Background:Human papillomavirus (HPV)-positive oropharyngeal cancer (OPSCC) is associated with improved survival compared with HPV-negative disease. However, a minority of HPV-positive patients have poor prognosis. Currently, there is no generally accepted strategy for identifying these patients.Methods:We retrospectively analysed 270 consecutively treated OPSCC patients from three centres for effects of clinical, pathological, immunological, and molecular features on disease mortality. We used Cox regression to examine associations between factors and OPSCC death, and developed a prognostic model for 3-year mortality using logistic regression analysis.Results:Patients with HPV-positive tumours showed improved survival (hazard ratio (HR), 0.33 (0.21–0.53)). High levels of tumour-infiltrating lymphocytes (TILs) stratified HPV-positive patients into high-risk and low-risk groups (3-year survival; HPV-positive/TILhigh=96%, HPV-positive/TILlow=59%). Survival of HPV-positive/TILlow patients did not differ from HPV-negative patients (HR, 1.01; P=0.98). We developed a prognostic model for HPV-positive tumours using a ‘training' cohort from one centre; the combination of TIL levels, heavy smoking, and T-stage were significant (AUROC=0·87). This model was validated on patients from the other centres (detection rate 67% false-positive rate 5.6% AUROC=0·82).Interpretation:Our data suggest that an immune response, reflected by TIL levels in the primary tumour, has an important role in the improved survival seen in most HPV-positive patients, and is relevant for the clinical evaluation of HPV-positive OPSCC.

Worldwide, approximately 405 000 cases of oral cancer (OSCC) are diagnosed each year, with a rising incidence in many countries. Despite advances in surgery and radiotherapy, which remain the standard treatment options, the mortality rate has remained largely unchanged for decades, with a 5-year survival rate of around 50%. OSCC is a heterogeneous disease, staged currently using the TNM classification, supplemented with pathological information from the primary tumour and loco-regional lymph nodes. Although patients with advanced disease show reduced survival, there is no single pathological or molecular feature that identifies aggressive, early-stage tumours. We retrospectively analysed 282 OSCC patients for disease mortality, related to clinical, pathological, and molecular features based on our previous functional studies [EGFR, αvβ6 integrin, smooth muscle actin (SMA), p53, p16, EP4]. We found that the strongest independent risk factor of early OSCC death was a feature of stroma rather than tumour cells. After adjusting for all factors, high stromal SMA expression, indicating myofibroblast transdifferentiation, produced the highest hazard ratio (3.06, 95% CI 1.65-5.66) and likelihood ratio (3.6; detection rate: false positive rate) of any feature examined, and was strongly associated with mortality, regardless of disease stage. Functional assays showed that OSCC cells can modulate myofibroblast transdifferentiation through αvβ6-dependent TGF-β1 activation and that myofibroblasts promote OSCC invasion. Finally, we developed a prognostic model using Cox regression with backward elimination; only SMA expression, metastasis, cohesion, and age were significant. This model was independently validated on a patient subset (detection rate 70%; false positive rate 20%; ROC analysis 77%, p < 0.001). Our study highlights the limited prognostic value of TNM staging and suggests that an SMA-positive, myofibroblastic stroma is the strongest predictor of OSCC mortality. Whether used independently or as part of a prognostic model, SMA identifies a significant group of patients with aggressive tumours, regardless of disease stage.

Understanding gene expression profiles during early human pancreas development is limited by comparison to studies in rodents. In this study, from the inception of pancreatic formation, embryonic pancreatic epithelial cells, approximately half of which were proliferative, expressed nuclear PDX1 and cytoplasmic CK19. Later, in the fetal pancreas, insulin was the most abundant hormone detected during the first trimester in largely nonproliferative cells. At sequential stages of early fetal development, as the number of insulin-positive cell clusters increased, the detection of CK19 in these cells diminished. PDX1 remained expressed in fetal beta cells. Vascular structures were present within the loose stroma surrounding pancreatic epithelial cells during embryogenesis. At 10 weeks post-conception (w.p.c.), all clusters containing more than ten insulin-positive cells had developed an intimate relationship with these vessels, compared with the remainder of the developing pancreas. At 12-13 w.p.c., human fetal islets, penetrated by vasculature, contained cells independently immunoreactive for insulin, glucagon, somatostatin and pancreatic polypeptide (PP), coincident with the expression of maturity markers prohormone convertase 1/3 (PC1/3), islet amyloid polypeptide, Chromogranin A and, more weakly, GLUT2. These data support the function of fetal beta cells as true endocrine cells by the end of the first trimester of human pregnancy.

Alström syndrome (OMIM 203800) is an autosomal recessive disease, characterized by cone-rod retinal dystrophy, cardiomyopathy and type 2 diabetes mellitus, that has been mapped to chromosome 2p13 (refs 1-5). We have studied an individual with Alström syndrome carrying a familial balanced reciprocal chromosome translocation (46, XY,t(2;11)(p13;q21)mat) involving the previously implicated critical region. We postulated that this individual was a compound heterozygote, carrying one copy of a gene disrupted by the translocation and the other copy disrupted by an intragenic mutation. We mapped the 2p13 breakpoint on the maternal allele to a genomic fragment of 1.7 kb which contains exon 4 and the start of exon 5 of a newly discovered gene (ALMS1); we detected a frameshift mutation in the paternal copy of the gene. The 12.9-kb transcript of ALMS1 encodes a protein of 4,169 amino acids whose function is unknown. The protein contains a large tandem-repeat domain comprising 34 imperfect repetitions of 47 amino acids. We have detected six different mutations (two nonsense and four frameshift mutations causing premature stop codons) in seven families, confirming that ALMS1 is the gene underlying Alström syndrome. We believe that ALMS1 is the first human disease gene characterized by autosomal recessive inheritance to be identified as a result of a balanced reciprocal translocation.Alström syndrome was initially mapped to an interval of 6.1 cM between loci D2S286 and D2S327 (refs 3-5). Although several candidate genes have been investigated, no mutations have previously been identified [6][7][8] . We have shown that the 2p13 breakpoint in the individual with the 46,XY,t(2;11)(p13;q21)mat translocation is between these loci by metaphase fluorescence in situ hybridization (FISH) analysis using the BACs RP11-355F16 (containing D2S286) and RP11-480F1 (located 150 kb proximal to D2S327) as probes. The BAC RP11-582H21 crosses the translocation breakpoint (Fig. 1a,b) and is overlapped by RP11-79N18, which contains CCT7, a member of a chaperonin gene

The initiation, growth, recurrence and metastasis of solid tumours, including squamous cell carcinoma of the head and neck region, have been related to the behaviour of a small subpopulation of 'tumour-initiating' cells. Cells with stem cell characteristics have also been identified in cell lines derived from cancers and the aim of the present work was to extend examination of such cells. Established cell lines were examined for their patterns of colony morphologies and staining, the presence of a Hoechst dye-excluding 'side population', expression of the putative stem cell markers CD44, CD133 and CD29, and their ability to grow as 'cancer spheroids'. Two cell lines, CaLH2 and CaLH3, recently generated from HNSCC tumour biopsies, were similarly examined. All cell lines showed a holoclone/meroclone/paraclone series of colony morphologies and cell sorting indicated that CD44 marker expression was related to clonogenicity. FACS analysis after exposure to Hoechst dye indicated that the CA1, H357 and UK1 cell lines contain a dye-excluding 'side population', a property associated with stem-like subpopulations. When held in suspension, all cell lines formed spheroids that could be re-passaged. These observations indicate that cell lines derived from HNSCC contain cells with stem cell properties and that such cell lines may provide experimental systems relevant to the behaviour of stem cells present in the tumours of origin and to their responses to therapy.

The Epstein-Barr virus (EBV) is associated with the development of several human tumors, including Hodgkin's lymphoma (HL) and EBV-positive undifferentiated nasopharyngeal carcinoma (NPC).1 In HL, the malignant Hodgkin's and Reed-Sternberg (HRS) cells constitute only a minority of the total tumor mass, and are surrounded by variable proportions of nonmalignant reactive cells. In approximately onehalf of HL, EBV can be detected in HRS cells, where the virus expresses a limited subset of genes; these include the Epstein-Barr nuclear antigen-1 (EBNA1) and the latent membrane proteins, LMP1 and LMP2.2 Although EBV-specific cytotoxic T cells (CTLs) can be detected in HL and NPC and have been shown to kill LMP1-and LMP2-expressing cells in vitro, they are unable to eliminate EBV-infected tumor cells in vivo. [3][4][5] This failure may be because of increased recruitment of regulatory T cells

Chronic graft-versus-host disease (cGVHD) is characterized by a state of profound immunodeficiency in association with alloreactive and autoimmune phenomena. These observations indicate an impairment of immunologic tolerance that could involve both central and peripheral mechanisms. Defective thymic function may contribute to dysregulation of central tolerance, but few studies have addressed peripheral tolerance. Recently a population of CD4 ؉ CD25 ؉ T cells (T reg cells) has been characterized, which controls immunologic reactivity in vivo and which on transfer can prevent experimental acute GVHD. We investigated the number and function of peripheral blood CD4 ؉ CD25 high T cells in patients more than 100 days after allogeneic hematopoietic stem cell transplantation. Patients with cGVHD had markedly elevated numbers of CD4 ؉ CD25 high T cells as compared to patients without GVHD. CD4 ؉ CD25 high T cells derived from patients in both groups were of donor origin, lacked markers of recent activation, and expressed intracellular CD152. In contrast to controls, CD4 ؉ CD25 high T cells derived from patients with cGVHD were characterized by lower surface CD62L expression. In vitro, CD4 ؉ CD25 high T cells were hyporesponsive to polyclonal stimulation and suppressed the proliferation and cytokine synthesis of CD4 ؉ CD25 ؊ cells, an effect that was independent of interleukin 10. These results indicate that chronic graft-versus-host injury does not occur as a result of T reg cell deficiency. IntroductionChronic graft-versus-host disease (cGVHD) remains the most frequent complication following allogeneic hematopoietic stem cell transplantation (HSCT), occurring in 30% to 70% of long-term survivors. 1 Profound immune dysregulation leads to both immunodeficiency and autoimmunity, suggesting defects in central or peripheral immunologic tolerance. 2 Impairment of thymic function observed in patients with cGVHD 3 could lead to the release into the periphery of T cells with autoreactive potential. 4 Although such perturbations in central tolerance may be important, data relating to experimental autologous GVHD induction 5 or to models in which thymic, negative selection is prevented, 6 suggest that autoreactive T cells only induce tissue injury when there are additional defects in peripheral tolerance.Recently a population of naturally occurring regulatory CD4 ϩ T cells (T reg cells) that constitutively express the ␣ chain of the receptor for interleukin 2 (IL-2; CD25) has been characterized that can suppress immune responses both in vitro and in vivo (for reviews, see Maloy and Powrie, 7 Shevach, 8 and Wood and Sakaguchi 9 ). In humans, T reg cells appear to be enriched within the 1% to 2% of peripheral blood CD4 ϩ T cells that are CD25 high10 and in vitro, inhibit the function of effector T cells via a mechanism that requires prior T-cell activation, involves cell-to-cell contact, and is independent of cytokine secretion. [11][12][13][14][15][16] In contrast, in vivo studies indicate that cytokines such as IL-10 or transfor...

Based on evidence suggesting similarities to human embryonic stem cells, human embryonic germ (hEG) cells have been advocated as an alternative pluripotent stem cell resource but have so far received limited attention. To redress this imbalance, human fetal gonads were collected for the isolation and culture of primordial germ cells at 7-9 weeks postconception. We provide evidence for the derivation, culture, and differentiation of hEG cells in vitro. This evidence includes the expression of markers characteristic of pluripotent cells, the retention of normal XX or XY karyotypes, and the demonstration of pluripotency, as suggested by the expression of markers indicative of differentiation along the three germ lineages (ectoderm, mesoderm, and endoderm) and an associated loss of pluripotent markers. In assessing this differentiation, however, we also demonstrate a hitherto unacknowledged overlap in gene expression profiles between undifferentiated and differentiated cell types, highlighting the difficulty in ascribing cell lineage by gene expression analyses. Furthermore, we draw attention to the problems inherent in the management of these cells in prolonged culture, chiefly the difficulty in preventing spontaneous differentiation, which hinders the isolation of pure, undifferentiated clonal lines. While these data advocate the pursuit of pluripotent hEG cell studies with relevance to early human embryonic development, culture limitations carry implications for their potential applicability to ambitious cell replacement therapies.