Produção de vetores recombinantes para análise das propriedades biológicas e cancerígenas da proteína K1 do herpesvírus associado ao sarcoma de Kaposi (KSHV/HHV-8)

Pós-graduação em Patologia - FMB

Pharyngocutaneous fistula following total laryngectomy

Pharyngocutaneous fistula (PCF) is the most common complication after total laryngectomy. Objectives: To establish the incidence of this complication and to analyze the predisposing factors. Method: This is a cross-sectional study of a historical cohort including 94 patients who underwent total laryngectomy. The following aspects were correlated to the occurrence of PCF: gender, age, tumor site, TNM staging, type of neck dissection, previous radiation therapy, previous tracheotomy, and use of stapler for pharyngeal closure. The following were considered in PCF cases: the day into postoperative care when the fistula was diagnosed, duration of occurrence, and proposed treatment. Results: Twenty (21.3%) patients had PCF. The incidence of PCF was statistically higher in T4 tumors when compared to T2 and T3 neoplasms (p = 0.03). The other analyzed correlations were not statistically significant. However, 40.9% of the patients submitted to tracheostomy previously had fistulae, against 21.1% of the patients not submitted to this procedure. Conclusion: Advanced primary tumor staging is correlated with higher incidences of PCF.

Cloning and characterization of human protease-activated receptor 4

Protease-activated receptors 1–3 (PAR1, PAR2, and PAR3) are members of a unique G protein-coupled receptor family. They are characterized by a tethered peptide ligand at the extracellular amino terminus that is generated by minor proteolysis. A partial cDNA sequence of a fourth member of this family (PAR4) was identified in an expressed sequence tag database, and the full-length cDNA clone has been isolated from a lymphoma Daudi cell cDNA library. The ORF codes for a seven transmembrane domain protein of 385 amino acids with 33% amino acid sequence identity with PAR1, PAR2, and PAR3. A putative protease cleavage site (Arg-47/Gly-48) was identified within the extracellular amino terminus. COS cells transiently transfected with PAR4 resulted in the formation of intracellular inositol triphosphate when treated with either thrombin or trypsin. A PAR4 mutant in which the Arg-47 was replaced with Ala did not respond to thrombin or trypsin. A hexapeptide (GYPGQV) representing the newly exposed tethered ligand from the amino terminus of PAR4 after proteolysis by thrombin activated COS cells transfected with either wild-type or the mutant PAR4. Northern blot showed that PAR4 mRNA was expressed in a number of human tissues, with high levels being present in lung, pancreas, thyroid, testis, and small intestine. By fluorescence in situ hybridization, the human PAR4 gene was mapped to chromosome 19p12.

Activation of latent Kaposi's sarcoma-associated herpesvirus by demethylation of the promoter of the lytic transactivator

Kaposi's sarcoma-associated herpesvirus (KSHV) is strongly linked to Kaposi's sarcoma, primary effusion lymphomas, and a subset of multicentric Castleman's disease. The mechanism by which this virus establishes latency and reactivation is unknown. KSHV Lyta (lytic transactivator, also named KSHV/Rta), mainly encoded by the ORF 50 gene, is a lytic switch gene for viral reactivation from latency, inasmuch as it is both essential and sufficient to drive the entire viral lytic cycle. Here we show that the Lyta promoter region was heavily methylated in latently infected cells. Treatment of primary effusion lymphoma-delivered cell lines with tetradecanoylphorbol acetate caused demethylation of the Lyta promoter and induced KSHV lytic phase in vitro. Methylation cassette assay shows demethylation of the Lyta promoter region was essential for the expression of Lyta. In vivo, biopsy samples obtained from patients with KSHV-related diseases show the most demethylation in the Lyta promoter region, whereas samples from a latently infected KSHV carrier remained in a methylated status. These results suggest a relationship among a demethylation status in the Lyta promoter, the reactivation of KSHV, and the development of KSHV-associated diseases.

Improved clonality assessment in germinal centre/post-germinal centre non-Hodgkin's lymphomas with high rates of somatic hypermutation.

BACKGROUND: PCR detects clonal rearrangements of the Ig gene in lymphoproliferative disorders. False negativity occurs in germinal centre/post-germinal centre lymphomas (GC/PGCLs) as they display a high rate of somatic hypermutation (SHM), which causes primer mismatching when detecting Ig rearrangements by PCR. AIMS: To investigate the degree of SHM in a group of GC/PGCLs and assess the rate of false negativity when using BIOMED-2 PCR when compared with previously published strategies. METHODS: DNA was isolated from snap-frozen tissue from 49 patients with GC/PGCL (23 diffuse large B cell lymphomas (DLBCLs), 26 follicular lymphomas (FLs)) and PCR-amplified for complete (VDJH), incomplete (DJH) and Ig kappa/lambda rearrangements using the BIOMED-2 protocols, and compared with previously published methods using consensus primers. Germinal centre phenotype was defined by immunohistochemistry based on CD10, Bcl-6 and MUM-1. RESULTS: Clonality detection by amplifying Ig rearrangements using BIOMED-2 family-specific primers was considerably higher than that found using consensus primers (74% DLBCL and 96% FL vs 69% DLBCL and 73% FL). Addition of BIOMED-2 DJH rearrangements increased detection of clonality by 22% in DLBCL. SHM was present in VDJH rearrangements from all patients with DLBCL (median (range) 5.7% (2.5-13.5)) and FL (median (range) 5.3% (2.3-11.9)) with a clonal rearrangement. CONCLUSIONS: Use of BIOMED-2 primers has significantly reduced the false negative rate associated with GC/PGCL when compared with consensus primers, and the inclusion of DJH rearrangements represents a potential complementary target for clonality assessment, as SHM is thought not to occur in these types of rearrangements.

Identification of novel target genes in different subtypes of cutaneous T-cell lymphoma

Ihon T-solulymfoomat (cutaneous T-cell lymphoma, CTCL) ovat ryhmä imukudossyöpiä, joiden esiintyvyys on nousussa erityisesti länsimaissa. Taudin syntymekanismit ovat suurelta osin tuntemattomat, diagnostiikka on vaikeaa ja siksi usein viivästynyttä eikä parantavaa hoitoa ole. CTCL ilmenee iho-oirein, vaikka syöpäsolut eivät ole iholla normaalisti esiintyviä soluja, vaan elimistön puolustusjärjestelmän soluja, jotka ovat tuntemattomasta syystä vaeltaneet iholle. Syöpäsolut ovat kypsiä T-auttajasoluja (Th-soluja) ja ilmentävät tyypin 2 immuunivasteelle ominaisia sytokiineja. Kromosomaalinen epästabiilius on tautiryhmän keskeinen piirre. CTCL-potilailla on lisääntynyt riski sairastua myös muihin syöpiin, erityisesti keuhkosyöpään ja non-Hodgkin –lymfoomiin. Väitöskirjatutkimuksen tavoitteena oli havaita CTCL:n syntymekanismeja selvittäviä kromosomi- ja geenimuutoksia. Erityisesti tavoitteena oli identifioida molekyylejä, jotka soveltuisivat diagnostisiksi merkkiaineiksi tai täsmähoidon kohteeksi. Työssä on tutkittu kahta tautiryhmän yleisintä muotoa, mycosis fungoidesta (MF) ja Sezaryn syndroomaa (SS) sekä harvinaisempaa vaikeasti diagnosoitavaa subkutaanista pannikuliitin kaltaista T-solulymfoomaa (SPTL). Lisäksi on tutkittu CTCL:ään liittyvää keuhkosyöpää ja verrattu sitä tavalliseen (primaariin) keuhkosyöpään. Tutkimusmenetelminä on käytetty esimerkiksi molekyylisytogeneettisiä metodeja ja mikrosiruja. Väitöskirjatyössä havaittiin ensimmäinen CTCL:lle ominainen toistuva geenitason muutos: puutos- tai katkoskohta NAV3-geenissä. Tämän geenipoikkeavuuden havaittiin esiintyvän useissa taudin alaryhmissä (MF, SS, SPTL). NAV3-geenipuutoksen osoittaminen FISH-tekniikalla on sovellettavissa kliiniseen diagnostiikkaan. Tutkimukset geenipuutoksen aiheuttamista toiminnallisista seurauksista ovat käynnissä. Työssä saatiin myös uutta tietoa taudin syntymekanismeista havaitsemalla useiden Th1-tyypin immuunivasteelle ominaisten geenien alentunut ilmeneminen CTCL-potilailla. Tämän lisäksi potilasnäytteissä havaittiin eräiden solun pinta-antigeenien lisääntynyt ilmeneminen, mikä luo pohjan uusien vasta-ainepohjaisten täsmähoitojen kehittämiselle. Väitöskirjatutkimuksessa todettiin myös CTCL:ään liittyvän keuhkosyövän eroavan kromosomi- ja geenimuutosten suhteen verrokkikeuhkosyövästä, mikä jatkossa antaa aiheen tutkia syöpäkantasolujen merkitystä CTCL:n ja sen liitännäiskasvainten kehittymisen taustalla.

Epitheliotropic cutaneous T-cell lymphoma associated with melanoma in a dog: case report

Several types of tumors affect dogs' skin. Simultaneously occurring neoplasms with different histological patterns might be rarely present in the same animal. This paper describes the occurrence of epitheliotropic cutaneous T-cell lymphoma and melanoma in a dog. The animal had nodular lesions in the abdominal region and serpiginous plaques on the dorsal region of the trunk. Cytology evidenced malignant fusiform cells from the abdominal lesions as well as few round cells from the dorsal. The histopathological examination of the abdominal lesions showed dermis with polygonal to spindle-shaped neoplastic cells. The lesion of the dorsal region evidenced neoplastic round cells with generally distinct cell borders and a moderate amount of eosinophilic cytoplasm. Abdominal lesions were positive for Melan A. Dorsal and forelimb lesions were positive for CD3. This study reports the occurrence of epitheliotropic cutaneous T-cell lymphoma and malignant melanoma in a crossbred Boxer dog and discusses the importance of performing immunohistochemical profile to confirm the phenotype of the tumor.

Chronic reparative changes in medium-sized vessels in a case of primary cutaneous anaplastic large-cell lymphoma with angioinvasive features and cytotoxic phenotype: new histopathological findings in line with indolent clinical behavior

Angioinvasion/angiodestruction has been reported in a small subset of primary cutaneous anaplastic large-cell lymphomas (PCALCL). Recently, PCALCL with angioinvasive features and cytotoxic phenotype has been characterized as a variant associated with good clinical outcomes despite worrisome histopathologic features. We report a case of PCALCL with angioinvasive features and cytotoxic phenotype associated with reparative changes on the wall of medium-sized vessels involved by the neoplasm, including intimal fibroblastic proliferation and luminal obliteration. This vascular pattern, although previously unreported in PCALCL, is in accordance with the indolent behavior observed in this entity and provides a further link with lymphomatoid papulosis type E.

T-cell lymphoma in the tongue of a dog with cutaneous and striated forelimb muscle involvement

Background: Primary tongue tumors rarely affect dogs and correspond to 4% of tumors involving the oropharynx. Until now, primary tongue lymphoma had not been reported. However, lymphoma involvement in the skeletal muscle, although quite unusual, was described in the literature in four cases. Cutaneous lymphoma is another rare extranodal manifestation. The objective of this report is to describe a case of T immunophenotype lymphoma occurrence, whose manifestation is atypical, not only because it is situated in the tongue muscle but also because of the subsequent involvement of the striated musculature of the left forelimb and the skin, which showed unfavorable evolution. Case: A female seven-year-old mongrel was seen showing a regular lump in the base of the tongue, 3 cm in diameter, not ulcerated and of fi rm consistency, with halitosis as the only clinical sign of the disease. Incisional biopsy of the lump was performed and histopathology verifi ed that it was large cell lymphoma. The material was sent for immunohistochemical evaluation and was characterized as T immunophenotype lymphoma by positive CD3 and negative CD79a marking. The CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone) chemotherapy protocol was established as treatment and after the fi rst chemotherapy session there was partial remission of the mass, measuring 2 cm in diameter. The lump, however, remained stable in the following sessions. Thirty days after the diagnosis of lymphoma, the animal began to show lameness of the left forelimb and swelling near the head of the left humerus. A muscle mass, fi rm in consistency, progressing fast, presented a signifi cant increase, just three weeks after its appearance. Two skin lesions, arcuate, erythematous and pruritic also appeared in the dorsocervical and ventral-abdominal region. Incisional biopsy of these lesions was performed and the histopathological diagnosis confi rmed muscle and cutaneous large cell lymphoma and immunophenotype compatible with T cells (positive CD3 and negative CD79a). Due to disease advance, even during chemotherapy, a rescue protocol of L-asparaginase administration followed by lomustine and prednisone was proposed. Even with the rescue protocol there was no remission of the tumors and the case was classifi ed as progressive. The animal of this report died after completing the fi rst cycle of chemotherapy protocol, with a survival of 92 days. Discussion: Despite the fact that clinical behavior of primary lymphoma in dogs’ skeletal muscle is unknown, it is believed that, as in humans, it can be associated with chronic infl ammation or neoplastic cell invasion by proximity of the tumor or metastasis, which could justify the dissemination of the lymphoma reported here from the tongue to other tissues. However, appearance of concurrent independent lymphomas cannot be ruled out. As observed in the three cases of primary muscular lymphoma, the dog of this report had low response to therapy and short survival. This report presents the fi rst case of lymphoma in tongue with subsequent skin and left forelimb skeletal muscle involvement described in the literature. The clinical outcome corroborates the aggressiveness of muscular lymphoma observed in the other reports and also suggests that both tongue and other skeletal muscle tumors should be included in the differential diagnosis of canine lymphoma.

High numbers of DC-SIGN+ dendritic cells in lesional skin of cutaneous T-cell lymphoma

The role of dendritic cells (DCs) in disease progression of primary cutaneous T-cell lymphoma (CTCL) is not well understood. With their unique ability to induce primary immune responses as well as immunotolerance, DCs play a critical role in mediation of anti-tumor immune responses. Tumor-infiltrating DCs have been determined to represent important prognostic factors in a variety of human tumors.

Specific nail alterations in cutaneous T-cell lymphoma: successful treatment with topical mechlorethamine

Cutaneous T-cell lymphoma can be associated with clinically significant nail alterations, the presentation of which can be protean and misleading. To date, only a few reports have demonstrated direct specific tumor infiltration of the nail bed, while little is known about the efficacy of topical treatments.

Telomerase-specific GV1001 peptide vaccination fails to induce objective tumor response in patients with cutaneous T cell lymphoma

There is currently no curative therapy for cutaneous T cell lymphoma (CTCL). New therapies are therefore needed. Telomerase, the enzyme that allows for unrestricted cell divisions of cancer cells, is a promising target for cancer therapy. The telomerase-specific peptide vaccination GV1001 has shown promising results in previous studies. Since telomerase is expressed in malignant cells of CTCL, GV1001 vaccination in CTCL is a promising new therapeutic approach.

Primary cutaneous CD4+ small-/medium-sized pleomorphic T-cell lymphoma: a cutaneous nodular proliferation of pleomorphic T lymphocytes of undetermined significance? A study of 136 cases

Patients with skin nodules characterized by the infiltrate of pleomorphic small/medium T lymphocytes are currently classified as "primary cutaneous CD4+ small-/medium-sized pleomorphic T-cell lymphoma" (SMPTCL) or as T-cell pseudolymphoma. The distinction is often arbitrary, and patients with similar clinicopathologic features have been included in both groups. We studied 136 patients (male:female = 1:1; median age: 53 years, age range: 3-90 years) with cutaneous lesions that could be classified as small-/medium-sized pleomorphic T-cell lymphoma according to current diagnostic criteria. All but 3 patients presented with solitary nodules located mostly on the head and neck area (75%). Histopathologic features were characterized by nonepidermotropic, nodular, or diffuse infiltrates of small- to medium-sized pleomorphic T lymphocytes. A monoclonal rearrangement of the T-cell receptor-gamma gene was found in 60% of tested cases. Follow-up data available for 45 patients revealed that 41 of them were alive without lymphoma after a median time of 63 months (range: 1-357 months), whereas 4 were alive with cutaneous disease (range: 2-16 months). The incongruity between the indolent clinical course and the worrying histopathologic and molecular features poses difficulties in classifying these cases unambiguously as benign or malignant, and it may be better to refer to them with a descriptive term such as "cutaneous nodular proliferation of pleomorphic T lymphocytes of undetermined significance," rather than forcing them into one or the other category. On the other hand, irrespective of the name given to these equivocal cutaneous lymphoid proliferations, published data support a nonaggressive therapeutic strategy, particularly for patients presenting with solitary lesions.

TH2 cytokines from malignant cells suppress TH1 responses and enforce a global TH2 bias in leukemic cutaneous T-cell lymphoma

PURPOSE In leukemic cutaneous T-cell lymphoma (L-CTCL), malignant T cells accumulate in the blood and give rise to widespread skin inflammation. Patients have intense pruritus, increased immunoglobulin E (IgE), and decreased T-helper (TH)-1 responses, and most die from infection. Depleting malignant T cells while preserving normal immunity is a clinical challenge. L-CTCL has been variably described as a malignancy of regulatory, TH2 and TH17 cells. EXPERIMENTAL DESIGN We analyzed phenotype and cytokine production in malignant and benign L-CTCL T cells, characterized the effects of malignant T cells on healthy T cells, and studied the immunomodulatory effects of treatment modalities in patients with L-CTCL. RESULTS Twelve out of 12 patients with L-CTCL overproduced TH2 cytokines. Remaining benign T cells were also strongly TH2 biased, suggesting a global TH2 skewing of the T-cell repertoire. Culture of benign T cells away from the malignant clone reduced TH2 and enhanced TH1 responses, but separate culture had no effect on malignant T cells. Coculture of healthy T cells with L-CTCL T cells reduced IFNγ production and neutralizing antibodies to interleukin (IL)-4 and IL-13 restored TH1 responses. In patients, enhanced TH1 responses were observed following a variety of treatment modalities that reduced malignant T-cell burden. CONCLUSIONS A global TH2 bias exists in both benign and malignant T cells in L-CTCL and may underlie the infectious susceptibility of patients. TH2 cytokines from malignant cells strongly inhibited TH1 responses. Our results suggest that therapies that inhibit TH2 cytokine activity, by virtue of their ability to improve TH1 responses, may have the potential to enhance both anticancer and antipathogen responses.