Panagiotis Kanavaros

Cytotoxic protein expression in natural killer cell lymphomas and in αβ and γδ peripheral T-cell lymphomas

Lymphomas with T‐cell phenotype represent a heterogeneous group of diseases differing in histopathology, tumour site, and cell origin. They include peripheral T‐cell lymphomas (PTCLs) derived from αβ cells, but also some recently recognized entities such as γδ hepatosplenic lymphomas and natural killer (NK) cell lymphomas. Only a few studies have investigated the possibility that at least some PTCLs could be derived from lymphocytes with cytotoxic potential. In order to investigate this possibility, 60 cases of PTCL, including 27 cases expressing the αβ T‐cell receptor (TCRαβ), 15 TCRγδ cases and 18 cases expressing neither TCR (TCR silent), as well as 14 cases of NK‐cell lymphomas, were studied by immunohistochemistry for the expression of TIA‐1, perforin, and granzyme B proteins. Expression of TIA‐1 is characteristic of cytotoxic cells regardless of their activation status, whereas expression of perforin and granzymes is highly increased in activated cytotoxic cells and correlates with the induction of cytolytic activity. All NK‐cell lymphomas (11 sinonasal, three systemic cases) expressed TIA‐1, perforin, and granzyme B in most tumour cells. All γδ PTCLs (15 cases) expressed TIA‐1 protein in most tumour cells, with a different cytotoxic antigen profile in hepatosplenic γδ PTCL (TIA‐1+, perforin−, granzyme B−) and in non‐hepatosplenic γδ PTCLs (three nasal, one skin, one lung), the latter expressing the three cytotoxic proteins. Of the 45 cases of αβ and TCR silent PTCL, 15 (33 per cent) were considered to be derived from cytotoxic lymphocytes with expression of at least one cytotoxic protein (TIA‐1, 15/45; perforin, 10/41; granzyme B, 14/38) in tumour cells. This cytotoxic protein expression appeared to be related to the site of localization, since 7/13 (54 per cent) extranodal and only 8/32 (25 per cent) nodal αβ and TCR silent PTCLs expressed TIA‐1, and to histology, since this pattern was observed in a proportion of anaplastic (6/8, 75 per cent) and pleomorphic (8/17, 47 per cent) lymphomas, but not in AILD‐type NHL (0/16). Taken together, our data suggest that NK‐cell lymphomas and non‐hepatosplenic γδ PTCLs represent tumours of activated cytotoxic NK cells and γδ T cells, respectively; that hepatosplenic γδ PTCLs represent tumours of non‐activated cytotoxic γδ T cells; and that a small proportion of αβ and TCR silent PTCLs, mostly extranodal cases, or nodal anaplastic lymphomas, represent tumours of cytotoxic T cells.

Expression of c-myc and bcl-2 oncogene products in Reed-Sternberg cells independent of presence of Epstein-Barr virus.

AIMS: To evaluate the expression of c-myc and bcl-2 oncogene products in Reed-Sternberg cells in Hodgkins disease, especially in relation to Epstein-Barr virus infection and expression of EBV encoded latent membrane protein (LMP). METHODS: Tissues from 33 cases of Hodgkins disease were studied for the presence of EBV DNA by polymerase chain reaction (PCR) and DNA in situ hybridisation (DISH), for the presence of EBER-1 and EBER-2 EBV RNA by RNA in situ hybridisation (RISH); and for the presence of LMP, bcl-2, and c-myc proteins by immunohistochemical staining. RESULTS: A substantial number of Reed-Sternberg cells expressed bcl-2 in 20 of 29 (69%) and c-myc in 30 of 32 (94%) Hodgkins disease samples. In 18 of the 25 (72%) cases Reed-Sternberg cells expressed both oncogene products. Of these 18 cases, 10 (56%) were EBV-PCR positive; eight (44%) were EBV-PCR negative. CONCLUSIONS: Reed-Sternberg cells in Hodgkins disease frequently express both bcl-2 and c-myc oncogene products, suggesting that these oncogenes may act in concert in the pathogenesis of the disease. Moreover, the expression of c-myc and bcl-2 proteins in Reed-Sternberg cells is independent of EBV and LMP status.

Expression of Cytotoxic Proteins in Peripheral T-Cell and Natural Killer-Cell (NK) Lymphomas: Association with Extranodal Site, NK or Tγδ Phenotype, Anaplastic Morphology and CD30 Expression

Most peripheral T-cell lymphomas (PTCL) express the αβ T-cell receptor (TCR) whereas rare PTCL express the γδ TCR. Most if not all γδ PTCL are extranodal lymphomas and among them, hepatosplenic γδ PTCL constitute a distinct clinicopathological entity. Besides αβ and γδ PTCL, there is a recently recognized group of extranodal, mainly nasal tumours, which display, in most instances, phenotypic and genotypic features of Natural-Killer cell non-Hodgkins lymphomas (NK-NHL). Cytotoxic cells, including NK cells and cytotoxic αβ and γδ T lymphocytes may induce lysis of the target by using granule-associated cytotoxic proteins such as the T-cell intracellular antigen-1 (TIA-1), perforin and granzyme B. Expression of TIA-1 can be detected in all cytotoxic cells whereas granzyme B and perforin expression can be detected in high levels only in activated cytotoxic cells. Recently, several studies showed that the expression of these cytotoxic proteins in tumour cells of PTCL and NK-NHL is associated with a) extranodal site of clinicopathological presentation b) NK or Tγδ -cell phenotype c) CD30 expression in cutaneous T-cell lymphoproliferations and d) anaplastic morphology in nodal PTCL. This latter finding contrasts with the data that only rare Hodgkin lymphomas (HL) express cytotoxic proteins in Hodgkin and Reed-Sternberg cells. Altogether the data of the literature indicate that most extranodal T and NK-NHL are activated cytotoxic lymphomas with the notable exception of hepatosplenic γδ PTCL which represent tumours of non-activated cytotoxic cells. On this basis, it is suggested that the expression of cytotoxic proteins may be useful for the identification and classification of extranodal T and NK-cell lymphomas and, to some extent, for the differential diagnosis between HL and CD30+ anaplastic large cell lymphomas. Cytotoxic lymphomas are preferentially localized in extranodal sites such as skin, lung, upper respiratory and gastrointestinal tracts, which are continuously exposed to various antigens. Since cytotoxic T and NK cells are regarded as first line of defense in these sites, and some cytotoxic tumours such as nasal lymphomas and enteropathy-type intestinal lymphomas are associated with EBV and gliadin, respectively, it is likely that chronic antigen exposure may play a role in the pathogenesis of cytotoxic lymphomas occurring in mucosa and/or skin. Besides chronic antigenic stimulation, chronic immunosuppression may also have pathogenetic significance in cytotoxic lymphomas in view of their increased incidence in immunocompromised patients.

Increased expression of the bcl6 and CD10 proteins is associated with increased apoptosis and proliferation in diffuse large B-cell lymphomas.

There is increasing evidence that bcl6 and CD10 expression may be related to apoptosis and cell cycle progression. Therefore, 79 cases of de novo diffuse large B-cell lymphomas were studied for the expression of bcl6 and CD10 proteins in relation to 1) the apoptotic index; 2) the proliferation-associated proteins Ki67, cyclin A, and cyclin B1; and 3) the expression of the bcl2, p53, Rb, p16, and p27 proteins. Expression of bcl6, CD10, and bcl2 proteins was found in 54/79 (68%), 28/79 (35%), and 47/74 (63%) cases, respectively. The bcl6/CD10 patterns were as follows: bcl6+/CD10+ (26 cases, 32%), bcl6+/CD10- (28 cases, 33%), bcl6−/CD10− (23 cases, 31%), and bcl6−/CD10+ (2 cases, 4%). Significant positive correlations were found between bcl6/Ki67 (r = .328, P = .003), bcl6/cyclin A (r = .265, P = .018), bcl6/apoptotic index (r = .327, P = .010), CD10/Ki67 (r = .296, P = .008), and CD10/apoptotic index (r = .397, P = .001). In addition, high expression of bcl6 showed significant correlation with negative (null/low) bcl2 expression (χ2 test, P = .002). The above findings indicate that increased expression of the bcl6 and CD10 proteins is associated with increased apoptosis and proliferation in diffuse large B-cell lymphomas. The association between increased bcl6 expression and enhanced apoptosis might be due, at least in part, to the null/low bcl2 expression because previous in vitro data showed that bcl6 overexpression induces apoptosis accompanied by bcl2 and bcl-xl downregulation. Moreover, significant correlation was found between increased apoptotic index and the bcl6+/CD10+ pattern (t test: P = .014, Mann-Whitney test: P = .046). This finding and the positive correlation of the apoptotic index with bcl6 and CD10 expression may be related to previous results showing that the expression of these proteins has favorable effects on the clinical outcome of diffuse large B-cell lymphomas.

Nuclear localization of human AP endonuclease 1 (HAP1/Ref-1) associates with prognosis in early operable non-small cell lung cancer (NSCLC)

The present study examined the immunohistochemical expression of human AP endonuclease 1 (HAP1/Ref‐1), the major endonuclease in the repair of apurinic/apyrimidinic (AP) sites in cellular DNA, in normal lung and lung carcinomas. Cellular expression of HAP1 was determined using a standard avidin–biotin–peroxidase complex (ABC) technique and an anti‐HAP1 rabbit polyclonal antibody on paraffin‐embedded tissue sections from normal lung and in 103 primary non‐small cell lung carcinomas (NSCLCs). In normal lung, the staining for HAP1 was found to be both nuclear and cytoplasmic in the pneumocytes of the alveoli. Superficial ciliated cells of the bronchial epithelium presented cytoplasmic staining, while staining for the basal cells was mostly nuclear. Bronchial glandular cells demonstrated mixed nuclear and cytoplasmic staining. Lung carcinomas showed all patterns of expression for HAP1. Loss of HAP1 expression was associated with low proliferation index ( p=0·01) and with squamous histology ( p=0·04). In squamous carcinomas, a significant correlation was observed between positive nuclear HAP1 and negative p53 expression ( p=0·03). A survival benefit was seen in patients presenting nuclear HAP1 expression and those presenting the nuclear HAP1+/p53− phenotype ( p=0·01 and 0·007, respectively). It is concluded that nuclear HAP1 localization may be relevant to its role as a DNA repair protein and/or to the recently proposed role as an activator of wild‐type p53, and thus to the better outcome seen in this group of patients. Copyright

Epstein-Barr virus in non-Hodgkin's lymphomas of the upper respiratory tract: association with sinonasal localization and expression of NK and/or T-cell antigens by tumour cells.

Fifty‐five cases of non‐Hodgkins lymphoma (NHL) of the upper respiratory tract, comprising 27 sinonasal (SN) and 28 Waldeyers ring (WR) NHL, were investigated for expression of Epstein–Barr virus (EBV)‐encoded EBER transcripts and latent membrane protein‐1 (LMP‐1) by RNA in‐situ hybridization (RISH) and immunohistochemistry, respectively. Thirty‐two cases were B‐cell tumours (10 SNHLs and 22 WRNHLs) and 23 cases expressed natural killer (NK) and/or T‐cell antigens (17 SNHLs and 6 WRNHLs). EBER transcripts were detected in tumour cells in 19 lymphomas expressing NK and/or T‐cell antigens (16/17 SHNHLs and 3/6 WRNHLs) but in only 2/32 B‐NHLs (1/10 SNHLs and 1/22 WRNHLs). LMP‐1 expression was found in tumour cells in the 19 EBER‐positive tumours expressing NK and/or T‐cell antigens but in none of the B‐cell lymphomas. All the LMP‐1‐positive lymphomas expressed the CD30 molecule in tumour cells. These results indicate that in lymphomas of the upper respiratory tract, EBV is strongly associated with sinonasal localization and expression of NK and/or T‐cell antigens by tumour cells. EBV can also be detected in some cases of WRNHLs expressing NK and/or T‐cell antigens, whereas it is rarely found in B‐cell SNHLs and WRNHLs. Furthermore, the detection of the LMP‐1 protein in tumour cells in most SNHLs and some WRNHLs expressing NK and/or T‐cell antigens, in view of the LMP‐1 transforming potential, suggests that EBV may play a role in the pathogenesis of these lymphomas.

Epstein-Barr Virus (EBV) in Extranodal T-cell Non-Hodgkin's Lymphomas (T-NHL). Identification of Nasal T-NHL as a Distinct Clinicopathological Entity Associated with EBV

T-cell Non-Hodgkins lymphomas (T-NHL) can be defined as clonal malignant proliferations related phenotypically and functionally to normal T-cell populations of the lymphoid tissue. There is increasing evidence that T-NHL with similar morphology but originating from different sites differ in their clinical behaviour, immunophenotypic features, oncogene expression and relation with oncogenic viruses such as HTLV-I and EBV. Indeed, it has been shown that the prevalence of EBV in T-NHL is related to the site of origin. Thus, EBV was found in nearly all nasal T-NHL but only in a proportion of primary nodal, lung, gastrointestinal and Waldeyers ring T-NHL while it was undetectable in most primary cutaneous T-NHL. Besides their constant association with EBV, nasal T-NHL display peculiar clinical, histological, immunophenotypic and genotypic features. They present clinically as lethal midline granuloma and histologically as pleomorphic malignant tumours variably associated with angiocentricity, angioinvasion and necrosis. Moreover, they frequently exhibit extensive loss of T-cell antigens, including CD3 and TCR alpha beta and gamma delta proteins, usually express the Natural Killer (NK)-related CD56 antigen and frequently show absence of clonal rearrangements of TCR beta, gamma and delta loci. Therefore, among T-NHL, nasal T-NHL can be regarded as a distinct clinicopathologic entity associated with EBV, which could be derived either from immature T-cells or from NK cells.

Signs and symptoms of temporomandibular joint disorders related to the degree of mouth opening and hearing loss.

BackgroundThe temporomandibular joint is a unique bi-condylar joint involved in mastication and speech. Temporomandibular joint disorders (TMD) have a range of symptoms, including aural symptoms, and are present in approximately 75% of normal populations. The present study examined the relationship between signs and symptoms of TMD and mouth opening, gender, joint and aural symptoms, and hearing loss.MethodsThe study involved 464 healthy Greek university students (156 men and 308 women) with a mean age of 19.6 years. Age, gender and maximum mouth opening was recorded. Mouth opening was measured using Vernier calipers. An anamnestic questionnaire was used to stratify the subjects into four groups based on TMD severity. Aural symptoms and an audiogram were recorded for each subject too. Data were analyzed using multifactor ANOVA, chi-square, t-test, Mann-Whitney and Kruskal-Wallis tests.ResultsThe overall incidence of TMD signs and symptoms was 73.3%. The incidence and severity was greater in females than males (p-value 0.0001 < 0.05). The number of aural symptoms was associated to the TMD severity (p-value 0.0001 < 0.05) as well as maximum mouth opening (p-value 0.004 < 0.05). Audiometry showed that moderate and severe TMD was associated with hearing loss of median and low tones respectively (p-value 0.0001 < 0.05). TMJ pain (p-value 0.0001 < 0.05), TMJ ankylosis (p-value 0.0001 < 0.05), bruxism (p-value 0.0001 < 0.05) and ear itching (p-value 0.0001 < 0.05) were also found to be statistically different between TMD and non-TMD subjects.ConclusionsTMD signs and symptoms were more common and severe in females than males. TMD severity is correlated with the degree of mouth opening and the number of aural symptoms. The absence or presence of mild TMD are associated with normal audiograms while moderate and severe TMD are related to hearing loss in median and low tones respectively. Bruxism, joint ankylosis, joint pain and ear itching were more common in TMD than non-TMD patients.

Diffuse large B-cell lymphomas with germinal center B-cell-like differentiation immunophenotypic profile are associated with high apoptotic index, high expression of the proapoptotic proteins bax, bak and bid and low expression of the antiapoptotic protein bcl-xl

The aim of this study was to analyze the relations between differentiation immunophenotypes and the status of apoptosis and proliferation in diffuse large B-cell lymphomas. Therefore, the bcl6/CD10/MUM1/CD138 differentiation immunophenotypic profiles were studied in relation to (a) the apoptotic index, (b) the apoptosis-associated bcl2 family proteins bcl2, bcl-xl, bax, bak, bad and bid, (c) the proliferation index (Ki67) and (d) the cell cycle proteins cyclin A, cyclin B1, cyclin D3, cyclin E, p53, Rb, p16 and p27 in 79 cases of diffuse large B-cell lymphomas. Two major differentiation immunophenotypic profiles were distinguished: the germinal center B-cell-like profile; 31 cases (bcl6+/CD10±/MUM1−/CD138−: 29 cases and bcl6−/CD10+/MUM1−/CD138−: two cases) and the nongerminal center B-cell-like profile (bcl6±/CD10−/MUM1+/CD138−); 48 cases. The expression of bax, bak and bid and the apoptotic index were significantly higher in the germinal center B-cell-like profile than in the nongerminal center B-cell-like profile (P=0.045, 0.018, 0.003 and 0.034, respectively). In contrast, the expression of bcl-xl was significantly lower in the germinal center B-cell-like profile than in the nongerminal center B-cell-like profile (P=0.026). The expression of bcl6 and CD10 showed significant positive correlation with the expression of bax (r=0.659, P<0.001 and r=0.240, P=0.033, respectively), bak (r=0.391, P<0.001 and r=0.233, P=0.039, respectively) and bid (r=0.652, P<0.001 and r=0.238, P=0.035, respectively) and significant negative correlation with the expression of bcl-xl (r=−0.536, P<0.001 and r=−0.250, P=0.029, respectively). The expression of MUM1 showed significant negative correlation with the expression of bax (r=−0.276, P=0.014) and bid (r=−0.266, P=0.018) and significant positive correlation with the expression of bcl-xl (r=0.238, P=0.037). The above findings indicate that diffuse large B-cell lymphomas with germinal center B-cell-like immunophenotypic profile are associated with increased apoptosis status, high expression of the proapoptotic proteins bax, bak and bid and low expression of the antiapoptotic protein bcl-xl.

Angiographic findings and clinical implications of persistent primitive hypoglossal artery.

BackgroundThe primitive hypoglossal artery (PHA) is a rare vascular anomaly, which belongs to the group of carotid-basilar anastomosis that may occur in adults.Case presentationHerein is presented a case of a patient with a PHA, who had undergone a cerebral angiography due to investigation of subarachnoid hemorrhage. Additionally, the diagnostic alternatives for detection and assessment of PHA and the spectrum of diseases related to its presence are discussed.ConclusionsThe presence of a persistent PHA can be recognized as an incidental finding in a cerebral angiography without any other clinical implication or may be associated with certain clinical entities such as aneurysm formation and atherosclerotic disease.