Determinação da relação de novo biomarcador (IMP3) no carcinoma de nasofaringe

Pós-graduação em Patologia - FMB

Investigação da evolução dos pólipos endometriais em câncer de endométrio

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Transforming growth factor alpha immunoreactivity. A study in hepatocellular carcinoma and in non-neoplastic liver tissue

Background. Transforming growth factor alpha (TGF alpha) is an important mitogen that binds to epidermal growth factor receptor and is associated with the development of several tumors. Aims. Assessment of the immunoexpression of TGF alpha in hepatocellular carcinoma (HCC) and in non-neoplastic liver tissue and its relationship to morphological patterns of HCC. Material and methods. The immunohistochemical expression of TGF alpha was studied in 47 cases of HCC (27 multinodular, 20 nodular lesions). Five lesions measured up to 5 cm and 15 lesions above 5 cm. Thirty-two cases were graded as I or II and 15 as III or IV. The non-neoplastic tissue was examined in 40 cases, of which 22 had cirrhosis. HBsAg and anti-HCV were positive in 5/38 and 15/37 patients, respectively. The statistical analysis for possible association of immunostaining of TGF alpha and pathological features was performed through chi-square test. Results. TGF alpha was detected in 31.9% of the HCC and in 42.5% of the non-neoplastic. There was a statistically significant association between the expression of TGF alpha and cirrhosis (OR = 8.75, 95% CI = [1.93, 39.75]). The TGF alpha was detected more frequently in patients anti-HCV(+) than in those HBsAg(+). The immunoexpression of TGF alpha was not found related to tumor size or differentiation. In conclusion the TGF alpha is present in hepatocarcinogenesis in HBV negative patients. Further analysis is needed to examine the involvement of TGF alpha in the carcinogenesis associated with HCV and other possible agents. In addition, TGF alpha has an higher expression in hepatocyte regeneration and proliferation in cirrhotic livers than in HCC.

Cellular Infiltrates and NF kappa B Subunit c-Rel Signaling in Kidney Allografts of Patients With Clinical Operational Tolerance

Background. Nuclear factor kappa B (NF kappa B) plays a potential role in tolerance by orchestrating onset and resolution of inflammation and regulatory T cell differentiation through subunit c-Rel. We characterized cellular infiltrates and expression of NF kappa B1, c-Rel and its upstream regulators phosphatidylinositol 3-kinase/RAC-alpha serine/threonine kinase, in allograft biopsies from patients with spontaneous clinical operational tolerance (COT). Methods. Paraffin-fixed kidney allograft biopsies from 40 patients with COT (n=4), interstitial rejection (IR; n=12), borderline changes (BC; n=12), and long-term allograft function without rejection (NR; n=12) were used in the study. Cellular infiltrates and immunohistochemical expression of key proteins of the NF kappa B pathway were evaluated in the cortical tubulointerstitium and in cellular infiltrates using digital image analysis software. Results were given as mean +/- SEM. Results. Biopsies from patients with COT exhibited a comparable amount of cellular infiltrate to IR, BC, and NR (COT, 191 +/- 81; IR, 291 +/- 62; BC, 178 +/- 45; and NR, 210 +/- 42 cells/mm(2)) but a significantly higher proportion of forkhead box P3-positive cells (COT, 11%+/- 1.7%; IR, 3.5%+/- 0.70%; BC, 3.4%+/- 0.57%; and NR, 3.7%+/- 0.78% of infiltrating cells; P=0.02). c-Rel expression in cellular infiltrates was significantly elevated in IR, BC, and NR when analyzing the number of positive cells per mm(2) (P=0.02) and positive cells per infiltrating cells (P=0.04). In contrast, tubular PI3K and c-Rel expression were significantly higher in IR and BC but not in NR compared with COT (P=0.03 and P=0.006, respectively). With RAC-alpha serine-threonine kinase, similar tendencies were observed (P=0.2). Conclusions. Allografts from COT patients show significant cellular infiltrates but a distinct expression of proteins involved in the NF kappa B pathway and a higher proportion of forkhead box P3-positive cells.

Prognostic significance of CD24 and claudin-7 immunoexpression in ductal invasive breast cancer

This study aimed to identify the CD24 and CD44 immunophenotypes within invasive ductal breast carcinoma (I DC) subgroups defined by immunohistochesmistry markers and to determine its influence on prognosis as well as its association with the expression of Ki-67, cytokeratins (CK5 and CK 18) and claudin-7. Immunohistochemical expression of CD44 and CD24 alone or in combination was investigated in 95 IDC cases arranged in a tissue microarray (TMA). The association with subgroups defined as luminal A and B; HER2 rich and triple negative, or with the other markers and prognosis was analyzed. CD44(+)/CD24(-) and CD44(-)/CD24(+) were respectively present in 8.4% and 16.8% of the tumors, a lack of both proteins was detected in 6.3%, while CD441(-)/CD24(+) was observed in 45.3% of the tumors. Although there was no significant correlation between subgroups and different phenotypes, the CD44(+)/CD24(-) phenotype was more common in the basal subgroups but absent in HER2 tumors, whereas luminal tumors are enriched in CD44(-)/CD24(+) and CD44(+)/CD24(+) cells. The frequency of CD44(+)/CD24(-) or CD44(-)/CD24(+) was not associated with clinical characteristics or biological markers. There was also no significant association of these phenotypes with the event free (DFS) and overall survival (OS). Single CD44(+) was evident in 57.9% of the tumors and was marginally associated to grading and not to any other tumor characteristics as well as OS and DFS. CD24(+) was positive in 74.7% of the tumors, showing a significant association with estrogen receptor, progesterone receptor and Ki-67 and a marginal association with CKI8 and claudin-7. Expression of claudin-7 and Ki-67 did not associate with the cancer subgroups, while a positive association between CK18 and the luminal subgroups was found (P=0.03). CK5, CK18 and Ki-67 expression had no influence in OS or DFS. Single CD24(+) (P=0.07) and claudin-7 positivity (P=0.05) were associated with reduced time of recurrence, suggesting a contribution of these markers to aggressiveness of breast cancer.

Matrix metalloproteinases, tissue inhibitors of metalloproteinases, and growth factors regulate the aggressiveness and proliferative activity of keratocystic odontogenic tumors

Objective. The objective of this preliminary study was to evaluate the expression of matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs) and growth factors in keratocystic odontogenic tumors (KOTs). Study Design. The expression of MMPs, TIMPs, growth factors, and the extracellular signal-regulated kinase (ERK) 1/2 signaling pathway were assessed by immunohistochemistry in 15 cases of KOT and 4 cases of calcifying cystic odontogenic tumor (CCOT). Results. KOT samples expressed significantly higher amounts of MMPs, TIMPs, growth factors, epidermal growth factor receptor (EGFR), and ERK compared with CCOT samples, with the exception of MMP-2 and TIMP-1. Conclusions. MMP-9, TIMP-2, EGF and transforming growth factor alpha act together and likely regulate the proliferation and aggressiveness of KOT. ERK-1/2 serves as the transducer of signals generated by these proteins, which signal through the common receptor, EGFR. This process may be related to the increased proliferation and aggressiveness observed in KOT. (Oral Surg Oral Med Oral Pathol Oral Radiol 2012;114:487-496)

Nitric Oxide Synthase in Heart and Thoracic Aorta After Liver Ischemia and Reperfusion Injury: An Experimental Study in Rats

Objectives: We tested the effects of liver reperfusion in the immunohistochemical expression of nitric oxide synthase on the thoracic aorta and the heart. Materials and Methods: We randomized 24 male Wistar rats into 3 groups: (1) control; (2) R2 group, with 60 minutes of partial (70%) liver ischemia and 2 hours of global liver reperfusion; (3) and R6 group, with 60 minutes of partial liver ischemia and 6 hours of global liver reperfusion. Results: In the heart, there was little, diffuse immunohistochemical endothelial staining; immunohistochemical inducible nitric oxide synthase staining was expressed in the adventitia layer of intramyocardial vessels in both cases, with a time-dependent but not statistically significant increase. In the thoracic aorta, a time-dependent decrease in endothelial nitric oxide synthase expression in the muscular layer after reperfusion, which was statistically significant in R6 versus the control. Positive immunostaining for inducible nitric oxide synthase was seen in the muscular and endothelial layers, and this varied from moderate in the control group, to light in the endothelium in groups R2 and R6. Conclusions: We observed changes that may be implicated in heart injury and impairment of aortal tone after liver ischemia and reperfusion injury.

Pamidronate for the treatment of osteoporosis secondary to chronic cholestatic liver disease in Wistar rats

Osteoporosis is a major complication of chronic cholestatic liver disease (CCLD). We evaluated the efficacy of using disodium pamidronate (1.0 mg/kg body weight) for the prevention (Pr) or treatment (Tr) of cholestasis-induced osteoporosis in male Wistar rats: sham-operated (Sham = 12); bile duct-ligated (Bi = 15); bile duct-ligated animals previously treated with pamidronate before and 1 month after surgery (Pr = 9); bile duct-ligated animals treated with pamidronate 1 month after surgery (Tr = 9). Rats were sacrificed 8 weeks after surgery. Immunohistochemical expression of IGF-I and GH receptor was determined in the proximal growth plate cartilage of the left tibia. Histomorphometric analysis was performed in the right tibia and the right femur was used for biomechanical analysis. Bone material volume over tissue volume (BV/TV) was significantly affected by CCLD (Sham = 18.1 ± 3.2 vs Bi = 10.6 ± 2.2%) and pamidronate successfully increased bone volume. However, pamidronate administered in a preventive regimen presented no additional benefit on bone volume compared to secondary treatment (BV/TV: Pr = 39.4 ± 12.0; Tr = 41.2 ± 12.7%). Moreover, the force on the momentum of fracture was significantly reduced in Pr rats (Sham = 116.6 ± 23.0; Bi = 94.6 ± 33.8; Pr = 82.9 ± 22.8; Tr = 92.5 ± 29.5 N; P < 0.05, Sham vs Pr). Thus, CCLD had a significant impact on bone histomorphometric parameters and pamidronate was highly effective in increasing bone mass in CCLD; however, preventive therapy with pamidronate has no advantage regarding bone fragility.

p16 (INK4a) has clinicopathological and prognostic impact on oropharynx and larynx squamous cell carcinoma

CDKN2A encodes proteins such as p16 (INK4a), which negatively regulate the cell-cycle. Molecular genetic studies have revealed that deletions in CDKN2A occur frequently in cancer. Although p16 (INK4a) may be involved in tumor progression, the clinical impact and prognostic implications in head and neck squamous cell carcinoma (HNSCC) are controversial. The objective of this study was to evaluate the frequency of the immunohistochemical expression of p16 (INK4a) in 40 oropharynx and 35 larynx from HNSCC patients treated in a single institution and followed-up at least for 10 years in order to explore potential associations with clinicopathological outcomes and prognostic implications. Forty cases (53.3%) were positive for p16 (INK4a) and this expression was more intense in non-smoking patients (P = 0.050), whose tumors showed negative vascular embolization (P = 0.018), negative lymphatic permeation (P = 0.002), and clear surgical margins (P = 0.050). Importantly, on the basis of negative p16 (INK4a) expression, it was possible to predict a probability of lower survival (P = 0.055) as well as tumors presenting lymph node metastasis (P = 0.050) and capsular rupture (P = 0.0010). Furthermore, increased risk of recurrence was observed in tumors presenting capsular rupture (P = 0.0083). Taken together, the alteration in p16 (INK4a) appears to be a common event in patients with oropharynx and larynx squamous cell carcinoma and the negative expression of this protein correlated with poor prognosis.

Ruolo dei recettori tirosinchinasici in oncologia animale

Disregolazioni dei recettori tirosinchinasici (RTK) sono di frequente riscontro nei tumori dell’uomo e in molti casi sono indicatori biologici che permettono di definire in maniera più accurata la prognosi dei pazienti. Possono rappresentare inoltre marker predittivi per la risposta a terapie antitumorali con farmaci a bersaglio molecolare. Numerosi inibitori tirosinchinasici (TKI) sono attualmente in corso di studio o già disponibili per l’utilizzo in oncologia umana, e molti di questi hanno dimostrato una significativa efficacia utilizzati singolarmente o in combinazione a terapie convenzionali. Studi recenti indicano che un quadro analogo di disregolazione dei recettori tirosinchinasici è presente anche nelle neoplasie dei piccoli animali, e ne suggeriscono in molti casi un’implicazione prognostica. Gli inibitori tirosinchinasi sono da poco entrati nell’arena dell’oncologia veterinaria, ma i primi risultati lasciano supporre che siano destinati ad essere integrati definitivamente nei protocolli terapeutici standard. La tesi consiste in una parte introduttiva in cui sono trattate le principali funzioni biologiche dei recettori tirosinchinasici, la loro struttura e il loro ruolo nell’oncogenesi e nella progressione tumorale in medicina umana e veterinaria. Si affrontano inoltre le principali metodiche di laboratorio per l’analisi molecolare in oncologia e i meccanismi d’azione dei farmaci inibitori tirosinchinasici, con un cenno ai prodotti maggiormente utilizzati e alle loro indicazioni. Segue la presentazione e la discussione dei risultati di quattro studi relativi alla valutazione delle disregolazioni del recettore tirosinchinasico Kit (espressione aberrante e mutazioni genomiche) nel mastocitoma cutaneo del gatto e del recettore del fattore di crescita epidermico (EGFR) nel carcinoma squamocellulare cutaneo del gatto e nei tumori polmonari primitivi del cane, con particolare attenzione al loro ruolo prognostico.

The clinical impact of p16 status in fine-needle aspirates of cervical lymph node metastasis of head and neck squamous cell carcinomas

Lymph node involvement is prognostically the most determinant clinical factor for patients with head and neck squamous cell carcinomas (HNSCCs). Ultrasound of the neck and fine-needle aspiration (FNA) cytology is one of the first diagnostic procedures and the most accurate diagnostic staging tool for the neck. Patients with HPV-positive oropharyngeal carcinomas (OPSCC) show a significantly better prognosis when compared with HPV-negative OPSCC. P16 overexpression is accepted as surrogate marker for HPV-positive in OPSCC. These HPV/p16-positive OPSCC are localized either in the palatal tonsils or the base of tongue and frequently present with lymph node metastases. We analyzed the correlation and reliability of p16 expression of the FNA of the lymph node metastasis with the immunohistochemical expression of p16 of the same lymph node metastasis and its corresponding primary tumor, as it could be of importance for determining the localization and different prognosis of the primary tumor. 54 HNSCC patients were evaluated, p16 expression of the primary tumors and their lymph node metastases correlated precisely. In 25 of the 54 HNSCC patients, a FNA of the lymph node metastases was taken before the treatment. The positive cytological and immunohistochemical p16 staining correlated exactly. Of the 17 histologically p16-negative lymph node metastases 15 FNA were p16-negative, whereas two samples were p16-positive. In our view, a cytological p16 analysis of cervical lymph node metastasis can facilitate the correct localization of the primary tumor and discriminate reliably HPV-positive OPSCC from HPV-negative HNSCC with their significantly diverse prognosis.

Matrix metalloproteinase-19 is a predictive marker for tumor invasiveness in patients with oropharyngeal squamous cell carcinoma

AIMS: To evaluate the expression of matrix metalloproteinase-19 (MMP-19) in oropharyngeal squamous cell carcinoma along with its association with structural features of invasiveness. To investigate whether MMP-19 expression correlates with lymphatic or systemic metastasis and prognosis in patients who have received definitive radiotherapy. METHODS AND RESULTS: The histological evaluation of the invasive front was based on Bryne's malignancy grading system. We correlated the immunohistochemical expression pattern with morphological parameters which characterize tumor invasiveness such as keratinization, nuclear polymorphism, invasion pattern, and the host inflammatory response. Local immunoreactivity for MMP-19 was positively correlated with tumor invasiveness as reflected in its structural characteristics and the degree of nuclear polymorphism, and negatively correlated with the inflammatory response of the host. No correlation existed between MMP-19 expression and clinicopathological features (TNM stage, grade of differentiation) or a patient''s outcome and prognosis. CONCLUSIONS: This latter finding probably reflects the unique change for MMPs from high immunoreactivity within healthy tissue areas and non-invasive tumor parts, through absence in the least invasive neoplastic regions, to strong re-expression at a highly invasive front of the same tumor. Our findings indicate that MMP-19 can be used as a marker for tumor invasiveness in patients with oropharyngeal squamous cell carcinoma.

Suburothelial myofibroblasts in the human overactive bladder and the effect of botulinum neurotoxin type A treatment

BACKGROUND: An increasing body of evidence suggests a possible role of suburothelial myofibroblasts (MFs) in bladder mechanosensation and in the pathophysiology of detrusor overactivity (DO). OBJECTIVE: To determine whether markers of MFs, including gap junction protein connexin43 (Cx43) and c-kit have altered immunohistochemical expression in the suburothelium of patients with neurogenic DO (NDO) or idiopathic DO (IDO) and whether this is affected by successful treatment of DO with botulinum neurotoxin type A (BoNTA). DESIGN, SETTING, AND PARTICIPANTS: Patients with NDO (n=10) or IDO (n=11) were treated in a single-centre, open-label study of intradetrusor BoNTA injections. Control tissue was obtained from 10 patients undergoing pelvic-floor repair procedures who had no overactive bladder (OAB) symptoms. This study is registered with ClinicalTrials.gov, number NCT00662064. INTERVENTIONS: Bladder biopsies performed with flexible cystoscopes were obtained from control subjects and from NDO and IDO patients before BoNTA treatment and at 4 wk and 16 wk after treatment. They were studied with quantitative immunofluorescence using antibodies to connexin 43 (Cx43), vimentin, and c-kit. MEASUREMENTS: Differences in Cx43, vimentin, and c-kit immunoreactivity between control subjects and NDO or IDO patients (primary outcomes). Changes in NDO or IDO, Cx43 immunoreactivity, and c-kit immunoreactivity after BoNTA treatment (secondary outcomes). RESULTS AND LIMITATIONS: Cx43 immunoreactivity was increased in both IDO and NDO patients compared to controls, but remained unchanged after BoNTA treatment. C-kit immunoreactivity was similar in NDO/IDO patients and controls and remained unchanged after BoNTA treatment. CONCLUSIONS: Increased gap junction formation in the suburothelium has been demonstrated in biopsies from humans with DO. It is hypothesised that this change could have a significant role in the pathogenesis of the detrusor abnormality. Successful treatment of NDO or IDO does not appear to be associated with changes in the expression of Cx43 or c-kit on suburothelial MFs.

Carcinoma of the lower uterine segment: A newly described association with Lynch Syndrome

Purpose. We performed a case-comparison study to describe the characteristics of LUS tumors and their association with risk factors for endometrial cancer. ^ Patients and Methods. From January 1996 through October 2007, 3,892 women were identified with a diagnosis of primary endometrial carcinoma or primary cervical adenocarcinoma. Pathology records from the 1,009 women who had a hysterectomy were reviewed. Subjects were included in the LUS group only if the tumor was clearly originating from the area between the lower corpus and upper cervix in the hysterectomy specimen. The LUS group was compared to all patients with endometrial corpus carcinoma who underwent hysterectomy at our institution in a 12-month period randomly selected from the study period. Risk factors for endometrial carcinoma such as body mass index (BMI) and Lynch Syndrome were assessed. Expression of estrogen receptor (ER), vimentin, carcinoembryonic antigen (CEA), p16, and human papilloma virus DNA (HPV DNA) was assessed; this panel is known to be effective in distinguishing adenocarcinomas of endometrial versus endocervical origin. Fisher's Exact, Chi-square, Mann-Whitney, and Student's t-tests were utilized for statistical analysis. ^ Results. Thirty-five of 1,009 women had endometrial carcinoma of the LUS (3.5%; 95% CI: 2–4%). Compared to patients with corpus tumors, LUS patients were younger (54.2 vs. 62.9 years, P = .001), had higher stage (P < .001), and more invasive tumors (P = .001). Preoperative diagnosis of the LUS tumors more frequently included the possibility of endocervical adenocarcinoma ( P < .001), leading to preoperative radiation therapy in 4 patients. Median BMI was similar in the LUS and corpus groups. Seventy-three percent of the available LUS tumors had a similar immunohistochemical expression pattern to conventional endometrioid adenocarcinoma. Because of the young median age for the LUS group, we performed immunohistochemistry for Lynch syndrome-associated DNA mismatch repair proteins MLH1, MSH2, MSH6, and PMS2. Microsatellite instability testing (MSI) and MLH1 promoter hypermethylation were performed when indicated. Thirty-six percent of the LUS tumors were MSI-high. Ten of thirty-five (29%) women with LUS tumors were either confirmed to have Lynch Syndrome or were strongly suspected to have Lynch Syndrome based on tissue-based molecular assays (95% CI, 16 to 45%). ^ Conclusions. Endometrial carcinoma arising in the LUS is a clinical and pathologic entity which can be diagnostically confused with cervical adenocarcinoma. In general, LUS tumors can be correctly identified as being endometrial carcinoma using the immunohistochemical panel noted above. The prevalence of Lynch Syndrome in patients with LUS tumors is much greater than that of the general endometrial cancer population (1.8%) or in endometrial cancer patients younger than 50 years of age (8–9%). Based on our results, the possibility of Lynch Syndrome should be considered in women with LUS tumors. ^

Induction of solid tumor differentiation by the peroxisome proliferator-activated receptor-γ ligand troglitazone in patients with liposarcoma

Agonist ligands for the nuclear receptor peroxisome proliferator-activated receptor-γ have been shown to induce terminal differentiation of normal preadipocytes and human liposarcoma cells in vitro. Because the differentiation status of liposarcoma is predictive of clinical outcomes, modulation of the differentiation status of a tumor may favorably impact clinical behavior. We have conducted a clinical trial for treatment of patients with advanced liposarcoma by using the peroxisome proliferator-activated receptor-γ ligand troglitazone, in which extensive correlative laboratory studies of tumor differentiation were performed. We report here the results of three patients with intermediate to high-grade liposarcomas in whom troglitazone administration induced histologic and biochemical differentiation in vivo. Biopsies of tumors from each of these patients while on troglitazone demonstrated histologic evidence of extensive lipid accumulation by tumor cells and substantial increases in NMR-detectable tumor triglycerides compared with pretreatment biopsies. In addition, expression of several mRNA transcripts characteristic of differentiation in the adipocyte lineage was induced. There was also a marked reduction in immunohistochemical expression of Ki-67, a marker of cell proliferation. Together, these data indicate that terminal adipocytic differentiation was induced in these malignant tumors by troglitazone. These results indicate that lineage-appropriate differentiation can be induced pharmacologically in a human solid tumor.