[Un- and dedifferentiated endometrial carcinoma : A rare entity with a wide range of differential diagnosis]. / Das un- und dedifferenzierte Endometriumkarzinom : Eine seltene Entität mit breitem differenzialdiagnostischem Spektrum.

Dedifferentiated endometrial carcinomas (ECs) are composed of undifferentiated EC and a FIGO grade 1 or 2 endometrioid carcinoma. The undifferentiated component represents a malignant epithelial neoplasm with no obvious differentiation and immunohistochemical loss of PAX8, E­cadherin and focal expression of EMA and/or CK18 and the predominant presence of nuclear staining for INI1 (SMARCB1) and BRG1 (SMARCA4). The main differential diagnoses include poorly differentiated endometrioid EC, neuroendocrine carcinoma, lymphoma, plasmocytoma, high-grade endometrial stromal sarcomas, undifferentiated uterine sarcomas (UUS), carcinosarcomas, and metastases to the endometrium. The histogenesis is not yet fully understood and molecular data are still limited. Some tumors represent a loss of MHL1 and PMS2 staining due to MLH1-promotor methylation. Rare cases are associated with Lynch syndrome or POLE mutation. The un- or dedifferentiated EC represents a high-grade endometrial carcinoma that requires extended surgery and indicates a poor prognosis. In cases with mismatch repair protein deficiency or POLE mutation, immuno-oncological treatment with checkpoint inhibitors are a therapeutic option.

[TILGen study-immunological targets in patients with breast cancer : Influence of tumor-infiltrating lymphocytes]. / TILGen-Studie ­ Immunologische Angriffspunkte bei Patientinnen mit Brustkrebs : Einfluss der tumorinfiltrierenden Lymphozyten.

BACKGROUND: The interaction of our immune system with breast cancer (BC) cells prompted the investigation of tumor-infiltrating lymphocytes (TILs) and targeted, tumor antigen-specific immunotherapy. OBJECTIVES: Correlation between TILs and pathological complete response (pCR) after neoadjuvant systemic therapy (NACT). Tumor-specific antigens (TSAs) in HER2+ and triple negative BC and establishment of TSA-specific therapies within the interdisciplinary TILGen study. METHODS: Illustration of the TILGen study design. Assessment of TILs and correlation with pCR within this BC study. RESULTS: pCR was achieved in 38.4% (56/146) and associated with estrogen receptor/progesterone receptor negative (ER-/PR-) and HER2+ tumors. Lymphocytic predominant BC (LPBC) was found in 16.4% (24/146), particularly in ER-/PR- (ER-: 27.3% vs. ER+: 9.9%, PR-: 22.3% vs. PR+: 8.2%), large, and poorly differentiated BC. TILs were significantly correlated with pCR in multivariate analysis. In LPBC, pCR was achieved in 66.7%, whereas it was 32.8% in non-LPBC. CONCLUSIONS: First results confirm the influence of the human immune system on the response to NACT in HER2+ and triple negative BC. TSA-specific immunotherapy might improve the outcome in BC patients but there is an urgent need for comprehensive studies to further investigate this issue.

Endometrial mRNA expression of selected pro-inflammatory factors and mucins in repeat breeder cows with and without subclinical endometritis.

Repeat breeder cows (RBC) are defined as cyclic cows without clinical abnormalities that fail to conceive after at least three subsequent inseminations. Previous studies have elucidated cellular defence mechanisms in the bovine uterus but detailed information on inflammatory events of endometrial cells in RBC is still lacking. Thus, the objective of this study was to analyse endometrial mRNA expression of selected transcripts associated with uterine inflammatory processes. Cytobrush samples from 91 RBC and 11 synchronised heifers with no history of gynaecological abnormalities (controls, CON) were collected. The proportion of polymorphonuclear neutrophils in these samples was used for the diagnosis of subclinical endometritis (SE). Ultrasonography and progesterone blood concentrations were used to determine ovarian activity and the stage of the oestrous cycle. Total RNA was isolated from the cytobrush samples and subjected to reverse transcription-quantitative PCR for interleukins (IL) 1A, IL1B, IL6, IL8, chemokine CXL ligand (CXCL) 3, CXCL5, prostaglandin-endoperoxide synthase 2 (PTGS2), tracheal antimicrobial peptide (TAP) and mucin (MUC) 4, MUC5, MUC6, MUC12 and MUC16. CXCL3 mRNA was higher (2-fold) and PTGS2 mRNA lower (6-fold) expressed in RBC compared with CON (P < 0.05). After subdivision of RBC in animals with (RBC-SE) and without SE (RBC-noSE), these differences remained significant between RBC-noSE and CON. Higher mRNA abundances of IL1A and IL1B were found in RBC-SE compared with RBC-noSE (3- and 4-fold; P < 0.05). No differences in the mRNA expression of IL6, IL8, CXCL5 and TAP were observed between RBC-SE, RBC-noSE and CON. MUC4 and MUC12 mRNA was more highly expressed in RBC than in CON (P < 0.05). In RBC-noSE, a 5- and 14-fold higher MUC4 and MUC12 mRNA expression was noticed compared with CON (P < 0.05). A significantly lower mRNA expression of MUC5 and MUC16 (7- and 4-fold) was detected in RBC in the luteal phase compared with RBC in the follicular phase, whereas such a down-regulation was not observed for MUC4 and MUC12. In conclusion, we demonstrated different PTGS2 and CXCL3 mRNA expression between RBC and control heifers, which might be related to subfertility in RBC. Further studies are required to confirm that an unregulated MUC4 and MUC12 mRNA expression may contribute to subfertility of RBC. These findings provide a valid basis for further research on regulatory mechanisms of mRNA expression in subfertile cows.

Comparison of prognostic and predictive impact of genomic or central grade and immunohistochemical subtypes or IHC4 in HR+/HER2- early breast cancer: WSG-AGO EC-Doc Trial.

INTRODUCTION: Potential prognostic and predictive markers in early, intermediate-risk breast cancer (BC) include histological grade, Ki-67, genomic signatures, e.g. genomic grade index (GGI), and intrinsic subtypes. Their prognostic/predictive impact in hormone receptor (HR: ER and/or PR) positive/HER2- BC is controversial. WSG-AGO EC-Doc demonstrated superior event-free survival (EFS) in patients with 1-3 positive lymph node receiving epirubicin/cyclophosphamide-docetaxel (EC-Doc) versus 5-fluoruracil/epirubicin/cyclophosphamide (FEC). METHODS: In a representative trial subset, we quantify concordance among factors used for clinical chemotherapy indication. We investigate the impact of central histology (n = 772), immunohistochemistry for intrinsic subtyping and IHC4, and dichotomous (GG) or continuous (GGI) genomic grade (n = 472) on patient outcome and benefit from taxane chemotherapy, focusing on HR+/HER2- patients (n = 459). RESULTS: Concordance of local grade (LG) with central (CG) or genomic grade was modest. In HR+/HER2- patients, low (GG-1: 16%), equivocal (GG-EQ: 17%), and high (GG-3: 67%) GG were associated with respective 5-year EFS of 100%, 93%, and 85%. GGI was prognostic for EFS within all LG subgroups and within CG3, whereas IHC4 was prognostic only in CG3 tumors.In unselected and HR+/HER2- patients, CG3 and luminal-A-like subtype entered the multivariate EFS model, but not IHC4 or GG. In the whole population, continuous GGI entered the model [hazard ratio (H.R.) of 75th versus 25th = 2.79; P = 0.01], displacing luminal-A-like subtype; within HR+/HER2- (H.R. = 5.36; P < 0.001), GGI was the only remaining prognostic factor.In multivariate interaction analysis (including central and genomic grade), luminal-B-like subtype [HR+ and (Ki-67 ≥20% or HER2+)] was predictive for benefit of EC-Doc versus FEC in unselected but not in HR+/HER2- patients. CONCLUSION: In the WSG-AGO EC-Doc trial for intermediate-risk BC, CG, intrinsic subtype (by IHC), and GG provide prognostic information. Continuous GGI (but not IHC4) adds prognostic information even when IHC subtype and CG are available. Finally, the high interobserver variability for histological grade and the still missing validation of Ki-67 preclude indicating or omitting adjuvant chemotherapy based on these single factors alone. TRIAL REGISTRATION: The WSG-AGO/EC-Doc is registered at ClinicalTrials.gov, NCT02115204.

The prevalence of subclinical endometritis and intrauterine infections in repeat breeder cows.

The objectives of this study were to assess the prevalence of subclinical endometritis and the presence of common uterine pathogens in repeat breeder cows. A total of 121 cows with three or more consecutive artificial inseminations without conception and no clinical signs of disease were defined as repeat breeder cows and were enrolled in this trial. Intrauterine samples were collected with the cytobrush technique to determine the prevalence of subclinical endometritis and bacteriologic infections. Blood samples were analyzed for concentrations of progesterone and estradiol in plasma to assess ovarian activity. Furthermore, breed, parity, history of calving and postpartum uterine infection, clinical findings of transrectal palpation, and backfat thickness were analyzed as potential factors for the prevalence of subclinical endometritis in repeat breeder cows. The prevalence of subclinical endometritis in repeat breeder cows was 12.7%; but common uterine pathogens, Escherichia coli and Trueperella pyogenes, were found in only one and three cows, respectively. Ovarian activity was determined in 95.0% of all cows. Recorded variables had no effect on the prevalence of subclinical endometritis in repeat breeder cows. In conclusion, subclinical endometritis and uterine infections linked to common pathogens were playing a minor role as a cause for repeat breeder cows in this study. Alternative reasons for failure to conceive in these cows are discussed.

Compression induces Ephrin-A2 in PDL fibroblasts via c-fos.

Ephrin-A2-EphA2 and ephrin-B2-EphB4 interactions have been implicated in the regulation of bone remodeling. We previously demonstrated a potential role for members of the Eph-ephrin family of receptor tyrosine kinases for bone remodeling during orthodontic tooth movement: compression-dependent upregulation of ephrin-A2 in fibroblasts of the periodontal ligament (PDL) attenuated osteogenesis in osteoblasts of the alveolar bone. However, factors affecting the regulation of ephrin-A2 expression upon the application of compressive forces remained unclear. Here, we report a mechano-dependent pathway of ephrin-A2 induction in PDL fibroblasts (PDLFs) involving extracellular signal-regulated kinases (ERK) 1/2 and c-fos. PDLF subjected to compressive forces (30.3 g/cm(2)) upregulated c-fos and ephrin-A2 mRNA and protein expression and displayed increased ERK1/2 phosphorylation. Inhibition of the MAP kinase kinase (MEK)/ERK1/2 pathway using the specific MEK inhibitor U0126 significantly reduced ephrin-A2 messenger RNA upregulation upon compression. Silencing of c-fos using a small interfering RNA approach led to a significant inhibition of ephrin-A2 induction upon the application of compressive forces. Interestingly, ephrin-A2 stimulation of PDLF induced c-fos expression and led also to the induction of ephrin-A2 expression. Using a reporter gene construct in murine 3T3 cells, we found that ephrin-A2 was able to stimulate serum response element (SRE)-dependent luciferase activity. As the regulation of c-fos is SRE dependent, ephrin-A2 might induce c-fos via SRE activation. Taken together, we provide evidence for an ERK1/2- and c-fos-dependent regulation of ephrin-A2 in compressed PDLF and suggest a novel pathway for ephrin-A2 induction emanating from ephrin-A2 itself. We showed previously that ephrin-A2 at compression sites might contribute to tooth movement by inhibiting osteogenic differentiation. The regulatory pathway of ephrin-A2 induction during tooth movement identified in this study might be accessible for pharmacological interventions.

Final analysis of the prospective WSG-AGO EC-Doc versus FEC phase III trial in intermediate-risk (pN1) early breast cancer: efficacy and predictive value of Ki67 expression.

BACKGROUND: Taxane-based adjuvant chemotherapy is standard in node-positive (N+) early breast cancer (BC). The magnitude of benefit in intermediate-risk N+ early BC is still unclear. WSG-AGO epiribicine and cyclophosphamide (EC)-Doc is a large trial evaluating modern taxane-based chemotherapy in patients with 1-3 positive lymph nodes (LNs) only. PATIENTS AND METHODS: A total of 2011 BC patients (18-65 years, pN1) were entered into a randomized phase III trial comparing 4 × E90C600 q3w followed by 4 × docetaxel 100 q3w (n = 1008) with the current standard: 6 × F500E100C500 q3w (n = 828) or C600M40F600 d1, 8× q4w (n = 175). Primary end point was event-free survival (EFS); secondary end points were overall survival (OS), toxicity, translational research, and quality of life. Central tumor bank samples were evaluable in a representative collective (n = 772; 40%). Ki-67 was assessed centrally in hormone receptor-positive disease as a surrogate marker for the distinction of luminal A/B-like tumors. RESULTS: Baseline characteristics were well balanced between study arms in both main study and central tumor bank subset. At 59-month median follow-up, superior efficacy of EC-Doc [versus FEC (a combination of 5-fluorouracil, epirubicin, and cyclophosphamide)] was seen in EFS and OS: 5-year EFS: 89.8% versus 87.3% (P = 0.038); 5-year OS: 94.5% versus 92.8% (P = 0.034); both tests one-tailed. EC-Doc caused more toxicity. In hormone receptor-positive (HR)+ disease, only high-Ki-67 tumors (≥ 20%) derived significant benefit from taxane-based therapy: hazard ratio = 0.39 (95% CI 0.18-0.82) for EC-Doc versus FEC (test for interaction; P = 0.01). CONCLUSION: EC-Doc significantly improved EFS and OS versus FEC in intermediate-risk BC (1-3 LNs) within all subgroups as defined by local pathology. In HR+ disease, patients with luminal A-like tumors may be potentially over-treated by taxane-based chemotherapy. CLINICAL TRIAL NUMBER: ClinicalTrials.gov, NCT02115204.

Physiological and behavioral stress parameters in calves in response to partial scrotal resection, orchidectomy, and Burdizzo castration.

Establishing artificial cryptorchids by partial scrotal resection without removing the testicles is a technique for castration of bull calves that recently has gained new interest. In contrast to orchidectomy and Burdizzo castration, the stress response of calves to shortening of the scrotum is unknown. In this study, partial scrotal resection in bull calves was compared with orchidectomy, Burdizzo castration, and controls without intervention (n=10 per group, ages 56 ± 3 d). Procedures were performed under xylazine sedation and local anesthesia. We hypothesized that partial scrotal resection is least stressful. Salivary cortisol, heart rate, heart rate variability, behavior, and locomotion were analyzed. Cortisol concentration peaked 60 min after start of the procedures. Cortisol release was at least in part xylazine induced and none of the experimental procedures released additional cortisol. Heart rate increased in calves of all groups with initial handling, but immediately after xylazine sedation decreased to 30% below initial values and was not modified by surgical procedures. The heart rate variability variables standard deviation of beat-to-beat interval and root mean square of successive beat-to-beat differences increased when calves were placed on the surgery table but effects were similar in calves submitted to surgeries and control calves. Locomotion increased, whereas lying time decreased in response to all surgeries. Locomotion increase was most pronounced after orchidectomy. Plasma fibrinogen concentrations increased after orchidectomy only. With adequate pain medication, orchidectomy, Burdizzo castration, and partial scrotal resection do not differ with regard to acute stress and, by inference, pain. Partial scrotal resection when carried out under xylazine sedation and local anesthesia thus is an acceptable castration technique in bull calves.

Human primary cementoblasts respond to combined IL-1ß stimulation and compression with an impaired BSP and CEMP-1 expression.

Cementoblasts are cells that produce, secrete and direct the production of cementum. Resorption lacunae occur in over 90% of teeth at the compression side of the periodontal ligament and might result in an irreversible loss of the original root length. We isolated and cultivated human primary cementoblasts and investigated their expression pattern concerning markers of the osteoblastogenic differentiation (RUNX2, OCN, ALP, and BSP) and CEMP-1. Compared to osteoblasts, cementoblasts displayed an expression pattern comparable to osteoblasts in an early stage of osteoblastogenic differentiation. Next, the human primary cementoblasts were stimulated with IL-1ß (1 and 10ng/ml) for 24 and 96h and subsequently subjected to compressive forces (30.3g/cm(2)) for 1 and 6h. Our in vitro data demonstrated that BSP and CEMP-1 expression significantly decreased when stimulation was accompanied by compression, while compression or stimulation alone led to increased levels of BSP and decreased levels of CEMP-1. We concluded that human primary cementoblasts subjected to compression and IL-1ß stimulation impeded BSP and CEMP-1 expression, proteins that are associated with cementogenesis.

Physiological and behavioural responses of young horses to hot iron branding and microchip implantation.

Branding is the traditional and well-established method used to mark horses, but recently microchip transponders for implantation have become available. In this study, behaviour, physiological stress variables and skin temperature in foals were determined in response to hot-iron branding (n=7) and microchip implantation (n=7). Salivary cortisol concentrations increased in response to branding (1.8 ± 0.2 ng/mL) and microchip implantation (1.4 ± 0.1ng/mL), but cortisol release over time did not differ. In response to both manipulations there was a transient increase in heart rate (P<0.001) and heart rate variability (P<0.01). Branding and microchip implantation induced a comparable aversive behaviour (branding, score 3.86 ± 0.85; microchip, score 4.00 ± 0.82). Both techniques thus caused similar physiological and behavioural changes indicative of stress. Acutely, implantation of a microchip was as stressful as branding in foals. Branding caused a necrotising skin burn lasting at least 7 days. Moreover branding, but not microchip implantation (P<0.001), was accompanied by a generalized increase in skin temperature which was comparable to low degree post-burn hypermetabolism in humans.

Compression-dependent up-regulation of ephrin-A2 in PDL fibroblasts attenuates osteogenesis.

Members of the ephrin/Eph family have recently been shown to be involved in the regulation of bone homeostasis in a murine model. The activation of the EphB4 receptor on osteoblasts by its ligand ephrin-B2 led to stimulation of osteoblastogenesis and therefore to bone formation. The activation of ephrin-A2-EphA2 signaling on osteoblasts inhibited the activation of osteoblast-specific gene expression, leading to bone resorption. Fibroblasts within the periodontal ligament periodontal ligament may be one of the first responders to orthodontic forces. Periodontal ligament fibroblasts (PDLF) are mechanoresponsive. Members of the ephrin/Eph family might link mechanical forces received by PDLF with the regulation of osteoblastogenesis on osteoblasts of the alveolar bone. To study whether ephrin-A2 is modulated upon compression, we subjected human primary PDLF to static compressive forces (30.3 g/cm(2)). Static compressive forces significantly induced the expression of ephrin-A2, while the expression of ephrin-B2 was significantly down-regulated. Moreover, osteoblasts of the alveolar bone stimulated with ephrin-A2 in vitro significantly suppressed their osteoblastogenic gene expression (RUNX2, ALPL) and decreased signs of osteoblastic differentiation, as demonstrated by a significantly reduced ALP activity. Together, these findings establish a role for this ligand/receptor system linking mechanical forces with the regulation of osteogenesis during orthodontic tooth movement.

Endothelial survival factors and spatial completion, but not pericyte coverage of retinal capillaries determine vessel plasticity.

Pericyte loss and capillary regression are characteristic for incipient diabetic retinopathy. Pericyte recruitment is involved in vessel maturation, and ligand-receptor systems contributing to pericyte recruitment are survival factors for endothelial cells in pericyte-free in vitro systems. We studied pericyte recruitment in relation to the susceptibility toward hyperoxia-induced vascular remodeling using the pericyte reporter X-LacZ mouse and the mouse model of retinopathy of prematurity (ROP). Pericytes were found in close proximity to vessels, both during formation of the superficial and the deep capillary layers. When exposure of mice to the ROP was delayed by 24 h, i.e., after the deep retinal layer had formed [at postnatal (p) day 8], preretinal neovascularizations were substantially diminished at p18. Mice with a delayed ROP exposure had 50% reduced avascular zones. Formation of the deep capillary layers at p8 was associated with a combined up-regulation of angiopoietin-1 and PDGF-B, while VEGF was almost unchanged during the transition from a susceptible to a resistant capillary network. Inhibition of Tie-2 function either by soluble Tie-2 or by a sulindac analog, an inhibitor of Tie-2 phosphorylation, resensitized retinal vessels to neovascularizations due to a reduction of the deep capillary network. Inhibition of Tie-2 function had no effect on pericyte recruitment. Our data indicate that the final maturation of the retinal vasculature and its resistance to regressive signals such as hyperoxia depend on the completion of the multilayer structure, in particular the deep capillary layers, and are independent of the coverage by pericytes.

Evidence that wild-type p53 in neuroblastoma cells is in a conformation refractory to integration into the transcriptional complex.

Neuroblastoma (NB) cells reportedly accumulate wild-type p53 exclusively in the cytoplasm. However, immunofluorescence assays with five different antibodies showed that p53 accumulates in the nucleus of up to 10% of NB cells. PAb1801 detected cytoplasmic 'punctate structures' which were also found in p53-null cells, rendering this antibody unsuitable for p53 detection. A comparison of DO-1 and PAb1801 staining in NB tissue sections confirmed the results obtained with NB cells. Nuclear accumulation of p53 was induced in NB cells using substances which disturb p53's tertiary structure at its zinc finger motif, or by treatment with mitomycin C. Constitutive nuclear accumulation was observed in an SK-N-SH variant, AW-1, which has a point mutation in p53 at Cys176>Ser, disturbing the same motif. Even though p53 showed DNA-binding capability after mitomycin C treatment of NB cells, the target gene products MDM2 and p21(WAF1,CIP1,SDI1) were not synthesized and no p53 transactivating activity measured in a reporter gene assay. Therefore we suggest that p53 in NB cells might be predominantly in a conformation refractory to integration into the transcriptional complex, resulting in at least partial transcriptional inactivity, hyperactive nuclear export and resistance to degradation by exogenously expressed MDM2.

TP53 DNA contact mutations are selectively associated with allelic loss and have a strong clinical impact in head and neck cancer.

Recent studies have suggested that different mutation types within the core domain of the tumour suppressor protein p53, i.e. DNA contact mutations and structural mutations, confer different biological properties. We have analysed in 86 head and neck squamous cell carcinomas (HNSCC), whether these p53 mutation types have a differential clinical impact. Thirty-seven missense mutations were identified. Thirteen of these (36%) were DNA contact mutations, occurring in the L3 loop, in the H2 loop sheet helix motif, in the S10 beta strand and in Zinc binding residues. Microsatellite marker analysis revealed a selective association between these mutations and the loss of wild-type alleles (100% LOH vs 50% LOH in tumours with structural mutations; P=0.0034, Fisher's exact, 2-tailed). In comparison to structural mutations or to the absence of mutations in the core domain, DNA contact mutations were associated with higher tumour stages (84.6% vs 62%), a higher incidence of lymph node metastasis (91.7% vs 56%; P=0.014, Fisher's exact, 2-tailed), a shortened recurrence-free survival (8.1 months vs 23.7 months, P=0.047, log rank test) and overall survival (11 months vs 29.2 months; P=0.003, log rank test). The latter was also the case when only stage IV tumours were analysed (P=0.0055, log rank test). These data indicate that in HNSCC, TP53 DNA contact mutations confer a strong selection pressure to eliminate wild-type alleles, and that they result in an accelerated tumour progression and reduced therapeutic responsiveness.

Etiological involvement of oncogenic human papillomavirus in tonsillar squamous cell carcinomas lacking retinoblastoma cell cycle control.

Two hundred eight primary squamous cell carcinomas of the head and neck have been analyzed with respect to the presence of the retinoblastoma tumor suppressor protein, pRb. Of these, 23 tumors (11%) that preferentially localized to the tonsils revealed complete absence or dramatic reduction in the amount of pRb. Other cell cycle components, cyclin D1 and p16INK4A, which are intimately related to pRb through an autoregulatory loop, were also dramatically decreased or overexpressed, respectively, in these pRb-defective tumors. On the other hand, the majority of the pRb-defective tumors contained the wild-type p53 gene. No evidence was found for genetic defects at the Rb locus in these tumors. Very significantly, in 11 of 12 pRb-defective tonsillar tumors, but in none of 9 pRb-positive tonsillar tumors (P < 10[-7]), DNA of oncogenic human papillomavirus types was identified, providing a strong indication for a human papillomavirus-associated etiology of these tumors and suggesting the functional inactivation of the pRb protein by the viral E7 gene product. In comparison to all head and neck squamous cell carcinomas studied, the pRb-defective tonsillar tumors were in general more poorly differentiated (P = 0.0059), and they were all metastatic at the time of resection. Of particular clinical interest, despite these adverse histopathological factors, the clinical outcome for these patients was relatively favorable, strongly implying that the pRb-defective tumors responded uniformly well toward postoperative radiation therapy.

Aberrant p21(CIP1/WAF1) protein accumulation in head-and-neck cancer.

p21(CIP1/WAF1) is an inhibitor of cyclin-dependent kinases and, in normal tissues including squamous epithelia, has been associated with cell-cycle exit and differentiation. As shown in this pilot study, however, the majority of head-and-neck squamous-cell carcinomas (HNSCC) display aberrant p21(CIP1/WAF1) expression: of 42 tumors analyzed by immunohistochemical staining, 28 (67%) over-expressed the p21(CIP1/WAF1) protein. Accumulation of p21(CIP1/WAF1) was independent of the histological grade of the tumors as well as the genetic status of the p53 gene. In many cases, most notably in poorly differentiated or undifferentiated HNSCC, p21(CIP1/WAF1)-positive cells were actively proliferating tumor cells, since they also expressed proliferating-cell nuclear antigen (PCNA) and Ki-67. Accumulation of p21(CIP1/WAF1) occurred through a post-transcriptional mechanism since, in contrast to immunohistochemical analysis of the p21(CIP1/WAF1) protein, in situ hybridization showed no increase of mRNA levels as compared with cells in normal mucosa (n = 25). Clinically, among the patients with p21(CIP1/WAF1)-over-expressing tumors, there was increased recurring disease (p = 0.03; chi2-test), shortened disease-free survival (p = 0.0019; log-rank test) and shortened overall survival (p = 0.0071; log-rank test). These in vivo data indicate that in many HNSCC, accumulated p21(CIP1/WAF1) is compatible with increased tumor-cell proliferation, and they provide preliminary evidence that p21(CIP1/WAF1) may be of prognostic and predictive significance.