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Background: Patients with human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinoma (OPSCC) have a better prognosis than those with HPV-negative tumours. There is interest in de-escalating their treatment but strategies are needed for risk stratification to identify subsets with a poor prognosis. This study investigated tumour-infiltrating lymphocytes (TILs) in relation to HPV tumour status and patient survival. Methods: Biopsies from 218 patients diagnosed with OPSCC between 2002 and 2011, who underwent chemo/radiotherapy were analysed for HPV by PCR, in-situ hybridisation and p16 immunohistochemistry (IHC). One hundred and thirty-nine samples with concordant HPV detection were analysed for CD3, CD4, CD8 and FoxP3 expression in tumour and stromal regions using multiplexIHC and multispectral image analysis. Labelling of smooth muscle actin (SMA) identified activated stroma. Results: Human papillomavirus-positive compared with HPV-negative OPSCC had higher infiltration in both tumour and stromal areas of CD4 and CD8 T cells but not FoxP3 T regulatory cells. Only CD3+CD8+ stromal and not tumour area infiltration was associated with increased survival ( P =0.02). There was significantly higher SMA expression in HPV-positive compared with -negative tumours, which did not correlate with survival. Conclusions: Studies of TILs for risk stratification in OPSCC should assess stromal infiltration.

Background: Vulval intraepithelial neoplasia (VIN) is a premalignant condition, which is frequently associated with type HPV16 infection, and multifocal disease has high rates of surgical treatment failure. Methods: We report a phase II clinical trial of the topical immunomodulator, imiquimod, for 8 weeks, followed by 3 doses (weeks 10, 14 and 18) of therapeutic human papillomavirus (HPV) vaccination (TA-CIN, fusion protein HPV16 E6E7L2) in 19 women with VIN grades 2 and 3. Histology and HPV testing of biopsies were performed at weeks 0, 10, 20 and 52. Intralesional infiltration of T-cell subsets and lymphocyte proliferation for HPV systemic immune responses were also assessed. Results: Lesion response (complete regression of VIN on histology) was observed in 32% (6 out of 19) of women at week 10, increasing to 58% (11 out of 19) at week 20 and 63% (12 out of 19) at week 52. At this time, 36% (5 out of 14) of lesions showed HPV16 clearance and 79% (15 out of 19) of women were symptom free. At week 20, after treatment with imiquimod and vaccination, there was significantly increased local infiltration of CD8 and CD4 T cells in lesion responders; in contrast, non-responders (persistent VIN by histology) showed an increased density of T regulatory cells. After vaccination, only lesion responders had significantly increased lympho-proliferation to the HPV vaccine antigens. Conclusion: The therapeutic effect of treatment depends on the differential immune response of responders and non-responders with affect locally and systemically.

Heterologous prime-boost vaccination schedules employing TA-HPV, a vaccinia virus encoding HPV 16/18 E6 and E7, in combination with TA-CIN, an HPV 16 L2E6E7 fusion protein, may offer advantages over the use of either agent alone for the immunotherapy of human papillomavirus (HPV) type 16-associated vulval intraepithelial neoplasia (VIN). In the present study, 10 women with HPV 16-positive high grade VIN, previously primed with TA-HPV, received three booster immunisations with TA-CIN. All but one demonstrated HPV 16-specific proliferative T-cell and/or serological responses following vaccination. Three patients additionally showed lesion shrinkage or symptom relief, but no direct correlation between clinical and immunological responses was seen.

Superantigens activate T-cells by linking the T-cell receptor to MHC class II on antigen-presenting cells, and novel reactivity can be introduced by fusing the superantigen to a targeting molecule. Thus, an antibody-targeted superantigen, which activates T cells to destroy tumour cells, might be used as cancer therapy. A suitable target is the 5T4 oncofetal antigen, which is expressed on many carcinomas. We constructed a fusion protein from a Fab of a monoclonal antibody recognizing the 5T4 antigen, and an engineered superantigen. The recombinant product 5T4FabV13-SEA D227A bound the 5T4 antigen expressed on the human non-small-cell lung cancer cell line Calu-1 with a K d of 1.2 nM while the substitution of Asp227 to Ala in the superantigen moiety reduced binding activity to MHC class II. 5T4FabV13-SEA D227A tumour reactivity was demonstrated in 7/7 NSCLC samples by immunohistochemistry, while normal tissue reactivity was low to moderate. 5T4FabV13-SEA D227A induced significant T-cell-dependent in vitro killing of sensitive 5T4 bearing Calu-1 cells, with maximum lysis at 10 –10 M, while the capacity to lyse MHC class II expressing cells was approximately 1000 times less effective. Immunotherapy of 5T4FabV13-SEA D227A against human NSCLC was investigated in SCID mice reconstituted with human peripheral blood mononuclear cells. Mice carrying intreperitoneally growing Calu-1 cells showed significant reduction in tumour mass and number after intravenous therapy with 5T4FabV13-SEA D227A . Thus, 5T4FabV13-SEA D227A has highly attractive properties for therapy of human NSCLC. © 2001 Cancer Research Campaign http://www.bjcancer.com

The 5T4 oncofoetal antigen is a heavily glycosylated cell surface protein found on human placental trophoblast and on diverse types of human cancer but is not expressed at significant levels on adult human tissues in health. It therefore satisfies the criteria for a tumour-associated antigen and is an ideal target for the immunotherapy of cancer. We report here that 5T4 is strongly expressed on the majority of renal cell carcinomas and therefore this population of patients is suitable for trials of 5T4-targeted therapies. In particular, we have shown that T cells from renal cell carcinoma patients can be genetically modified to kill 5T4 expressing renal cancer cell lines by introduction of a chimeric-signalling protein. This protein consists of a single chain antibody fragment capable of binding antigen directly at the cell surface and then activating the T cell by virtue of a CD3 ζ- signalling domain. This is a powerful tool that bypasses a number of mechanisms that allow tumours to escape T-cell killing and can be readily scaled up for clinical use.

Although the overall prognosis in childhood acute lymphoblastic leukemia (ALL) is good, outcome after relapse is poor. Recurrence is frequently characterized by the occurrence of disease at extramedullary sites, such as the central nervous system and testes. Subpopulations of blasts able to migrate to such areas may have a survival advantage and give rise to disease recurrence. Gene expression profiling of 85 diagnostic pre-B-ALL bone marrow samples revealed higher 5T4 oncofetal antigen transcript levels in cytogenetic high-risk subgroups of patients ( P <0.001). Flow cytometric analysis determined that bone marrow from relapse patients have a significantly higher percentage of 5T4-positive leukemic blasts than healthy donors ( P =0.005). The high-risk Sup-B15 pre-B-ALL line showed heterogeneity in 5T4 expression, and the derived, 5T4 + (Sup5T4) and 5T4 − (Sup) subline cells, displayed differential spread to the omentum and ovaries following intraperitoneal inoculation of immunocompromised mice. Consistent with this, Sup5T4 compared with Sup cells show increased invasion in vitro concordant with increased LFA-1 and VLA-4 integrin expression, adhesion to extracellular matrix and secretion of matrix metalloproteases (MMP-2/-9). We also show that 5T4-positive Sup-B15 cells are susceptible to 5T4-specific superantigen antibody-dependent cellular toxicity providing support for targeted immunotherapy in high-risk pre-B-ALL.

In a phase II study, 43 renal cell carcinoma patients were treated with individualised doses of ABR-214936; a fusion of a Fab recognising the antigen 5T4, and Staphylococcal enterotoxin A. Drug was given intravenously on 4 consecutive days, treatment was repeated 1 month later. Treatment was associated with moderate fever and nausea, but well tolerated. Of 40 evaluable patients, 28 had disease control at 2 months, and at 4 months, one patient showed partial response (PR) and 16 patients stable disease. Median survival, with minimum follow-up of 26 months was 19.7 months with 13 patients alive to date. Stratification by the Motzer's prognostic criteria highlights prolonged survival compared to published expectation. Patients receiving higher drug exposure had greater disease control and lived almost twice as long as expected, whereas the low-exposure patients survived as expected. Sustained interleukin-2 (IL-2) production after a repeated injection appears to be a biomarker for clinical effect, as the induced-IL-2 level on the day 2 of treatment correlated with survival. The high degree of disease control and the prolonged survival suggest that this treatment can be effective. These findings will be used in the trial design for the next generation of drug, with reduced antigenicity and toxicity.

Vulval intraepithelial neoplasia (VIN) is defined histopathologically by distinctive abnormalities of cellular maturation and differentiation. To investigate the functional properties of VIN, the expression of several proteins involved in the regulation of the cell cycle as well as in situ DNA replication competence was analysed by immunohistochemistry. Snap-frozen vulval biopsies were graded as normal squamous epithelium ( n =6), undifferentiated HPV positive VIN 1 ( n =3), VIN 2 ( n =8) and VIN 3 ( n =20). Immunohistochemistry was performed using the following markers: cyclin D1 (expressed in middle/late G1), cyclin B1 (expressed in G2/early M), phosphorylated histone H3 (expressed during mitosis) and minichromosome maintenance (Mcm) proteins 2 and 5 (expressed during the cell cycle, but not in differentiated or quiescent cells). In situ DNA replication competence was used to identify S-phase cells. The percentage of positively stained nuclei in three representative microscopic fields was calculated per biopsy. In normal vulva, the expression of all markers was restricted to the proliferative compartment of the basal layer of the epithelium. In contrast in high-grade VIN, the majority of epithelial cells expressed the Mcm proteins from basal to superficial layer. The detection of cyclins B1 and D1, phospho-histone H3 and in situ DNA replication was also found through the full thickness of these lesions but by a lower proportion of the cells. This is consistent with these markers providing a series of ‘snapshots’ of the cell cycle status of individual cells. The low-grade VIN showed reduced expression of the cell cycle markers in relation to the level of dysplasia. The combination of these analyses establishes that the majority of VIN cells remain in a functional replicative or prereplicative state of the cell cycle. Clinical application of these analyses may provide a basis for improved diagnosis of VIN.

Pseudomyxoma peritonei (PMP) is a rare neoplasm of mainly appendiceal origin, characterised by excess intra-abdominal mucin production leading to high morbidity and mortality. While histological features are frequently indolent, this tumour disseminates aggressively throughout the abdominal cavity, yet seldom metastasises. This study determined the expression of several markers of colorectal differentiation (carcinoembryonic antigen (CEA), cytokeratins (CK20 and CK7), epithelial membrane antigen), mucin production (MUC-2, interleukin-9 (IL-9), IL-9 receptor (IL-9R α )), and cell adhesion (N- and E-cadherin, vimentin) in PMP tissue ( n =26) compared with expressions in normal colonic mucosa ( n =19) and colorectal adenocarcinoma ( n =26). Expressions of CEA and cytokeratins were similar for PMP as those in colorectal adenocarcinomas with the exception that the CK20−/CK7− pattern was rare in PMP (Fisher's exact test: P =0.001). Similarly, expressions of mucin-related proteins were comparable for adenocarcinoma and PMP, with the exception that IL-9 expression was uncommon in adenocarcinoma ( P =0.009). Pseudomyxoma peritonei demonstrated a specific pattern of adhesion-related protein expressions of increased N-cadherin, reduced E-cadherin, and increased vimentin ( P =0.004), a phenotype suggesting a possible epithelial–mesenchymal transition state. Primary PMP cell cultures were successfully maintained and demonstrated marker expressions similar to those seen in in vivo tissues. These early characterisation studies demonstrate similarities between PMP and colorectal adenocarcinoma, but also reveal a specific cadherin phenotype that may characterise the distinct non-metastasising behaviour of PMP, and form the basis for future mechanistic and therapy-targeting research.

The identification of dendritic cells (DC) as the major antigen-presenting cell type of the immune system, combined with the development of procedures for their ex vivo culture, has opened possibilities for tumour immunotherapy based on the transfer of recombinant tumour antigens to DC. It is anticipated that the most effective type of response would be the stimulation of specific, MHC class I restricted cytotoxic T lymphocytes capable of recognising and destroying tumour cells. In order to make this approach possible, methods must be developed for the transfer of recombinant antigen to the DC in such a way that they will initiate an MHC class I restricted response. Here, we demonstrate that murine DC infected with a recombinant fowlpox virus (rFWPV) vector stimulate a powerful, MHC class I restricted response against a recombinant antigen. A rFWPV containing the OVA gene was constructed and used to infect the DC line DC2.4. The infected DC2.4 cells were found to stimulate the T-T cell hybridoma line RF33. 70, which responds specifically to the MHC class I restricted OVA peptide SIINFEKL. The stimulatory ability of the rFWPV-infected DC2.4 cells lasted for at least 72 h after infection and was eventually limited by proliferation of uninfected cells. By comparison, DC2.4 cells pulsed with synthetic SIINFEKL peptide stimulated RF33.70 well initially, but the stimulatory ability had declined to zero by 24 h after pulsing. FWPV infection of DC2.4 up-regulated MHC and costimulatory molecule expression. rFWPV was also found to infect both immature and mature human DC derived from cord blood CD34+ progenitors and express transgenes for up to 20 days after infection. We conclude that rFWPV shows promise as a vector for antigen gene transfer to DC in tumour immunotherapy protocols.