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Background: Fulfilling the promise of cancer immunotherapy requires novel predictive biomarkers to characterise the host immune microenvironment. Deciphering the complexity of immune cell interactions requires an automated multiplex approach to histological analysis of tumour sections. We tested a new automatic approach to select tissue and quantify the frequencies of cell-cell spatial interactions occurring in the PD1/PD-L1 pathway, hypothesised to reflect immune escape in oropharyngeal squamous cell carcinoma (OPSCC).Methods: Single sections of diagnostic biopsies from 72 OPSCC patients were stained using multiplex immunofluorescence (CD8, PD1, PD-L1, CD68). Following multispectral scanning and automated regions-of-interest selection, the Hypothesised Interaction Distribution (HID) method quantified spatial proximity between cells. Method applicability was tested by investigating the prognostic significance of co-localised cells (within 30 μm) in patients stratified by HPV status.Results: High frequencies of proximal CD8+ and PD-L1+ (HR 2.95, p = 0.025) and PD1+ and PD-L1+ (HR 2.64, p = 0.042) cells were prognostic for poor overall survival in patients with HPV negative OPSCC (n = 31).Conclusion: The HID method can quantify spatial interactions considered to reflect immune escape and generate prognostic information in OPSCC. The new automated approach is ready to test in additional cohorts and its applicability should be explored in research and clinical studies. [ABSTRACT FROM AUTHOR]

Background: Recurrence occurs in over 75% of women with epithelial ovarian cancer despite optimal treatment. Selectively killing tumour cells thought to initiate relapse using an antibody-drug conjugate could prolong progression-free survival and offer an improved side-effect profile. A1mcMMAF is an antibody-drug conjugate designed to target cells expressing the tumour-associated antigen 5T4. It has shown to be efficacious in various cell line models and have a greater impact when combined with routine chemotherapeutic regimes.Objectives: This study aims to explore the potential for the use of a 5T4 antibody-drug conjugate in women with ovarian cancer both as a monotherapy and in combination with platinum-based chemotherapy.Methods: Immunohistochemical analysis was used to assess 5T4 expression in tumours from patients with ovarian cancer. Effectiveness of A1mcMMAF therapy as a single agent and in combination with carboplatin was assessed in vitro in the ovarian cancer cell line SKOV3 and confirmed in vivo using a serial bioluminescence assay in a SKOV3 xenograft model of ovarian cancer.Results: 5T4 is confirmed as suitably expressed in epithelial ovarian cancers prior to adjuvant therapy and is an independent predictor of poor survival. A1mcMMAF showed specific activity, both in vitro and in vivo, against SKOV3 ovarian cancer cells. When used in combination with carboplatin, in vivo tumour growth was inhibited resulting in prolonged survival in a SKOV3 xenograft model.Conclusions: These data support further investigation of A1mcMMAF in combination with platinum-based chemotherapy in ovarian and other cancer treatments. [ABSTRACT FROM AUTHOR]

Background: Multicellular taste buds located within taste papillae on the tongue mediate taste sensation. In taste papillae, taste bud cells (TBCs), such as taste receptor cells and taste precursor cells, and the surrounding lingual epithelium including epithelial progenitors (also called taste stem/progenitor cells) are maintained by continuous cell turnover throughout life. However, it remains unknown how the cells constituting taste buds proliferate and differentiate to maintain taste bud tissue. Based on in situ hybridization (ISH) screening, we demonstrated that the oncofetal antigen 5T4 (also known as trophoblast glycoprotein: TPBG) gene is expressed in the adult mouse tongue. Results: In immunohistochemistry of coronal tongue sections, 5T4 protein was detected at a low level exclusively in the basal part of the lingual epithelium in developing and adult mice, and at a high level particularly in foliate papillae and circumvallate papillae (CVPs). Furthermore, immunohistochemistry of the basal part of CVPs indicated that the proliferation marker PCNA (proliferating cell nuclear antigen) co-localized with 5T4. 5T4 was strongly expressed in Krt5+ epithelial progenitors and Shh+ taste precursor cells, but weakly in mature taste receptor cells. The number of proliferating cells in the CVP was higher in 5T4- knockout mice than in wild-type (WT) mice, while neither cell differentiation nor the size of taste buds differed between these two groups of mice. Notably, X-ray irradiation enhanced cell proliferation more in 5T4 -knockout mice than in WT mice. Conclusion: Our results suggest that 5T4, expressed in epithelial progenitors (taste stem/progenitor cells), and taste precursor cells, may influence the maintenance of taste papillae under both normal and injury conditions. [ABSTRACT FROM AUTHOR]

Neural circuits that undergo reorganization by newborn interneurons in the olfactory bulb (OB) are necessary for odor detection and discrimination, olfactory memory, and innate olfactory responses, including predator avoidance and sexual behaviors. TheOBpossesses many interneurons, including various types of granule cells (GCs); however, the contribution that each type of interneuron makes to olfactory behavioral control remains unknown. Here, we investigated the in vivo functional role of oncofetal trophoblast glycoprotein 5T4, a regulator for dendritic arborization of 5T4-expressing GCs (5T4 GCs), the level of which is reduced in the OB of 5T4 knock-out (KO) mice. Electrophysiological recordings with acute OB slices indicated that external tufted cells (ETCs) can be divided into two types, bursting and nonbursting. Optogenetic stimulation of 5T4 GCs revealed their connection to both bursting and nonbursting ETCs, as well as to mitral cells (MCs). Interestingly, nonbursting ETCs received fewer inhibitory inputs from GCs in 5T4 KO mice than from those in wild-type (WT) mice, whereas bursting ETCs and MCs received similar inputs in both mice. Furthermore, 5T4 GCs received significantly fewer excitatory inputs in 5T4 KO mice. Remarkably, in olfactory behavior tests, 5T4 KO mice had higher odor detection thresholds than the WT, as well as defects in odor discrimination learning. Therefore, the loss of 5T4 attenuates inhibitory inputs from 5T4 GCs to nonbursting ETCs and excitatory inputs to 5T4 GCs, contributing to disturbances in olfactory behavior. Our novel findings suggest that, among the various types of OB interneurons, the 5T4 GC subtype is required for odor detection and discrimination behaviors. [ABSTRACT FROM AUTHOR]

Antibodies specific for neutralizing epitopes in either Human papillomavirus (HPV) capsid protein L1 or L2 can mediate protection from viral challenge and thus their accurate and sensitive measurement at high throughput is likely informative for monitoring response to prophylactic vaccination. Here we compare measurement of L1 and L2-specific neutralizing antibodies in human sera using the standard Pseudovirion-Based Neutralization Assay (L1-PBNA) with the newer Furin-Cleaved Pseudovirion-Based Neutralization Assay (FC-PBNA), a modification of the L1-PBNA intended to improve sensitivity towards L2-specific neutralizing antibodies without compromising assay of L1-specific responses. For detection of L1-specific neutralizing antibodies in human sera, the FC- PBNA and L1-PBNA assays showed similar sensitivity and a high level of correlation using WHO standard sera (n = 2), and sera from patients vaccinated with Gardasil (n = 30) or an experimental human papillomavirus type 16 (HPV16) L1 VLP vaccine (n = 70). The detection of L1-specific cross-neutralizing antibodies in these sera using pseudovirions of types phylogenetically-related to those targeted by the L1 virus-like particle (VLP) vaccines was also consistent between the two assays. However, for sera from patients (n = 17) vaccinated with an L2-based immunogen (TA-CIN), the FC-PBNA was more sensitive than the L1-PBNA in detecting L2-specific neutralizing antibodies. Further, the neutralizing antibody titers measured with the FC-PBNA correlated with those determined with the L2-PBNA, another modification of the L1-PBNA that spacio-temporally separates primary and secondary receptor engagement, as well as the protective titers measured using passive transfer studies in the murine genital-challenge model. In sum, the FC-PBNA provided sensitive measurement for both L1 VLP and L2-specific neutralizing antibody in human sera. Vaccination with TA-CIN elicits weak cross-protective antibody in a subset of patients, suggesting the need for an adjuvant. [ABSTRACT FROM AUTHOR]

Intrinsic radiosensitivity is an important factor underlying radiotherapy response, but there is no method for its routine assessment in human tumours. Gene signatures are currently being derived and some were previously generated by expression profiling the NCI-60 cell line panel. It was hypothesised that focusing on more homogeneous tumour types would be a better approach. Two cell line cohorts were used derived from cervix [n = 16] and head and neck [n = 11] cancers. Radiosensitivity was measured as surviving fraction following irradiation with 2 Gy (SF2) by clonogenic assay. Differential gene expression between radiosensitive and radioresistant cell lines (SF2 median) was investigated using Affymetrix GeneChip Exon 1.0ST (cervix) or U133A Plus2 (head and neck) arrays. There were differences within cell line cohorts relating to tissue of origin reflected by expression of the stratified epithelial marker p63. Of 138 genes identified as being associated with SF2, only 2 (1.4%) were congruent between the cervix and head and neck carcinoma cell lines (MGST1 and TFPI), and these did not partition the published NCI-60 cell lines based on SF2. There was variable success in applying three published radiosensitivity signatures to our cohorts. One gene signature, originally trained on the NCI-60 cell lines, did partially separate sensitive and resistant cell lines in all three cell line datasets. The findings do not confirm our hypothesis but suggest that a common transcriptional signature can reflect the radiosensitivity of tumours of heterogeneous origins. [ABSTRACT FROM AUTHOR]

CXCL12 is a pleiotropic chemokine capable of eliciting multiple signal transduction cascades and functions, via interaction with either CXCR4 or CXCR7. Factors that determine CXCL12 receptor preference, intracellular signalling route and biological response are poorly understood but are of central importance in the context of therapeutic intervention of the CXCL12 axis in multiple disease states. We have recently demonstrated that 5T4 oncofoetal glycoprotein facilitates functional CXCR4 expression leading to CXCL12 mediated chemotaxis in mouse embryonic cells. Using wild type (WT) and 5T4 knockout (5T4KO) murine embryonic fibroblasts (MEFs), we now show that CXCL12 binding to CXCR4 activates both the ERK and AKT pathways within minutes, but while these pathways are intact, they are non-functional in 5T4KO cells treated with CXCL12. Importantly, in the absence of 5T4 expression, CXCR7 is upregulated and becomes the predominant receptor for CXCL12, activating a distinct signal transduction pathway with slower kinetics involving transactivation of the epidermal growth factor receptor (EGFR), eliciting proliferation rather than chemotaxis. Thus the surface expression of 5T4 marks the use of the CXCR4 rather than the CXCR7 receptor, with distinct consequences for CXCL12 exposure, relevant to the spread and growth of a tumour. Consistent with this hypothesis, we have identified human small cell lung carcinoma cells with similar 5T4/CXCR7 reciprocity that is predictive of biological response to CXCL12 and determined that 5T4 expression is required for functional chemotaxis in these cells. [ABSTRACT FROM AUTHOR]

Sensory input has been shown to regulate development in a variety of species and in various structures, including the retina, cortex, and olfactory bulb (OB). Within the mammalian OB specifically, the development of dendrites in mitral/tufted cells is well known to be odor-evoked activity dependent. However, little is known about the developmental role of sensory input in the other majorOBpopulation of the GABAgenic interneurons, such as granule cells and periglomerular cells. Here, we identified, with DNA microarray and in situ hybridization screenings, a trophoblast glycoprotein gene, 5T4, whose expression in a specific subtype of OB interneurons is dependent on sensory input. 5T4 is a type I membrane protein, whose extracellular domain contains seven leucine-rich repeats (LRR) flanked by characteristic LRR-N-flanking and C-flanking regions, and a cytoplasmic domain. 5T4 overexpression in the newborn OB interneurons facilitated their dendritic arborization even under the sensory input-deprived condition. By contrast, both 5T4 knockdown with RNAi and 5T4 knockout with mice resulted in a significant reduction in the dendritic arborization of 5T4+granule cells. Further, we identified the amino acid sequence in the 5T4 cytoplasmic domain that is necessary and sufficient for the sensory input-dependent dendritic shaping of specific neuronal subtypes in the OB. Thus, these results demonstrate that 5T4 glycoprotein contributes in the regulation of activity-dependent dendritic development of interneurons and the formation of functional neural circuitry in the OB. [ABSTRACT FROM AUTHOR]

Regulatory T cells (Treg) are a sub-population of T cells that suppress self-reactivity and are implicated in immune tolerance towards malignant cells. Circulating Treg cells are increased in several cancers. In endometrial cancer Treg cells have been investigated only in tumour tissues and, in contrast to some other tumours, fewer Treg cells were reported in endometrial cancer compared with benign controls. Flow cytometry was used to determine the frequency of circulating Treg cells in women undergoing hysterectomy for either endometrial cancer (n = 24) or non- cancer-related conditions (n = 21). Circulating Treg cells were more abundant in women with cancer compared to those without (4.68% vs. 3.66%, p = 0.05, Mann-Whitney test). This relationship disappeared, however, when only data from post-menopausal women were included in the analysis. Mean Treg cell frequency was 4.65% in postmenopausal women with cancer (n = 23) and 4.73% in postmenopausal controls (n = 5) ( p = 0.9). In women without cancer we found that mean Treg cell frequency was higher in postmenopausal women (4.73%, n = 5) in comparison to premenopausal controls (3.33%, n = 16) ( p = 0.02). These results suggest that the increased proportion of Treg cells seen in endometrial cancer patients might be, at least in part, attributed to their postmenopausal status or age. [ABSTRACT FROM AUTHOR]

The author explains why the Australian government should consider replacing quadrivalent human papillomavirus (HPV) vaccines into bivalent vaccines. He argues that the claims of both the quadrivalent vaccine Garadasil from Merck and the bivalent vaccine Cervarix from GlaxoSmithKline (GSK) lies in the principle of the induction of neutralizing antibodies that can prevent the virus from damaging its target cell. He considers the argument of the cost effectiveness of quadrivalent HPV vaccine.

5T4 oncofetal molecules are highly expressed during development and upregulated in cancer while showing only low levels in some adult tissues. Upregulation of 5T4 expression is a marker of loss of pluripotency in the early differentiation of embryonic stem (ES) cells and forms an integrated component of an epithelial-mesenchymal transition, a process important during embryonic development and metastatic spread of epithelial tumors. Investigation of the transcriptional changes in early ES differentiation showed upregulation of CXCL12 and down-regulation of a cell surface protease, CD26, which cleaves this chemokine. CXCL12 binds to the widely expressed CXCR4 and regulates key aspects of development, stem cell motility and tumour metastasis to tissues with high levels of CXCL12. We show that the 5T4 glycoprotein is required for optimal functional cell surface expression of the chemokine receptor CXCR4 and CXCL12 mediated chemotaxis in differentiating murine embryonic stem cells and embryo fibroblasts (MEF). Cell surface expression of 5T4 and CXCR4 molecules is colocalized in differentiating ES cells and MEF. By contrast, differentiating ES and MEF derived from 5T4 knockout (KO) mice show only intracellular CXCR4 expression but infection with adenovirus encoding mouse 5T4 restores CXCL12 chemotaxis and surface co-localization with 5T4 molecules. A series of chimeric constructs with interchanged domains of 5T4 and the glycoprotein CD44 were used to map the 5T4 sequences relevant for CXCR4 membrane expression and function in 5T4KO MEF. These data identified the 5T4 transmembrane domain as sufficient and necessary to enable CXCR4 cell surface expression and chemotaxis. Furthermore, some monoclonal antibodies against m5T4 can inhibit CXCL12 chemotaxis of differentiating ES cells and MEF which is not mediated by simple antigenic modulation. Collectively, these data support a molecular interaction of 5T4 and CXCR4 occurring at the cell surface which directly facilitates the biological response toCXCL12. The regulation of CXCR4 surface expression by 5T4 molecules is a novel means to control responses to the chemokine CXCL12 for example during embryogenesis but can also be selected to advantage the spread of a 5T4 positive tumor from its primary site. [ABSTRACT FROM AUTHOR]

We have recently reported the results of a phase II trial in which two TroVax [modified vaccinia ankara (MVA) encoding the tumour antigen 5T4] vaccinations were given to patients both pre- and post-surgical resection of liver metastases secondary to colorectal cancer (CRC). 5T4-specific cellular responses were assessed at the entry and 2 weeks after each vaccination by proliferation of fresh lymphocytes and ELISA for antibody responses; 18 from the 19 CRC patients mounted a 5T4-specific cellular and/or humoral response. Here, we present a comparison of individual and between patient responses over the course of the treatments using cryopreserved peripheral blood mononuclear cells (PBMC) samples from the baseline until after the fourth vaccination at 14 weeks. Assays used were proliferation assay with 5T4-Fc fusion protein, overlapping 32mer 5T4 peptides, MVA-LacZ and MVA-5T4 infected autologous monocytes. Responses to 5T4 protein or one or more peptide pools were pre-existing in 12/20 patients and subsequently 10 and 12 patients showed boosted and/or de novo responses, respectively. Cumulatively, 13/20 patients showed proliferative responses by week 14. We also assessed the levels of systemic T regulatory cells, plasma cytokine levels, phenotype of tumour-infiltrating lymphocytes including T regulatory cells and tumour HLA class I loss of expression. More than half of the patients showed phenotypes consistent with relative immune suppression and/or escape highlighting the complexity of positive and negative factors challenging any simple correlation with clinical outcome. [ABSTRACT FROM AUTHOR]

The human 5T4 (h5T4) oncofoetal antigen is expressed by a wide variety of human carcinomas including colorectal, ovarian, gastric and renal, but rarely on normal tissues. Its restricted expression on tumour tissues as well as its association with tumour progression and bad prognosis has driven the development of a MVA-based vaccine (TroVax) which has been tested in several early phase clinical trials and these studies have led to the start of a phase III trial in renal cell carcinoma patients. We have recently shown that CD8+ T cells recognizing h5T4 can be generated in the absence of CD4+ T cells from peripheral blood lymphocytes of human healthy individuals. We report the existence and expansion of human CD4+ T cells against h5T4 by stimulation with autologous monocyte-derived dendritic cells infected with a replication defective adenovirus encoding the h5T4 cDNA (Ad-h5T4). The h5T4-specific T-cell responses in normal individuals are enhanced by initial depletion of CD25+ cells (putative T regulatory cells) prior to the in vitro stimulation. We have identified a novel h5T4-derived 15-mer peptide recognized by CD4+ T cells in HLA-DR4 positive healthy individuals. Interestingly, CD4+ T cells spontaneously recognizing a different 5T4 epitope restricted by HLA-DR were identified in tumour-infiltrating lymphocytes isolated from a regressing renal cell carcinoma lung metastasis. Our data show that CD4+ T cells recognizing h5T4 can be expanded and detected in healthy individuals and a renal cell carcinoma patient. Such h5T4-specific CD4+ T cells boosted or induced by vaccination could act to modulate both cell or antibody mediated anti-tumour responses. [ABSTRACT FROM AUTHOR]

CD4+CD25+ regulatory T (Treg) cells are present in increased numbers in patients with advanced cancer and CD25+ T cell depletion potentiates tumour immunity in animal models. The aim of this study was to assess the feasibility and safety of adoptive transfer of CD25+ depleted autologous T cells in patients with advanced renal cell carcinoma and to examine resulting changes in lymphocyte subsets. Six patients with advanced renal cell carcinoma underwent leukapheresis followed by conditioning chemotherapy with cyclophosphamide and fludarabine. The autologous leukapheresis product was depleted of CD25+ cells using CliniMACS® System then re-infused into the patient. Efficient CD25+ depletion from all leukapheresis products was achieved and 0.55–5.87 × 107/kg CD3+ cells were re-infused. Chemotherapy related haematological toxicity was observed, but blood counts recovered in all patients allowing discharge after a mean inpatient stay of 21 days. One patient subsequently developed a rapidly progressive neurological syndrome. A transient reduction in CD25+ subset was noted in the peripheral blood of 5 out of 6 patients with evidence of increased T cell responses to PHA in 4 out of 6 patients. One patient showed increased specific proliferative responses to the tumour associated antigen h5T4 coinciding with the nadir of Treg cells. Given the transient nature of the reduction in CD25+ subset and the observed toxicity there is a need to explore further strategies to improve the safety and efficacy of this approach. Nevertheless, the results provide proof of concept in potentiation of tumour antigen T cell responses when Treg cell levels are depleted. [ABSTRACT FROM AUTHOR]

Regulatory T cells are important in maintaining immune homeostasis, mediating peripheral tolerance and preventing autoimmunity. Increased frequencies of CD4+CD25high T regulatory (TReg) cells have been documented in the peripheral blood of patients with several types of cancer consistent with a role in tumour escape from immunological control. We have investigated the presence of TReg cells systemically and in situ in previously untreated patients with renal cell carcinoma (RCC). We have shown that there is a significant increased frequency of CD4+CD25high T cells in RCC patients ( n = 49) compared to normal donors ( n = 38), respectively, 2.47% versus 1.50%; P < 0.0001. We confirmed these data using the FOXP3 marker of TReg cells in a subset of these patients and normal donors. The population of TReg cells identified showed the expected phenotype with CD4+CD25high population in both RCC patients and normal donors contained higher proportions of CD45RO and GITR than CD4+CD25−/low populations and exhibiting suppressive activity in an anti-CD3 and anti-CD28 induced proliferation assay. CD4+FOXP3+ T cells were detected in the tumour microenvironment by immunofluorescence and the numbers enumerated in lymphocytes recovered following enzymatic disaggregations of biopsies; their frequency was higher in the tumour than the peripheral blood of the same patients. The early follow up data show an association between higher peripheral blood regulatory T-cell count and adverse overall survival. These data confirm the increase of TReg cells in RCC patients and provide impetus to further investigate modulation of TReg activity in RCC patients as part of therapy. [ABSTRACT FROM AUTHOR]

We have investigated the tumour therapeutic efficacy of homologous and heterologous prime-boost vaccine strategies against the 5T4 oncofoetal antigen, using both replication defective adenovirus expressing human 5T4 (Ad5T4), and retrovirally transduced DC lines (DCh5T4) in a subcutaneous B16 melanoma model (B16h5T4). In naïve mice we show that all vaccine combinations tested can provide significant tumour growth delay. While DCh5T4/Adh5T4 sequence is the best prophylactic regimen ( P > 0.0001), it does not demonstrate any therapeutic efficacy in mice with established tumours. In active therapy the Adh5T4/DCh5T4 vaccination sequence is the best treatment regimen ( P = 0.0045). In active therapy, we demonstrate that B16h5T4 tumour growth per se induces Th2 polarising immune responses against 5T4, and the success of subsequent vaccination is dependant on altering the polarizing immune responses from Th2 to Th1. We show that the first immunization with Adh5T4 can condition the mice to induce 5T4 specific Th1 immune responses, which can be sustained and subsequently boosted with DCh5T4. In contrast immunisation with DCh5T4 augments Th2 immune responses, such that a subsequent vaccination with Adh5T4 cannot rescue tumour growth. In this case the depletion of CD25+ regulatory cells after tumour challenge but before immunization can restore therapeutic efficacy. This study highlights that all vaccine vectors are not equal at generating TAA immune responses; in tumour bearing mice the capability of different vaccines to activate the most appropriate anti-tumour immune responses is greatly altered compared to what is found in naïve mice. [ABSTRACT FROM AUTHOR]

The 5T4 oncofetal antigen is expressed by a wide variety of human carcinomas, including colorectal, ovarian and gastric carcinomas. The restricted expression of 5T4 on tumor tissues as well as its implication in tumor progression and bad prognosis makes 5T4 a promising new candidate for immunotherapy. An MVA vaccine encoding 5T4 antigen has been successfully evaluated in preclinical studies in a murine tumor model. Here, we report the generation of human CD8 T cells specific for the 5T4 antigen by stimulation with autologous monocyte derived DC infected with a replication defective adenovirus encoding the 5T4 cDNA (Ad5T4). Analysis of several donors confirms a repertoire of such CD8 responses. In a parallel approach, incorporating the results of proteasome-mediated digestion of 5T4 derived 35-mer peptides and the potential high affinity epitopes predicted by a computer-based algorithm, we identified 8 putative HLA-A*0201-presented CD8 MHC class I epitopes of 5T4 antigen. Two of these generated specific CD8 T cells after restimulation with peptide loaded autologous DC and assay by cytotoxicity and IFNγ ELISPOT. Moreover these particular peptide generated T cells recognized naturally 5T4 positive tumor cells only if they expressed HLA-A*0201 as judged by IFNγ ELISPOT or ELISA. Also, HLA-A*0201 CD8 T cells recognized these peptides in a DC-Ad5T4 polyclonal response. In conclusion, there is a repertoire of CD8 T cell recognition of 5T4 in normal human donors and some candidate HLA-A*0201 epitopes have been identified. © 2006 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Background: Cervical carcinogenesis is initiated by infection with high-risk (i.e., carcinogenic) human papillomavirus (HPV) types. The subsequent progression from premalignant cervical intraepithelial neoplasia (CIN) to invasive cancer is driven by both genetic and epigenetic processes. We assessed the role of the gene encoding the adhesion molecule tumor suppressor in lung cancer 1 (TSLC1) in this progression.Methods: We analyzed TSLC1 gene expression by real-time quantitative reverse transcription-polymerase chain reaction, promoter methylation by sodium bisulfite genomic DNA sequencing, and allelic loss by microsatellite analysis in primary keratinocytes, in four non-tumorigenic HPV-immortalized human keratinocyte cell lines, and in 11 human cervical cancer cell lines that were positive for a high-risk HPV DNA type and in normal cervical epithelial cells. We transfected cervical cancer SiHa cells that did not express TSLC1 mRNA with an expression vector containing the TSLC1 complementary DNA (cDNA) or an empty vector and analyzed transfectants for anchorage-independent growth and tumorigenicity in nude mice. We also examined TSLC1 promoter methylation in premalignant cervical lesions and in cervical carcinomas and smears. All statistical tests were two-sided.Results: TSLC1 mRNA was strongly reduced, relative to levels in primary keratinocytes, or absent in 10 (91%) of 11 cervical carcinoma cell lines but in none (0%) of the four HPV-immortalized cell lines (difference = 91%, 95% confidence interval [CI] = 74% to 100%; P =.004). The TSLC1 promoter was hypermethylated, relative to normal foreskin and cervical epithelial cells, in nine (82%) of the 11 cervical carcinoma cell lines but in none (0%) of the four HPV-immortalized cell lines (difference = 82%, 95 CI = 59% to 100%; P =.01). Seven (88%, 95% CI = 47% to 100%) of the eight SiHa/TSLC1 transfectants displayed a marked reduction in anchorage-independent growth (i.e., 0-100 colonies per 5000 cells) compared with none of the four (0%, 95% CI = 0% to 60%) SiHa transfectants bearing the empty vector (i.e., SiHa/hygro transfectants; difference = 88%, 95% CI = 65% to 100%; P =.01) or untransfected SiHa cells. All seven mice (100%, 95% CI = 59% to 100%) injected with untransfected SiHa cells or SiHa/hygro transfectants displayed tumors of at least 50 mm(3) by 2-6 weeks after injection compared with none of eight mice (0%, 95% CI = 0% to 37%) injected with the SiHa/TSLC1 transfectants (difference = 100%, 95% CI = 68% to 100%; P<.001). We detected TSLC1 promoter hypermethylation in seven (35%, 95% CI = 15% to 59%) of 20 high-grade CIN lesions (i.e., CIN II and III) and in 30 (58%, 95% CI = 43% to 71%) of 52 cervical squamous cell carcinomas compared with none (0%, 95% CI = 0% to 34%) of nine normal cervical epithelial biopsy samples and none (0%, 95% CI = 0% to 22%) of 12 CIN I lesions (P<.001 for cervical squamous cell cancer versus normal epithelial biopsy samples plus CIN I lesions).Conclusions: TSLC1 gene silencing via promoter hypermethylation is a frequent event in the progression from high-risk HPV-containing, high-grade CIN lesions to invasive cervical cancer. [ABSTRACT FROM AUTHOR]

5T4 oncotrophoblast antigen is a transmembrane glycoprotein expressed by trophoblast and many carcinomas but not most normal adult tissues. Results from overexpression of human and mouse 5T4 cDNA in cell lines are consistent with it having an influence on adhesion, shape and motility. We show that murine embryonic stem cell lines are 5T4 negative but that there is rapid up regulation of protein and transcripts upon differentiation, including derivatives of each primary germ layer, as evidenced by cell surface FACS, western and RT-PCR analyses. The kinetics of differentiation and 5T4 expression are closely correlated, with early events linking 5T4 expression to changes in motility and morphology. Comparison of 5T4 expression with other ES cell transcript (Oct 3/4; Rex-1) and antigen markers (Forsmann, SSEA-1) establishes 5T4 as a useful marker for the non-destructive detection of early differentiation of ES cells. For example, 'undifferentiated' ES phenotype defined as SSEA-1 positive and 5T4 negative is seven times more efficient at chimera formation than SSEA-1-positive/5T4-positive cells. Thus, 5T4 glycoprotein expression is associated with early differentiative events of ES cells involving altered motility, and it has useful practical consequences for assessing ES potency and studying similar processes in development and metastasis. [ABSTRACT FROM AUTHOR]

Studies the targeting of immune effector molecules to human tumor cells through the genetic delivery of 5T4-specific single-chain Fv fusion proteins. Key issues of interest; Analysis of pertinent topics and relevant issues; Implications on gene therapy for cancer.

It is now clear that many human tumour antigens can be recognised by the immune system. These tumour antigens can be classified into several groups including cancer-testis, differentiation, tissue specific, over-expressed, and viral-associated antigens. In many cases, there is a known molecular basis of carcinogenesis which provides the explanation for the differentiated expression of these antigens in tumours compared with normal cells. Improved understanding of the biology of the immune response, particularly of immune recognition and activation of T-cells, allow better design of vaccines. Pre-clinical comparative studies allow evaluation of optimal vaccine strategies which can then be delivered to the clinic. Currently, a range of cancer vaccines are being tested including those using tumour cells, proteins, peptides, viral vectors, DNA or dendritic cells. Ultimately, this research should give rise to an entirely new modality of cancer treatments. [ABSTRACT FROM PUBLISHER]

While head and neck squamous cell carcinomas (HNSCC) are marginally decreasing due to the reduction in exposure to the major risk factors, tobacco and alcohol, the incidence of high-risk human papillomavirus (HPV)-positive oropharynx squamous cell carcinomas (OPSCC), especially those in the tonsil and base of tongue subsites, are increasing. Patients with the latter are younger, display a longer overall survival, and show a lower recurrence rate after standard-of-care treatment than those with HPV-negative OPSCC. This may reflect an important role for immune surveillance and control during the natural history of the virally driven tumour development. Immune deviation through acquisition of immune-suppressive factors in the tumour microenvironment (TME) is discussed in relation to treatment response. Understanding how the different immune factors are integrated in the TME battleground offers opportunities for identifying prognostic biomarkers as well as novel therapeutic strategies. OPSCC generally receive surgery or radiotherapy for early-stage tumour treatment, but many patients present with locoregionally advanced disease requiring multimodality therapies which can involve considerable complications. This review focuses on the utilization of newly emerged immune checkpoint inhibitors (PD-1/PD-L1 pathway) for treatment of HNSCC, in particular HPV-positive OPSCC, since they could be less toxic and more efficacious. PD-1/PD-L1 expression in the TME has been extensively investigated as a biomarker of patient response but is yet to provide a really effective means for stratification of treatment. Extensive testing of combinations of therapeutic approaches by types and sequencing will fuel the next evolution of treatment for OPSCC. [ABSTRACT FROM AUTHOR]

The article Combination Treatment with an Antibody-Drug Conjugate (A1mcMMAF) Targeting the Oncofetal Glycoprotein 5T4 and Carboplatin Improves Survival in a Xenograft Model of Ovarian Cancer, written by Y. Louise Wan, Puja Sapra, James Bolton, Jia Xin Chua, Lindy G. Durrant and Peter L. Stern, was originally published under a CC BY-NC 4.0 license, but has now been made available under a CC BY 4.0 license. [ABSTRACT FROM AUTHOR]