Patients with oral tongue squamous cell carcinoma and co­existing diabetes exhibit lower recurrence rates and improved survival: Implications for treatment.

Locoregional recurrences and distant metastases are major problems for patients with squamous cell carcinoma of the head and neck (SCCHN). Because SCCHN is a heterogeneous group of tumours with varying characteristics, the present study concentrated on the subgroup of squamous cell carcinoma of the oral tongue (SCCOT) to investigate the use of machine learning approaches to predict the risk of recurrence from routine clinical data available at diagnosis. The approach also identified the most important parameters that identify and classify recurrence risk. A total of 66 patients with SCCOT were included. Clinical data available at diagnosis were analysed using statistical analysis and machine learning approaches. Tumour recurrence was associated with T stage (P=0.001), radiological neck metastasis (P=0.010) and diabetes (P=0.003). A machine learning model based on the random forest algorithm and with attendant explainability was used. Whilst patients with diabetes were overrepresented in the SCCOT cohort, diabetics had lower recurrence rates (P=0.015 after adjusting for age and other clinical features) and an improved 2-year survival (P=0.025) compared with non-diabetics. Clinical, radiological and histological data available at diagnosis were used to establish a prognostic model for patients with SCCOT. Using machine learning to predict recurrence produced a classification model with 71.2% accuracy. Notably, one of the findings of the feature importance rankings of the model was that diabetics exhibited less recurrence and improved survival compared with non-diabetics, even after accounting for the independent prognostic variables of tumour size and patient age at diagnosis. These data imply that the therapeutic manipulation of glucose levels used to treat diabetes may be useful for patients with SCCOT regardless of their diabetic status. Further studies are warranted to investigate the impact of diabetes in other SCCHN subtypes.

Reiterative modeling of combined transcriptomic and proteomic features refines and improves the prediction of early recurrence in squamous cell carcinoma of head and neck.

BACKGROUND: Patients with squamous cell carcinoma of the head and neck (SCCHN) have a high-risk of recurrence. We aimed to develop machine learning methods to identify transcriptomic and proteomic features that provide accurate classification models for predicting risk of early recurrence in SCCHN patients. METHODS: Clinical, genomic, transcriptomic and proteomic features distinguishing recurrence risk were examined in SCCHN patients from The Cancer Genome Atlas (TCGA). Recurrence within one year after treatment was classified as high-risk and no recurrence as low-risk. RESULTS: No significant differences in individual clinicopathological characteristics, mutation profiles or mRNA expression patterns were seen between the groups using conventional statistical analysis. Using the machine learning algorithm, extreme gradient boosting (XGBoost), ten proteins (RAD50, 4E-BP1, MYH11, MAP2K1, BECN1, NF2, RAB25, ERRFI1, KDR, SERPINE1) and five mRNAs (PLAUR, DKK1, AXIN2, ANG and VEGFA) made the greatest contribution to classification. These features were used to build improved models in XGBoost, achieving the best discrimination performance when combining transcriptomic and proteomic data, providing an accuracy of 0.939 and an Area Under the ROC Curve (AUC) of 0.951. CONCLUSIONS: This study highlights machine learning to identify transcriptomic and proteomic factors that play important roles in predicting risk of recurrence in patients with SCCHN and to develop such models by iterative cycles to enhance their accuracy, thereby aiding the introduction of personalized treatment regimens.

Comparison of Quality of Life among Patients with Oro-Hypopharyngeal Cancer after Tonsillectomy and Panscopy Using Transoral Robotic Surgery: A Pilot Study.

Studies have shown lower treatment-related morbidity when using transoral robotic surgery (TORS) compared to conventional surgery. Patients investigated for oro- and hypopharyngeal cancer (T1, T2) were compared concerning quality of life (QoL) after tonsillectomy and TORS using validated QoL questionnaires: QLQ-C30 and QLQ-H&N35. The patients treated with TORS showed a higher pain score and thus also a higher need for painkillers, whereas they had lower values on self-assessment of anxiety/depression using the Hospital Anxiety and Depression Scale score. The pre- and postoperative information given did not meet the expectations of the patients treated with conventional surgery. The present data show advantages of the TORS technique from the patients' perspective. Even if patients treated with TORS are in need of more painkilling treatment, they cope better with the long-term effects of treatment, as judged by self-assessment of anxiety and depression.

Comparison of Preoperative Positron Emission Tomography/Computed Tomography with Panscopy and Ultrasound in Patients with Head and Neck Cancer.

INTRODUCTION: To compare data from preoperative positron emission tomography/computed tomography (PET/CT) with results of panscopy with biopsy and ultrasound with fine needle aspiration cytology (US-FNAC) on the same patients. METHODS: In this retrospective (2014-2016) study, we compared PET/CT results with the results from panscopy with biopsy and US-FNAC in patients suspected of head and neck malignancy treated at the University Hospital in Umeå, Sweden. RESULTS: A 91.3% concordance was seen between results from PET/CT and panscopy with biopsy, whereas between PET/CT and US-FNAC the concordance was 89.1%. CONCLUSIONS: The present data show the usefulness of PET/CT in the diagnosis of head and neck malignancies.

Levels of MUC1 in tumours and serum of patients with different sub-types of squamous cell carcinoma of the head and neck.

Mucin 1 (MUC1) is a membrane-bound and secreted glycoprotein that has a protective role in surface epithelia. We recently demonstrated that MUC1 mRNA expression was upregulated in tumour-free tongue tissues adjacent to squamous cell carcinoma of the oral tongue (SCCOT) compared with that in the tumour tissues. The present study investigated MUC1 protein in SCCOT tissue and serum from patients with squamous cell carcinoma of the head and neck (SCCHN) at different sub-sites. The results from immunohistochemistry demonstrated that all SCCOT tissues expressed MUC1; however, the protein levels were not correlated with MUC1 mRNA levels in the same tumours. Furthermore, serum MUC1 level was lower in patients with SCCOT, tonsil SCC and gingival SCC compared with that in healthy subjects; however, the difference was only significant for patients with SCCOT (P=0.0421). No correlation was seen between MUC1 level in tumour tissues and MUCI level in serum from the same patients. The absence of correlation between MUC1 protein and mRNA levels in SCCOT tissues emphasized the importance of validating genomic data in clinical samples. Although significant MUC1 downregulation was observed in the serum of patients with SCCOT, there was a large variation within the groups, suggesting that MUC1 may not be used as a biomarker for these types of tumors.

PD-L1 in squamous cell carcinoma of the oral tongue shows gender-specific association with prognosis.

OBJECTIVE: To use alternative quantitation approaches to clarify the clinical implication of programmed cell death ligand 1 (PD-L1) in squamous cell carcinoma of the oral tongue (SCCOT). MATERIALS AND METHODS: Ventana SP263 immunohistochemistry assay and a multiplicative QuickScore method were applied to quantify PD-L1 in tumor and surrounding immune cells from 101 patients with SCCOT. Tumor-infiltrating immune cells were estimated from bulk tissue transcriptional profiles of 25 patients. Circulating PD-L1 levels were measured in serum from 30 patients using an electrochemiluminescence assay platform. RESULTS: We found higher tumor cell PD-L1 levels in females than males (p = .019). For patients with low PD-L1 in tumor cells, better survival was seen in males than females (overall survival p = .021, disease-free survival p = .020). Tumor-infiltrating natural killer T cells, immature dendritic cells, and M1 macrophages were positively associated with tumor cell PD-L1 (p < .05). CONCLUSIONS: Our data confirmed the significance of gender on tumor cell PD-L1 expression and demonstrated combined effects of gender and PD-L1 levels on clinical outcome in patients with SCCOT. The data also indicated the involvement of specific immune cell types in PD-L1-regulated immune evasion.

High podoplanin and low E-cadherin levels correlate with better prognosis in adenoid cystic carcinoma.

Objectives: As tumour spread is a complicating event for malignant salivary gland tumours, we decided to study factors related to cell adhesion and lymph vessel formation in two of the three most common malignant salivary gland tumours, mucoepidermoid carcinoma (MEC) and adenoid cystic carcinoma (ACC), to clarify the clinical relevance and potential usefulness of these factors. We also included a group of polymorphous adenocarcinoma (PAC) as this tumour, in common with ACC often shows perineural growth, but in contrast to ACC has an overall good prognosis. Material and methods: Eighteen patients with ACC, 15 with MEC, and six with PAC were included. Protein expression of podoplanin and E-cadherin was evaluated as percentage of cells expressing the protein and intensity of expression. Ki-67 expression was included in the study as a marker of proliferative activity. Results: Looking at podoplanin, significantly more ACCs were high expressing compared with both MECs (P = .001) and PACs (P = .028). Also when looking at Ki-67 expression, significantly more ACCs were high expressing compared with MECs (P = .003). Significantly better survival was also seen for ACCs with high podoplanin (P = .022) and low E-cadherin expression (P = .021), respectively. Conclusions: Our findings show that ACCs express significantly higher levels of podoplanin compared with both MECs and PACs and that high levels are correlated to better survival. Even though the group of PACs analysed was small, these tumours, despite their tendency to perineural spread, which they have in common with ACC, differ from ACCs concerning expression of factors with a known connection to tumour spread.

High immune cytolytic activity in tumor-free tongue tissue confers better prognosis in patients with squamous cell carcinoma of the oral tongue.

Immune cells and cytolytic activity within the tumor microenvironment are being intensively studied. Through transcriptome profiling, immune cell enumeration using the xCell tool and cytolytic activity quantification according to granzyme A (GZMA) and perforin (PRF1) mRNA levels, we investigated immunoreactivity in tumor and/or tumor-free tongue tissue samples from 31 patients with squamous cell carcinoma of the oral tongue and 14 healthy individuals (control tongue tissues). We found significantly altered immune cell compositions (p < 0.001) and elevated cytolytic activity (p < 0.001) in tumor compared to tumor-free samples, and altered infiltration of a subset of immune cells (e.g. CD8+ T cells, p < 0.01) as well as increased cytolytic activity (p < 0.001) in tumor-free compared to control samples. Controlling for patient age at diagnosis and tumor stage, Cox regression analysis showed that high cytolytic activity in tumor-free samples associated with improved disease-free survival (hazard ratio= 4.20, 95% CI = 1.09-16.20, p = 0.037). However, the degree of cytolytic activity in tumor samples did not provide prognostic information. Taken together, our results show the presence of cancer-related immune responses in clinically tumor-free tongue in patients with squamous cell carcinoma of the oral tongue. Measuring cytolytic activity in tumor-free tongue samples contralateral to tumor might thus be an effective approach to predict clinical outcome.

Copy number variation: A prognostic marker for young patients with squamous cell carcinoma of the oral tongue.

BACKGROUND: The incidence of squamous cell carcinoma of the oral tongue (SCCOT) is increasing in people under age 40. There is an urgent need to identify prognostic markers that help identify young SCCOT patients with poor prognosis in order to select these for individualized treatment. MATERIALS AND METHODS: To identify genetic markers that can serve as prognostic markers for young SCCOT patients, we first investigated four young (≤40 years) and five elderly patients (≥50 years) using global RNA sequencing and whole-exome sequencing. Next, we combined our data with data on SCCOT from the cancer genome atlas (TCGA), giving a total of 16 young and 104 elderly, to explore the correlations between genomic variations and clinical outcomes. RESULTS: In agreement with previous studies, we found that SCCOT from young and elderly patients was transcriptomically and also genomically similar with no significant differences regarding cancer driver genes, germline predisposition genes, or the burden of somatic single nucleotide variations (SNVs). However, a disparate copy number variation (CNV) was found in young patients with distinct clinical outcome. Combined with data from TCGA, we found that the overall survival was significantly better in young patients with low-CNV (n = 5) compared to high-CNV (n = 11) burden (P = 0.044). CONCLUSIONS: Copy number variation burden is a useful single prognostic marker for SCCOT from young, but not elderly, patients. CNV burden thus holds promise to form an important contribution when selecting suitable treatment protocols for young patients with SCCOT.

Ethnicity based variation in expression of E-cadherin in patients with squamous cell carcinoma of the oral tongue.

The oral tongue is the most common site for tumours within the oral cavity. Despite intense research, there has been no improvement in the survival rate for patients with oral tongue squamous cell carcinoma (OTSCC) during the last decades. Differences between oral cancer patients based on ethno-geographical distribution have been reported. The present study used immunohistochemistry to evaluate commonly used markers of cancer cell phenotypes, E-cadherin, ß-catenin and cytokeratins 5 and 19, in 120 patients with OTSCC. To evaluate the impact of ethnicity, patients from Sweden and Italy were included. A higher proportion of Swedish patients exhibited high expression of E-cadherin in their tumours (P=0.039), and high levels of E-cadherin in Swedish OTSCC patients that had succumbed to their disease were associated with poor prognosis. These data demonstrated differences in the pathological characteristics of OTSCC between two different European populations. The findings emphasise the need to take ethnicity/geographical location of patients into account when comparing results from different studies of OTSCC.

Transoral Robotic Surgery in the Nordic Countries: Current Status and Perspectives.

Background: The five Nordic countries with a population of 27 M people form a rather homogenous region in terms of health care. The management of head and neck cancer is centralized to the 21 university hospitals in these countries. Our aim was to gain an overview of the volume and role of transoral robotic surgery (TORS) and to evaluate the need to centralize it in this area as the field is rapidly developing. Materials and Methods: A structured questionnaire was sent to all 10 Departments of Otorhinolaryngology-Head and Neck Surgery in the Nordic countries having an active programme for TORS in December 2017. Results: The total cumulative number of performed robotic surgeries at these 10 Nordic centers was 528 and varied between 5 and 240 per center. The median annual number of robotic surgeries was 38 (range, 5-60). The observed number of annually operated cases remained fairly low (<25) at most of the centers. Conclusions: The present results showing a limited volume of performed surgeries call for considerations to further centralize TORS in the Nordic countries.

Evidence that circulating proteins are more promising than miRNAs for identification of patients with squamous cell carcinoma of the tongue.

Despite intense research, squamous cell carcinoma of the tongue remains a devastating disease with a five-year survival of around 60%. Late detection and recurrence are the main causes for poor survival. The identification of circulating factors for early diagnosis and/or prognosis of cancer is a rapidly evolving field of interest, with the hope of finding stable and reliable markers of clinical significance. The aim of this study was to evaluate circulating miRNAs and proteins as potential factors for distinguishing patients with tongue squamous cell carcinoma from healthy controls. Array-based profiling of 372 miRNAs in plasma samples showed broad variations between different patients and did not show any evidence for their use in diagnosis of tongue cancer. Although one miRNA, miR-150, was significantly down-regulated in plasma from patients compared to controls. Surprisingly, the corresponding tumor tissue showed an up-regulation of miR-150. Among circulating proteins, 23 were identified as potential markers of squamous cell carcinoma of the tongue. These findings imply that circulating proteins are a more promising source of biomarkers for tongue squamous cell carcinomas than circulating miRNAs. The data also highlight that circulating markers are not always directly associated with tumor cell properties.

No evidence for the presence of Epstein-Barr virus in squamous cell carcinoma of the mobile tongue.

Squamous cell carcinoma of the head and neck (SCCHN) comprises a large group of cancers in the oral cavity and nasopharyngeal area that typically arise in older males in association with alcohol/tobacco usage. Within the oral cavity, the mobile tongue is the most common site for tumour development. The incidence of tongue squamous cell carcinoma (TSCC) is increasing in younger people, which has been suggested to associate with a viral aetiology. Two common human oncogenic viruses, human papilloma virus (HPV) and Epstein-Barr virus (EBV) are known causes of certain types of SCCHN, namely the oropharynx and nasopharynx, respectively. EBV infects most adults worldwide through oral transmission and establishes a latent infection, with sporadic productive viral replication and release of virus in the oral cavity throughout life. In view of the prevalence of EBV in the oral cavity and recent data indicating that it infects tongue epithelial cells and establishes latency, we examined 98 cases of primary squamous cell carcinoma of the mobile tongue and 15 cases of tonsillar squamous cell carcinoma for the presence of EBV-encoded RNAs (EBERs), EBV DNA and an EBV-encoded protein, EBNA-1. A commercially available in situ hybridisation kit targeting EBER transcripts (EBER-ISH) showed a positive signal in the cytoplasm and/or nuclei of tumour cells in 43% of TSCCs. However, application of control probes and RNase A digestion using in-house developed EBER-ISH showed identical EBER staining patterns, indicating non-specific signals. PCR analysis of the BamH1 W repeat sequences did not identify EBV genomes in tumour samples. Immunohistochemistry for EBNA-1 was also negative. These data exclude EBV as a potential player in TSCC in both old and young patients and highlight the importance of appropriate controls for EBER-ISH in investigating EBV in human diseases.

Patients with high c-MYC-expressing squamous cell carcinomas of the tongue show better survival than those with low- and medium-expressing tumours.

BACKGROUND: c-MYC is a potent oncoprotein with roles in a wide range of cellular processes such as differentiation, apoptosis and growth control. Deregulation of the MYC gene is commonly seen in human tumours resulting in overexpression of the protein. Here we studied expression of c-MYC in correlation to clinical outcome in patients with primary squamous cell carcinoma of the mobile tongue. METHODS: Immunohistochemistry was used to identify c-MYC in a group of 104 tongue squamous cell carcinomas with an antibody directed against the N-terminal part of the protein. Staining was evaluated by multiplying the percentage of c-MYC-expressing cells with staining intensity, giving a quick score for each tumour. RESULTS: All 104 tumours expressed c-MYC at varying levels. Quantitation according to per cent of positive cells and staining intensity revealed that most (15/21; 71%) high-expressing tumours were seen in males. Within the group of high c-MYC-expressing tumours, the majority were alive 2 and 5 years after treatment. CONCLUSIONS: The present findings show that expression of c-MYC has prognostic value in squamous cell carcinoma of the tongue, and could be useful in choice of therapy.

Gene expression changes in tumor free tongue tissue adjacent to tongue squamous cell carcinoma.

Due to the high frequency of loco-regional recurrences, which could be explained by changes in the field surrounding the tumor, patients with squamous cell carcinoma of head and neck show poor survival. Here we identified a total of 554 genes as dysregulated in clinically tumor free tongue tissue in patients with tongue tumors when compared to healthy control tongue tissue. Among the top dysregulated genes when comparing control and tumor free tissue were those involved in apoptosis (CIDEC, MUC1, ZBTB16, PRNP, ECT2), immune response (IFI27) and differentiation (KRT36). Data suggest that these are important findings which can aid in earlier diagnosis of tumor development, a relapse or a novel squamous cell carcinoma of the tongue, in the absence of histological signs of a tumor.

Epigenetic regulation of OAS2 shows disease-specific DNA methylation profiles at individual CpG sites.

Epigenetic modifications are essential regulators of biological processes. Decreased DNA methylation of OAS2 (2'-5'-Oligoadenylate Synthetase 2), encoding an antiviral protein, has been seen in psoriasis. To provide further insight into the epigenetic regulation of OAS2, we performed pyrosequencing to detect OAS2 DNA methylation status at 11 promoter and first exon located CpG sites in psoriasis (n = 12) and two common subtypes of squamous cell carcinoma (SCC) of the head and neck: tongue (n = 12) and tonsillar (n = 11). Compared to corresponding controls, a general hypomethylation was seen in psoriasis. In tongue and tonsillar SCC, hypomethylation was found at only two CpG sites, the same two sites that were least demethylated in psoriasis. Despite differences in the specific residues targeted for methylation/demethylation, OAS2 expression was upregulated in all conditions and correlations between methylation and expression were seen in psoriasis and tongue SCC. Distinctive methylation status at four successively located CpG sites within a genomic area of 63 bp reveals a delicately integrated epigenetic program and indicates that detailed analysis of individual CpGs provides additional information into the mechanisms of epigenetic regulation in specific disease states. Methylation analyses as clinical biomarkers need to be tailored according to disease-specific sites.

Number of negative points: a novel method for predicting radiosensitivity in head and neck tumor cell lines.

The present study was aimed at establishing a method that combines multiple factors of protein and genetic changes that enables prediction of radiosensitivity in the head and neck squamous cell carcinoma (HNSCC) cell lines. In nine HNSCC cell lines, the quantity of protein expression and the type of genetic alterations were translated into a point system, called the Number of Negative Points. The expression of 14 proteins involved in growth control and/or apoptosis was quantified using a densitometric assessment of Western blots. The blots were adjusted to actin and standardised to normal oral keratinocytes classifying them into four groups depending on the amount of protein expressed (0-3 points). Mutations of the p53 gene were classified into three groups and each mutation was given one point. Since the cell lines each had a known intrinsic radiosensitivity, a multivariate statistical calculation could then be performed to select for the combination of factors having the strongest correlation to radiosensitivity. The strongest correlation of the investigated factors was the combination of epidermal growth factor receptor, survivin and splice site/missense p53 mutations (R=0.990 and P<0.0001). No single factor had a significant correlation to the intrinsic radiosensitivity. Since a significant correlation to the intrinsic radiosensitivity was achieved only when two or more factors were combined, we conclude that a method such as the Number of Negative Points is necessary for prediction of treatment response. We present a novel method to combine factors which enables the prediction of radiosensitivity of HNSCC cell lines.

Multiple genotypic aberrances associate to terminal differentiation-deficiency of an oral squamous cell carcinoma in serum-free culture.

Oral squamous cell carcinoma (OSCC) lines proliferative in the serum-free conditions devised for normal oral keratinocytes (NOK) are virtually absent, complicating studies of carcinogenesis. A tongue squamous cell carcinoma generated under conditions for normal cell culture an apparently immortal line (termed LK0412) that has undergone >or=200 population doublings from over a year in culture. LK0412 exhibited epithelial morphology, intermediate filaments, desmosomes, and cytokeratin. Soft agar growth and tumorigenicity in athymic nude mice indicated the malignant phenotype. Compared with NOK, LK0412 exhibited increased indices for proliferation and apoptosis, and a decreased terminal differentiation index. Fetal bovine serum inhibited growth and increased apoptosis but failed to induce terminal differentiation of LK0412; the latter outcome differed clearly from that in NOK. Gene ontology assessment of transcript profiles implicated multiple alterations in biological processes, molecular functions, and cellular components in LK0412. Genetic changes, some that were confirmed to the protein level, included previously proposed OSCC markers, i.e., BAX, CDC2, and TP53, as well as multiple cancer-associated genes not considered for OSCC, e.g., BST2, CRIP1, ISG15, KLRC1, NEDD9, NNMT, and TWIST1. Elevation of p53 protein agreed with a missense mutation detectable in both the LK0412 line and the original tumor specimen. Moderate differentiation characterized the original tumor as well as tumors generated from inoculation of LK0412 in mice. Overall, the results suggest that the LK0412 cell line represent a subgroup of OSCC with unique genomic and phenotypic profiles. LK0412 should be useful to exploration of OSCC development, particularly the deregulated growth and differentiation responsiveness to serum factors.

Radiotherapy response in oral squamous carcinoma cell lines: evaluation of apoptotic proteins as prognostic factors.

BACKGROUND: In this study, we investigated the importance of apoptosis for cell death after radiotherapy, and whether the expression of pro- and anti-apoptotic proteins has any correlation to the radiosensitivity. METHODS: Three oral squamous cell carcinoma cell lines, UT-SCC-2, UT-SCC-9 and UT-SCC-24A, were subjected to radiotherapy. After irradiation, viable and dead cells were counted to determine radiation sensitivity and apoptosis was analyzed by measurement of caspase-3 activity. The expressions of pro- and anti-apoptotic proteins were assessed using western blot analyses. RESULTS AND CONCLUSION: After irradiation, apoptotic morphology and caspase-3 activity were only detected in cell lines exhibiting high or moderate radiosensitivity. Western blot analysis indicates that survivin, epidermal growth factor receptor, cyclooxygenase-2, and Bcl-x(L) are critical components in irradiation resistance of the investigated cell lines. Moreover, our results suggest that apoptotic cell death and the balance between pro- and anti-apoptotic proteins are of importance for the outcome of radiotherapy.

Role of lysosomal cathepsins in naphthazarin- and Fas-induced apoptosis in oral squamous cell carcinoma cells.

CONCLUSION: Intracellular cysteine cathepsins are pro-apoptotic factors involved in activation of caspases in two oral squamous cell carcinoma (SCC) cell lines. OBJECTIVE: To study the possible involvement of lysosomal cathepsins in oral SCC cell apoptosis. MATERIAL AND METHODS: Apoptosis was induced in the two human oral SCC cell lines UT-SCC-20A and UT-SCC-24A using naphthazarin or anti-Fas antibodies, and was studied by analysis of caspase activity and nuclear morphology. Involvement of lysosomal cathepsins was investigated using the cysteine cathepsin inhibitor z-FA-FMK and the cathepsin D inhibitor pepstatin A. The amounts of cellular and soluble Fas death receptor were determined by ELISA. RESULTS: Release of cathepsins from the lysosomes to the cytosol was observed early in apoptosis. Cysteine cathepsins were found to be involved in activation of caspases in response to treatment with naphthazarin or anti-Fas antibodies, but inhibition of cysteine cathepsin activity was not sufficient to prevent cell death. Moreover, inhibition of cysteine cathepsin activity resulted in increased expression of the Fas death receptor, suggesting involvement of extracellular cysteine cathepsins in death receptor shedding.