously one, two, or three times with approximately 10(7) irradiated, autologous, IL-2-secreting tumor cells. Treatment was well tolerated, with local reactions at 11 of 24 injection sites and minor systemic symptoms of fever and headache after 6 injections. One patient developed anti-tumor DTH after the first vaccination and showed an increased response after the second vaccination. Anti-autologous tumor CTLs could be detected prevaccination in the peripheral blood of seven patients and their activity increased after vaccination in four patients. No UICC-defined clinical responses were seen, but three patients had stable disease for 7-15 months, one of whom has not yet progressed (15+ months). Thus, patient vaccination with autologous, genetically engineered tumor cells is feasible and safe. Anti-tumor DTH and CTLs can be induced in some patients with such a vaccine.

MHC restricted and non-restricted killer lymphocytes.

Cytotoxic lymphocytes are either MHC-restricted (cytotoxic T-cells) or nonrestricted (natural killer NK-cells), although cells of the monocyte/macrophage lineage are also cytotoxic, and lymphocytes or phagocytic cells expressing Fc-receptors for immunoglobulin can function as antibody-dependent killer cells (referred to as antibody-dependent cellular cytotoxicity: ADCC). Antigen-specific T-lymphocytes recognise their target antigen in the context of MHC class I components, focusing their attack only against those cells expressing the relevant antigen specificity on their cell surface. A more primitive and alternative mechanism exists whereby NK-cells, classified as large granular lymphocytes (LGL), are able to kill in a non-specific manner, not requiring prior sensitisation to antigen. Both antigen-specific T-cells and LGL mediate their cytotoxicity through the release of cytotoxic molecules at the target-effector cell interface. LGL also have a regulatory role in the immune system through the release of cytokines, and can be distinguished from T-lymphocytes by the expression of distinct phenotypic markers (CD16+, CD56+) and they lack CD3 antigen expression or rearranged alpha/beta or gamma/delta T-cell receptor gene products. Cytotoxic activity is positively regulated by interleukin-2 (IL-2) and interferon (IFN), whilst prostaglandins and transforming growth factor-beta (TGF beta) diminish activation and effector pathways. Cytotoxicity mediated by NK- and T-cell populations are principally involved in the defence against microbial infections and neoplasia; the abrogation of cytotoxicity either by direct interaction of 'suppressor factors' with effector cells, or indirectly by reducing cytokine production can inevitably lead to the proliferation of the disease.

Immunological similarities between cytosolic and particulate tissue transglutaminase.

At the present time it is uncertain whether or not the cytosolic and particulate forms of tissue transglutaminase are distinct and discrete enzymes. In this study a number of physical and immunological similarities between the two forms are demonstrated, indicating that they share some common epitopes, although their native confirmations may differ.

Thrombopoietin (TPO) induces tyrosine phosphorylation and activation of STAT5 and STAT3.

The growth and differentiation of megakaryocytes are regulated by thrombopoietin (TPO), a recently characterized cytokine which exerts its effects via a member of the hematopoietin receptor superfamily, c-Mpl. Since many cytokines which bind hematopoietin receptors activate the STAT family of transcription factors, we investigated whether STAT proteins were activated by TPO. TPO induced the formation of a DNA-binding complex recognizing a known STAT-binding sequence. STAT5 was a major component of this DNA-binding complex, and STAT5 was tyrosine phosphorylated in response to TPO. Additionally, TPO-induced the tyrosine phosphorylation and DNA-binding activity of STAT3. Together with the recent demonstration of JAK2 activation in response to TPO, the data presented here define a rapid signaling pathway likely to be important in TPO-induced gene regulation.

A positive association between agonist-induced cyclic AMP production in vitro and metastatic potential in murine B16 melanoma and hamster fibrosarcoma.

A positive association between agonist-stimulated cyclic AMP production in vitro and both experimentally induced (B16 melanoma) and spontaneous (fibrosarcoma) metastases were found. Five B16 melanoma cell lines producing varying degrees of lung colonization following intravenous injection and three hamster fibrosarcoma cell lines producing a varying number of metastases in lungs and regional lymph nodes after removal of the primary tumour were studied. Agonist-stimulated (forskolin and melanocyte-stimulating hormone), but not basal cyclic AMP accumulation, increased with increasing metastatic potential. This relationship did not extend to other intracellular signalling systems as determined by investigation of basal or foetal-calf stimulated phosphatidylinositol hydrolysis for either tumour type. Intracellular free calcium was also similar in B16 melanoma cell lines of varying metastatic potential.