[Lung cancer staging by endobronchial ultrasound with transbronchial needle aspiration]. / Endobronchiale ultrasonografie met transbronchiale naaldaspiratie bij stadiëring van longcarcinomen.

OBJECTIVE: To determine the diagnostic yield of endobronchial ultrasound with transbronchial needle aspiration (EBUS-TBNA) and to investigate the number of cervical mediastinoscopies that could be avoided when this technique was used as the initial modality in the invasive mediastinal staging of lung cancer. DESIGN: Retrospective cohort study. METHOD: At the St. Antonius Hospital, Nieuwegein, the Netherlands, results from all patients who had undergone EBUS-TBNA for mediastinal staging in lung cancer from September 2008 to January 2011 were collected. If metastases in the mediastinal lymph nodes had been demonstrated by EBUS-TBNA, no indication for additional mediastinoscopy ensued. The diagnostic yield of EBUS-TBNA as well as the number of mediastinoscopies that had been avoided, were calculated. RESULTS: EBUS-TBNA had been used for mediastinal staging in lung cancer in 77 patients. In 39 of these 77 patients (51%), mediastinal lymph node metastases was found using EBUS-TBNA and mediastinoscopy could thus be avoided. In 9 out of 38 (24%) patients whose EBUS-TBNA cytology results were found to be either benign or not representative, mediastinoscopy or endoscopic ultrasound eventually did reveal mediastinal lymph node metastases. In 13 of these 38 patients (34%), no additional cytologic or histologic testing was performed. Diagnostic yield was calculated for the two scenarios. The sensitivity and negative-predictive values for EBUS-TBNA were 64-81% and 42-76%, respectively. CONCLUSION: In more than 50% of lung cancer patients with suspected mediastinal lymph node metastases, cervical mediastinoscopy can be avoided when EBUS-TBNA is used. This examination is the technique of first choice for the invasive staging of the mediastinum in lung cancer, but it can not replace mediastinoscopy completely.

Human Papillomavirus and Oropharyngeal Squamous Cell Carcinoma: A Case-Control Study regarding Tobacco and Alcohol Consumption.

We aimed to determine the role of HPV in the pathogenesis and outcome of oropharyngeal squamous cell carcinoma (OSCC) in lifelong nonsmoking and nondrinking patients. A case-case analysis was performed to compare the presence of HPV-DNA in tumor cells of 16 nonsmoking and nondrinking with 16 matched smoking and drinking patients (matching criteria: age at incidence, gender, tumor sublocation, tumor stage). HPV was detected using 2 PCR tests, FISH analysis, and p16(INK4A) immunostaining. Nonsmoking and nondrinking patients had more HPV-positive tumors than smoking and drinking patients (n = 12; 75% versus n = 2; 13%; P < 0.001). All HPV-positive tumors showed p16(INK4A) overexpression, and 1 HPV-negative tumor had p16(INK4A) overexpression, (P < 0.001). Overall survival and disease-specific survival were higher for HPV-positive compared to HPV-negative cases (P = 0.027, P = 0.039, resp.). In conclusion, HPV is strongly associated with OSCC of nonsmoking and nondrinking patients. Specific diagnostic and therapeutic actions should be considered for these patients to achieve a better prognosis.

Hypertrophic differentiation and calcification during intervertebral disc degeneration.

BACKGROUND: In degenerative intervertebral discs (IVDs) collagen type X expression and calcifications have been demonstrated, resembling advanced osteoarthritis (OA), which is associated with hypertrophic differentiation, characterized by the production of collagen type X, Runt-related transcription factor 2 (Runx2), osteoprotegerin (OPG), alkaline phosphatase (ALP) and calcifications. OBJECTIVE: The aim of this study was to determine if hypertrophic differentiation occurs during IVD degeneration. METHODS: IVDs from all Thompson degeneration grades were prepared for histology, extraction of nucleus pulposus (NP) and annulus fibrosis (AF) tissue (N=50) and micro-CT (N=27). The presence of collagen type X, OPG and Runx2 was determined by immunohistochemistry, with OPG levels also determined by Enzyme-linked immunosorbent assay (ELISA). The presence of calcification was determined by micro-CT, von Kossa and Alizarin Red staining. RESULTS: Immunohistochemical staining for collagen type X, OPG, Runx2 appeared more intense in the NP of degenerative compared to healthy IVD samples. OPG levels correlated significantly with degeneration grade (NP: P<0.000; AF: P=0.002) and the number of microscopic calcifications (NP: P=0.002; AF: P=0.008). The extent of calcifications on micro-CT also correlated with degeneration grade (NP: P<0.001, AF: P=0.001) as did von Kossa staining (NP: P=0.015, AF: P=0.016). ALP staining was only incidentally seen in the transition zone of grades IV and V degenerated IVDs. CONCLUSION: This study for the first time demonstrates that hypertrophic differentiation occurs during IVD degeneration, as shown by an increase in OPG levels, the presence of ALP activity, increased immunopositivity of Runx2 and collagen type X.

Ear piercing techniques and their effect on cartilage, a histologic study.

BACKGROUND: The popularity of high ear piercing has led to an increased incidence of perichondritis. Damage to the relatively avascular cartilage will make the ear prone to infection. The literature suggests that a piercing gun, mainly used by jewellers to pierce the lobule, may give excessive cartilaginous damage. Therefore some authors favour the piercing needle, as used in piercing studios. But until now, no comparative histological studies have been performed. PURPOSE OF STUDY: To evaluate the extent of damage to ear cartilage using different piercing techniques. METHODS: Twenty-two fresh human cadaver ears were pierced using two spring loaded piercing guns (Caflon and Blomdahl), one hand force system (Studex) and a piercing needle (16G i.v. catheter). Extent of damage to the perichondrium and cartilage was quantified using a transverse section along the pin tract and compared between the different methods. RESULTS: The pattern of injury was similar in all techniques, showing perichondrium stripped from the cartilage around the pin tract, with most damage present on the exit site (mean length of 0.43 mm). Cartilage fractures and loose fragments were present over a mean length of 0.21 mm. No significant difference in the amount of injury between the different techniques was observed. CONCLUSIONS: In contradiction with assumptions in the literature, all piercing methods give the same extent of damage to cartilage and perichondrium. Each method is expected to have the same risk for perichondritis, thus in the prevention of post-piercing perichondritis focus should be on other factors such as hygiene and after-care.

Expression of c-FLIP is primarily detected in diffuse large B-cell lymphoma and Hodgkin's lymphoma and correlates with lack of caspase 8 activation.

AIMS: Inhibition of apoptosis is important in the pathogenesis of lymphomas. c-FLIP, a regulator of caspase 8-mediated apoptosis, plays an important role in protecting normal B and T cells from apoptosis and possibly also in lymphomas. Because of contradictory reports about immunohistochemical detection of c-FLIP expression, the aim was to test the specificity of four antibodies in c-FLIP-transfected cells and subsequently to investigate expression of c-FLIP in different types of lymphoma. METHODS AND RESULTS: Two of four antibodies were specific. In primary lymphomas c-FLIP expression was restricted to Hodgkin's lymphomas (> 90%) and diffuse large B-cell lymphomas (44%). Burkitt lymphomas and indolent B-cell lymphomas were negative in all cases. No expression was detected in primary T-cell lymphomas, although expression was observed in one relapsed ALK+ anaplastic large cell lymphoma. Expression of c-FLIP was inversely correlated with caspase 8 activation. CONCLUSIONS: c-FLIP is important in escape of B cells from apoptosis during normal follicle centre cell reaction and may thus be an important early event in the development of B-cell-derived lymphomas. Moreover, non-specific staining of frequently used antibodies might explain discrepancies in different reports of c-FLIP expression.

DNA copy number status is a powerful predictor of poor survival in endocrine pancreatic tumor patients.

The clinical behavior of endocrine pancreatic tumors (EPTs) is difficult to predict in the absence of metastases or invasion to adjacent organs. Several markers have been indicated as potential predictors of metastatic disease, such as tumor size > or =2 cm, Ki67 proliferative index > or =2%, cytokeratin (CK) 19 status, and recently in insulinomas, chromosomal instability (CIN). The goal of this study was to evaluate the value of these markers, and in particular of the CIN, to predict tumor recurrence or progression and tumor-specific death, using a series of 47 insulinomas and 24 non-insulinoma EPTs. From these EPT cases, a genomic profile has been generated and follow-up data have been obtained. The proliferative index has been determined in 68 tumors and a CK19 expression pattern in 50 tumors. Results are statistically analyzed using Kaplan-Meier plots and the log-rank statistic. General CIN, as well as specific chromosomal alterations such as 3p and 6q loss and 12q gain, turned out to be the most powerful indicators for poor tumor-free survival (P< or =0.0004) and tumor-specific death (P< or =0.0113) in insulinomas. The CIN, chromosome 7q gain, and a proliferative index > or =2% were reliable in predicting a poor tumor-free survival in non-insulinoma EPTs (P< or =0.0181, whereas CK19 expression was the most optimal predictor of tumor-specific death in these tumors. In conclusion, DNA copy number status is the most sensitive and efficient marker of adverse clinical outcome in insulinomas and of potential interest in non-insulinoma EPTs. As a consequence, this marker should be considered as a prognosticator to improve clinical diagnosis, most practically as a simple multi-target test.

Ectopic expression of the serine protease inhibitor PI9 modulates death receptor-mediated apoptosis.

Apoptosis is a highly controlled process, whose triggering is associated with the activation of caspases. Apoptosis can be induced via a subgroup of the tumor necrosis factor (TNF) receptor superfamily, which recruit and activate pro-caspase-8 and -10. Regulation of apoptosis is achieved by several inhibitors, including c-FLICE-inhibitory protein, which prevents apoptosis by inhibiting the pro-apoptotic activation of upstream caspases. Here we show that the human intracellular serine protease inhibitor (serpin), protease inhibitor 9 (PI9), inhibits TNF-, TNF-related apoptosis-inducing ligand- and Fas ligand-mediated apoptosis in certain TNF-sensitive cell lines. The reactive center P1 residue of PI9 was required for this inhibition since PI9 harboring a Glu --> Ala mutation in its reactive center failed to impair death receptor-induced cell death. This suggests a classical serpin-protease interaction. Indeed, PI9 inhibited apoptotic death by directly interacting with the intermediate active forms of caspase-8 and -10. This indicates that PI9 can regulate pro-apoptotic apical caspases.

Prognostic significance of activated cytotoxic T-lymphocytes in primary nodal diffuse large B-cell lymphomas.

Clinical outcome in diffuse large B-cell lymphoma (DLBCL) remains unpredictable, despite the identification of clinical prognostic parameters. Here, we investigated in pretreatment biopsies of 70 patients with DLBCL whether numbers of activated cytotoxic T-lymphocytes (CTLs), as determined by the percentage of CD3-positive lymphocytes with granzyme B (GrB) expression, have similar prognostic value as found earlier in Hodgkin's lymphoma and anaplastic large-cell lymphoma and whether loss of major histocompatibility complex (MHC)-I molecules or expression of the GrB antagonist protease inhibitor 9 (PI9) may explain immune escape from CTL-mediated cell death. Independent of the International Prognostic Index (IPI), the presence of >/=15% activated CTLs was strongly associated with failure to reach complete remission, with a poor progression-free and overall survival time. Downregulation of MHC-I light- and/or heavy-chain expression was found in 41% of interpretable cases and in 19 of 56 interpretable cases PI9 expression was detected. We conclude that a high percentage of activated CTLs is a strong, IPI independent, indicator for an unfavorable clinical outcome in patients with primary nodal DLBCL. Although in part of DLBCL expression of PI9 and loss of MHC-I expression was found, providing a possible immune-escape mechanism in these cases, no correlation with clinical outcome was found.

[Diagnostic image (97). A woman with septicemia due to the small intestinal infarction]. / Diagnose in beeld (97). Een vrouw met sepsis door dunnedarminfarcering.

A 69-year-old woman who was treated for a spindle cell carcinoma of the upper jaw developed tumour emboli causing ischaemia and infarction of fingertips, small intestine, spleen, pancreas and kidneys.

Expression of the serpin serine protease inhibitor 6 protects dendritic cells from cytotoxic T lymphocyte-induced apoptosis: differential modulation by T helper type 1 and type 2 cells.

Dendritic cells (DCs) play a central role in the immune system as they drive activation of T lymphocytes by cognate interactions. However, as DCs express high levels of major histocompatibility complex class I, this intimate contact may also result in elimination of DCs by activated cytotoxic T lymphocytes (CTLs) and thereby limit induction of immunity. We show here that immature DCs are indeed susceptible to CTL-induced killing, but become resistant upon maturation with anti-CD40 or lipopolysaccharide. Protection is achieved by expression of serine protease inhibitor (SPI)-6, a member of the serpin family that specifically inactivates granzyme B and thereby blocks CTL-induced apoptosis. Anti-CD40 and LPS-induced SPI-6 expression is sustained for long periods of time, suggesting a role for SPI-6 in the longevity of DCs. Importantly, T helper 1 cells, which mature DCs and boost CTL immunity, induce SPI-6 expression and subsequent DC resistance. In contrast, T helper 2 cells neither induce SPI-6 nor convey protection, despite the fact that they trigger DC maturation with comparable efficiency. Our data identify SPI-6 as a novel marker for DC function, which protects DCs against CTL-induced apoptosis.

Persistence of genetically altered fields in head and neck cancer patients: biological and clinical implications.

In 1953, Slaughter et al. [D. P. Slaughter et al., Cancer (Phila.), 6: 963-968, 1953] proposed the concept of field cancerization in patients with squamous cell carcinoma of the head and neck (HNSCC) and discussed its clinical significance for the development of second primary tumors and local recurrences. To define the process of field cancerization and its putative clinical implications, we analyzed genetic aberrations in HNSCC and the accompanying macroscopically normal mucosa. In 28 HNSCC patients, loss of heterozygosity was determined in tumor and five noncontiguous mucosal biopsies using eight microsatellite markers at 9p, 3p, and 17p. For patients who showed loss of heterozygosity in their mucosal biopsies, all margins of the surgical specimen were subsequently analyzed to determine the extension of the field. In these cases, additional markers at 8p, 13q, and 18q as well as p53 mutations were included to determine subclonal differences between field and tumor. Genetically altered fields were detected in 36% (10 of 28) of the HNSCC patients. The field varied in size between patients and consisted of genetically different subclones. In 7 of 10 cases, the field extended into the surgical margins. One particular patient with a genetically altered field in a surgical margin developed a local recurrence after 28 months of follow-up. Microsatellite analysis showed that this recurrence had more molecular markers in common with the nonresected premalignant field than with the original tumor, suggesting that this persistent field has progressed further into a new malignancy. Our data show that genetically altered mucosa remains after treatment in a significant proportion of HNSCC patients, which may explain in part the high frequency of local recurrences and second primary tumors. Adequate identification and risk assessment of these genetically altered fields may have profound implications for future patient management.

Biological evidence that human papillomaviruses are etiologically involved in a subgroup of head and neck squamous cell carcinomas.

High-risk human papillomaviruses (HPVs) have been proposed to be associated with a subset of head and neck cancers (HNSCCs). However, clear biological evidence linking HPV-mediated oncogenesis to the development of HNSCC is hardly available. An important biological mechanism underlying HPV-mediated carcinogenesis is the inactivation of p53 by the HPV E6 oncoprotein. In the present study we investigated this biological relationship between HPV and HNSCC. In total 84 HNSCC tumors were analyzed for the presence of high-risk HPV nucleic acids by DNA polymerase chain reaction-enzyme immunoassay (PCR-EIA) and E6 reverse transcriptase (RT)-PCR as well as for the presence of mutations in the p53 gene. We found 20/84 HPV16 DNA-positive cases with one or more DNA assays, 10 of which were consistently positive with all assays. Only 9/20 cases showed E6 mRNA expression, indicative for viral activity. Only these nine E6 mRNA-positive cases all lacked a p53 mutation, whereas both the other HPV DNA-positive and HPV-DNA negative tumors showed p53 mutations in 36% and 63% of the cases, respectively. Moreover, only in lymph node metastases of HPV E6 mRNA-positive tumors both viral DNA and E6 mRNA were present. Our study provides strong biological evidence for a plausible etiological role of high-risk HPV in a subgroup of HNSCC. Analysis of E6 mRNA expression by RT-PCR or alternatively, semiquantitative analyses of the viral load, seem more reliable assays to assess HPV involvement in HNSCC than the very sensitive DNA PCR analyses used routinely.

Molecular assays for the diagnosis of minimal residual head-and-neck cancer: methods, reliability, pitfalls, and solutions.

The prognosis of cancer patients is determined by the radicalness of treatment: residual tumor cells will grow out and develop in manifest local recurrences, regional recurrences, and distant metastases. Classical diagnostic methods such as radiology and histopathology have limited sensitivities, and only by molecular techniques can minimal residual disease be detected. In tissue samples containing the normal tissue counterpart of a tumor, only tumor-specific markers can be exploited, whereas in other samples, tissue-specific markers can be used. At present, there are two main methodologies in use, one based on antigen-antibody interaction and the other based on amplified nucleic acids. The most commonly used nucleic acid markers are mutations or alterations in tumor DNA (tumor-specific markers) or differentially expressed mRNA (tissue-specific markers). Many reports and reviews have been published on the assessment of minimal residual disease by molecular markers, showing either positive or negative clinical correlations. One of the main reasons for these contradictory findings is the technical difficulty in finding the small numbers of tumor cells in the large number of normal cells, which necessitates sensitivities of the assays up to 1 tumor cell in 2 x 10(7) normal cells. These assays often are complex, demand considerable experience, and usually are laborious. In this review, we will address a number of the technical issues related to molecular assays for tumor cell detection that make use of nucleic acids as markers. Many difficulties in data interpretation are at least in part because of technical details that might have been solved by the incorporation of one or more appropriate controls. We hope that this review clarifies a number of these issues and help clinicians and investigators interested in this field to understand and weigh the contradictory findings in the published studies. This will help move the field forward and facilitate clinical implementation.

Immunoreactivity of B-cell markers (CD79a, L26) in rare cases of extranodal cytotoxic peripheral T- (NK/T-) cell lymphomas.

The monoclonal antibodies L26 (CD20) and CD79a are very useful reagents for the immunohistochemical assessment of B-cell lineage in lymphoproliferative disorders. Although very few CD20-positive peripheral T-cell lymphomas (PTL) have been reported, comprehensive analyses of CD79a reactivity in extranodal PTL and NK/T-cell lymphomas have not been performed previously. This study investigated CD79a (clone JCB117) and CD20 reactivity in 94 extranodal non-B-cell lymphomas (enteropathy-type intestinal T-cell lymphoma [n = 52], nasal NK/T-cell lymphoma [n = 11], and primary cutaneous PTL [n = 31]) and in 17 cases of nodal PTL, unspecified. In four cases (enteropathy-type intestinal T-cell lymphoma [n = 3] and nasal NK/T-cell lymphoma [n = 1]), the majority of tumor cells stained for CD79a (all CD20 negative) and one cutaneous PTL, unspecified, was CD20 positive (CD79a negative). Extensive immunophenotyping and polymerase chain reaction-based molecular analyses revealed that all five B-cell marker-positive extranodal lymphomas had a cytotoxic phenotype and did indeed represent monoclonal peripheral T-cell proliferations. To minimize the risk of misinterpretation of lymphoma cell lineage, especially in cases of extranodal, lymphoproliferative disease, we suggest the use of both CD79a and CD20 in combination with a panel of antibodies reactive to T cells, such as betaF1 and CD5, and to T cells and NK cells, such as CD3, CD2, CD56, and TIA-1.

Differential levels of granzyme B, regulatory cytokines, and apoptosis in Crohn's disease and ulcerative colitis at first presentation.

The mechanisms of tissue damage in ulcerative colitis and Crohn's disease may reflect disordered humoral or cell-mediated effector mechanisms, respectively. Mucosal biopsies from untreated inflammatory bowel disease patients and normal controls were analysed for the expression of granzyme B, a cytotoxic effector molecule specifically associated with cell-mediated immunity, and for regulatory cytokines. Messenger RNA (mRNA) was analysed by reverse transcription-polymerase chain reaction and enzyme-linked oligonucleotide chemiluminescence assay. Mucosal biopsies were analysed by immunohistochemistry for granzyme B protein and lymphocyte markers and for the presence of apoptotic cells by terminal deoxynucleotidyl transferase end labelling. Granzyme B mRNA was elevated in Crohn's disease, but not in ulcerative colitis or control mucosal biopsies. Granzyme B mRNA levels correlated with interferon gamma mRNA levels in Crohn's disease. Granzyme B was expressed in CD3+, CD8+ T cells in the lamina propria of Crohn's disease mucosa and there were significantly more apoptotic cells in the lamina propria in Crohn's disease. In conclusion, granzyme B-expressing T lymphocytes are present in the focal mucosal lesions of Crohn's disease, together with spatially related apoptotic cell death. These results support the hypothesis that T-cell-mediated cytotoxic effector mechanisms may play a role in Crohn's disease.

Case report of a patient with an intimal sarcoma of the pulmonary trunk presenting as a pulmonary embolism.

A fatal case of an 89-year-old woman with an intimal sarcoma obstructing the pulmonary trunk and an open foramen ovale is presented. Clinical symptoms, physical examination and further evaluation originally raised suspicion of a pulmonary embolism. Recent classification systems, specific radiological and pathological characteristics of sarcomas of the pulmonary trunk are discussed.

Human papillomavirus is a necessary cause of invasive cervical cancer worldwide.

A recent report that 93 per cent of invasive cervical cancers worldwide contain human papillomavirus (HPV) may be an underestimate, due to sample inadequacy or integration events affecting the HPV L1 gene, which is the target of the polymerase chain reaction (PCR)-based test which was used. The formerly HPV-negative cases from this study have therefore been reanalyzed for HPV serum antibodies and HPV DNA. Serology for HPV 16 VLPs, E6, and E7 antibodies was performed on 49 of the 66 cases which were HPV-negative and a sample of 48 of the 866 cases which were HPV-positive in the original study. Moreover, 55 of the 66 formerly HPV-negative biopsies were also reanalyzed by a sandwich procedure in which the outer sections in a series of sections are used for histological review, while the inner sections are assayed by three different HPV PCR assays targeting different open reading frames (ORFs). No significant difference was found in serology for HPV 16 proteins between the cases that were originally HPV PCR-negative and -positive. Type-specific E7 PCR for 14 high-risk HPV types detected HPV DNA in 38 (69 per cent) of the 55 originally HPV-negative and amplifiable specimens. The HPV types detected were 16, 18, 31, 33, 39, 45, 52, and 58. Two (4 per cent) additional cases were only HPV DNA-positive by E1 and/or L1 consensus PCR. Histological analysis of the 55 specimens revealed that 21 were qualitatively inadequate. Only two of the 34 adequate samples were HPV-negative on all PCR tests, as against 13 of the 21 that were inadequate ( p< 0.001). Combining the data from this and the previous study and excluding inadequate specimens, the worldwide HPV prevalence in cervical carcinomas is 99.7 per cent. The presence of HPV in virtually all cervical cancers implies the highest worldwide attributable fraction so far reported for a specific cause of any major human cancer. The extreme rarity of HPV-negative cancers reinforces the rationale for HPV testing in addition to, or even instead of, cervical cytology in routine cervical screening.

Expression of cytotoxic proteins by neoplastic T cells in mycosis fungoides increases with progression from plaque stage to tumor stage disease.

Granzyme B (GrB) and T-cell-restricted intracellular antigen (TIA-1) are cytotoxic proteins that are specifically expressed by cytotoxic CD4 or CD8 positive T cells and natural killer cells. Recent studies demonstrated frequent expression of GrB and TIA-1 by neoplastic cells in primary cutaneous CD30(+) large T-cell lymphomas and lymphomatoid papulosis but not in CD30(-) large T-cell lymphomas. In the present study, 74 biopsies from 54 patients with mycosis fungoides (MF) were investigated for the expression of GrB and TIA-1 using immunohistochemistry on paraffin sections. Staining of more than 10% of the neoplastic T cells for GrB or TIA-1 was considered positive. All but two follow-up biopsies had been obtained from patients without extracutaneous disease at the time of biopsy. Expression of TIA-1 and GrB was found in 33 (45%) and 14 (19%) of 74 MF biopsies, respectively. Comparison of biopsies from T3NoMo-stage MF (n = 27) and T2NoMo-stage MF (n = 45) showed increased expression of TIA-1 (55 versus 37%) and GrB (33 versus 9%) in T3NoMo-stage MF. Evaluation of multiple sequential biopsies from successive stages of MF also revealed an increase in the GrB/TIA-1 expression with tumor progression in five of eight cases. A clearcut relation between the expression of TIA-1 and/or GrB and the type of skin lesion biopsied was found. Considering all 74 biopsies, expression of TIA-1 and GrB was found in 18 of 50 (35%) and 5 of 50 (10%) patches or plaques, 9 of 16 (55%) and 3 of 16 (20%) tumors without blastic transformation, and 6 of 8 (75%) and 6 of 8 (75%) tumors with blastic transformation (defined as >50% blast cells). Correlation between GrB/TIA-1 expression in first diagnostic biopsies from patches or plaques from 40 patients with T2NoMo-stage MF and clinical follow-up data did not reveal differences in clinical behavior and survival between patients with (n = 14) or without (n = 26) expression of cytotoxic proteins, indicating that MF expressing cytotoxic proteins should not be considered as a separate group.

Extracellular granzymes A and B in humans: detection of native species during CTL responses in vitro and in vivo.

Activated CTLs and NK cells induce apoptosis via multiple mechanisms, including that termed granule exocytosis. The latter pathway consists of vectorial secretion of perforin and a family of granule-associated serine proteases (granzymes) to the target cell. To establish whether granzymes are released extracellularly during cytolytic reactions in vivo, ELISAs that measure the native enzymes were developed and were found to specifically detect granzyme A (GrA) and granzyme B (GrB) at picogram concentrations. Low levels of GrA and GrB were present in plasma of healthy individuals (GrA, 33.5 pg/ml (median); GrB, 11.5 pg/ml (median)), whereas significantly higher levels were present in patients with ongoing CTL response, i.e., patients suffering from infections by EBV or HIV type 1. Markedly elevated levels were also noted in synovial fluid of patients with active rheumatoid arthritis. The measurement of soluble granzymes should be useful to assess clinical disorders associated with activated CTL and NK cells. Furthermore, these results suggest that granzymes mediate biologic effects beyond their described role in apoptotic cell death.