Hypoxia-inducible factor 1-alpha and vascular endothelial growth factor in cartilage tumors.

Neoangiogenesis has been demonstrated in chondrosarcoma. Anti-angiogenic therapies are being tested in clinical trials for chondrosarcomas. Studies of the underlying mechanisms have been performed almost exclusively in cell lines. We immunostained 20 samples of chondrosarcoma and 20 samples of enchondromas with antibodies against hypoxia-inducible factor 1-alpha (HIF-1-alpha) and vascular endothelial growth factor (VEGF). The immunohistochemical staining of HIF-1-alpha and VEGF were highly correlated. Enchondromas were HIF-1-alpha and VEGF negative, whereas all chondrosarcoma exhibited HIF-1-alpha and VEGF immunostaining. HIF-1-alpha/VEGF double positive cases were almost exclusively chondrosarcomas with a high tumor grade. We suggest that HIF-1-alpha is a marker of malignancy in chondrosarcomas that correlates with tumor neo-angiogenesis. Our findings also suggest that a HIF-1-alpha/VEGF angiogenic pathway may exist in chondrosarcoma in vivo as in other malignant tumors. The inclusion of novel inhibitors to HIF-1-alpha and other factors may optimize anti-angiogenic interventions in chondrosarcoma.

A real-world study focused on the long-term efficacy of mycophenolate mofetil as first-line treatment of autoimmune hepatitis.

BACKGROUND: Front-line therapy with mycophenolate mofetil (MMF) in autoimmune hepatitis (AIH) has shown high on-treatment remission rates. AIM: To study prospectively in a real-world fashion the long-term outcome of a large group of consecutive treatment-naïve AIH patients. METHODS: Between 2000 and 2014, 158 patients were recruited but only 131 were eligible for treatment (109 MMF/prednisolone; 22 prednisolone ± azathioprine). Long-term data on outcome after drug withdrawal were evaluated. Patients stopped treatment after having achieved complete response (normal transaminases and IgG) for at least the last 2 years. RESULTS: At diagnosis, 31.6% of patients had cirrhosis and 72.8% insidious presentation. A total of 102 of 109 (93.6%) responded initially to MMF within 2 (1-18) months. A total of 78 of 109 (71.6%) had complete response on treatment and 61 of 78 (78.2%) maintained remission off prednisolone. MMF-treated patients had increased probability of complete response compared to those receiving azathioprine (P = 0.03). Independent predictors of complete response were lower ALT at 6 months (P = 0.001) and acute presentation (P = 0.03). So far, treatment withdrawal was feasible in 40/109 patients and 30 (75%) are still in remission after 24 (2-129) months. Remission maintenance was associated with longer MMF treatment (P = 0.005), higher baseline ALT (P < 0.02), lower IgG on 6 months (P = 0.004) and histological improvement. CONCLUSIONS: Mycophenolate mofetil proved to be an efficient first-line treatment for AIH, achieving so far the highest rates of remission maintenance off treatment (75%) ever published for at least a median of 2 years, although the remission criteria used were strict. However, the risk of potential bias and overestimation of intervention benefits from MMF cannot be completely excluded as this is a real world and not a randomised controlled trial.

Aurora B kinase in Hodgkin lymphoma: immunohistochemical pattern of expression in neoplastic Hodgkin and Reed-Sternberg cells.

Aurora B is a member of the chromosomal passenger complex, which is essential for proper completion of mitosis and cell division (cytokinesis). Inappropriate chromosomal segregation and cytokinesis due to deregulated expression of chromosome passenger proteins may lead to aneuploidy and cancer including lymphomas. According to our knowledge there are extremely limited studies investigating the immunohistochemical expression of Aurora B in tumor specimens of Hodgkin lymphoma. Our purpose was to characterize the expression of Aurora B in biopsies of Hodgkin lymphomas, and to evaluate the pattern of immunoreactivity in neoplastic Hodgkin and Reed-Sternberg cells (RS cells). We examined Aurora B immunoreactivity in paraffin sections of 15 samples of Hodgkin lymphomas, obtained from 15 patients, 8 men and 7 women. Ten were of nodular sclerosis type and five were of mixed cellularity. Our results showed immunoexpression of Aurora B in mononuclear lymphoid cells as well as in bi- and multinucleated RS cells. In addition, positive neoplastic cells in mitosis were observed, whereas a subpopulation without evidence of immunoreaction was also detected in each case. Taken together our results point to a possible association between Aurora B expression and mitotic deregulation in Hodgkin lymphoma, which may provide novel targets for treatment.

Review article: autoimmune hepatitis -- current management and challenges.

BACKGROUND: Autoimmune hepatitis (AIH) is a disease of unknown aetiology characterised by interface hepatitis, hypergammaglobulinaemia, circulating autoantibodies and a favourable response to immunosuppression. AIM: To review recent advancements in understanding aetiopathogenesis, clinical, serological and histological features, diagnostic criteria and treatment strategies of AIH. METHODS: Published studies on AIH extracted mainly from PubMed during the last 15 years. RESULTS: Autoimmune hepatitis has a global distribution affecting any age, both sexes and all ethnic groups. Clinical manifestations are variable ranging from no symptoms to severe acute hepatitis and only seldom to fulminant hepatic failure. Autoimmune attack is perpetuated, possibly via molecular mimicry mechanisms, and favoured by the impaired control of regulatory T-cells. A typical laboratory finding is hypergammaglobulinaemia with selective elevation of IgG, although in 15-25% of patients - particularly children, elderly and acute cases - IgG levels are normal. Liver histology and autoantibodies, although not pathognomonic, still remain the hallmark for diagnosis. Immunosuppressive treatment is mandatory and life-saving; however, to meet strict response criteria, the conventional therapy with prednisolone with or without azathioprine is far from ideal. CONCLUSIONS: Autoimmune hepatitis remains a major diagnostic and therapeutic challenge. The clinician, the hepato-pathologist and the laboratory personnel need to become more familiar with different expressions of the disease, interpretation of liver histology and autoimmune serology. According to the strict definition of treatment response issued by the 2010 AASLD guidelines, many patients are nonresponders to conventional treatment. Newer immunosuppressive agents targeting pathogenetic mechanisms can improve patient management, which needs to be tailored on a case-by-case basis.

Comparative study of spatial localization of HER-2 and CEP17 signals and of HER-2/CEP17 ratios, in "thin" and "thick" tissue sections.

One presumed drawback of performing fluorescence in situ hybridization on routine tissue sections for HER-2 status evaluation in breast carcinomas is nuclear truncation. Therefore, HER-2/CEP 17 ratios were compared in routine (4 µm) vs. thicker (15 µm) tissue sections. Additionally, the distances of both signals from the nuclear center were measured by three-dimensional image analysis. HER-2 and CEP 17 signals' number increased in thick sections; however, HER-2/CEP 17 ratios were decreased. This could be attributed to a preferential increase in CEP17 signals explained by their more peripheral localization and apparent "loss" in truncated nuclei. The aforementioned decrease of HER-2 ratios did not alter HER-2 status except in cases in the equivocal category where it changed from equivocal to non-amplified. Thus, at least a subset of the equivocal cases could represent an artifactual increase of HER-2 ratio related to nuclear truncation and loss of peripheral CEP 17 signals in routine sections.

Hypoxia inducible factor-1 alpha and vascular endothelial growth factor in biopsies of small cell lung carcinoma.

Neoangiogenesis has been documented in small cell lung carcinoma (SCLC). In addition, antiangiogenic therapies are being tested in clinical trials that involve SCLC. However, study of the underlying mechanisms has been performed almost exclusively in cell lines. In the current study, we immunostained 30 biopsy samples of SCLC with antibodies to hypoxia inducible factor-1 alpha (HIF-1 alpha), vascular endothelial growth factor (VEGF), vascular endothelial growth factor-receptor 1 (VEGF-R1/flt-1) and vascular endothelial growth factor-receptor 2 (VEGF-R1/flk-1). The immunoreactivity was analyzed using a bivariate Spearman correlation test and linear regression analysis. We found significant correlation between HIF-1 alpha nuclear staining and VEGF staining. Moreover HIF-1 alpha+/VEGF+ cases were associated with poor survival. We also found a positive correlation between VEGF and VEGF-R2 expression. We suggest that a HIF-1 alpha/VEGF angiogenic pathway may exist in vivo in SCLC, similar to that in non-SCLC. Our data also suggest a potential VEGF/VEGFR-2 autocrine pathway in SCLC. The inclusion of novel inhibitors to HIF-1 alpha and other factors may optimize antiangiogenic interventions in SCLC.

A case of membranous nephropathy associated with Sjögren syndrome, polymyositis and autoimmune hepatitis.

Sjögren syndrome (SS) is a chronic systemic autoimmune disease characterized by lymphocytic infiltration of exocrine glands, especially lacrimal and salivary. The immunologic process which occurs in this syndrome is B cell hyperactivity, which results in production of autoantibodies and immune complexes. SS can exist as a primary disorder or in association with other autoimmune processes. A usually mild, proximal and insidious inflammatory myopathy can occur in patients with SS with a broad clinical and pathological spectrum. Interstitial nephritis with mild proteinuria and tubular dysfunction is the most common renal manifestation of SS, but glomerular involvement due to immune complex deposition may also rarely occur [Goules et al. 2000]. There is an association of SS with hepatic abnormalities, as evidenced by abnormal liver biochemical tests or histological characteristics of primary biliary cirrhosis (PBC), portal tract fibrosis, or autoimmune hepatitis [Abraham et al. 2004]. The pathogenetic mechanism of liver involvement in SS is not clear, but it is possible that hepatic and salivary gland damage share a similar pathology. The combination of Sjögren syndrome with kidney, liver and muscle involvement in one entity is extremely rare and data in the literature are remarkably sparse. We present a case of a 43-year-old female patient suffering from SS accompanied by polymyositis, membranous nephropathy and autoimmune hepatitis.

High-risk human papillomavirus (HPV) in parotid lesions.

Although several studies have reported that oropharyngeal infection with HPV may predispose to tumorigenesis, little is known about the etiological factors of salivary gland tumors and the presence of HPV. We studied 9 parotid lesions for HPV infection including an oncocytoma, an acinic cell carcinoma, a high-grade adenocarcinoma, a low-grade polymorphous adenocarcinoma, a Warthin's tumor and 2 pleomorphic adenomas, a lymphoepithelial cyst and a lipoma of the parotid gland. DNA was extracted from formalin-fixed and paraffin-embedded tissue sections. Solution PCR for HPV detection was performed using the GP5+/GP6+ primers, while HPV typing was carried out by multiplex PCR for HPV6, 11, 16, 18, and 33; positive samples were recorfirmed by PCR with specific primers for each type. Quantitative real-time PCR for the high-risk HPV genotypes 16, 18, 31, 33, 35, 52, 58 and 67 was also performed to quantitate the viral load. Finally, in situ PCR was employed with HPV16-specific primers by direct-detection method. Seven of the 9 parotid lesions were HPV positive while 6 of these 7 had been infected by HPV16 and/or HPV18 oncogenic types. High viral load of highrisk genotypes of HPV was found in the oncocytoma, in one of the pleomorphic adenomas, and in the Warthin's tumor. Finally, in situ PCR indicated that HPV16 amplification occurred in the salivary gland tumors. This is the first time that highrisk HPV genotypes are detected in these histological types of parotid lesions, suggesting the possible involvement of the virus in the disease.

Clinical significance of organ- and non-organ-specific autoantibodies on the response to anti-viral treatment of patients with chronic hepatitis C.

BACKGROUND: Development of organ- and non-organ-specific autoantibodies has been reported in hepatitis C virus patients treated with interferon-alpha plus/minus ribavirin. AIMS: To address whether prevalence and the titre of gastric parietal autoantibodies and non-organ-specific autoantibody in hepatitis C virus-treated patients were affected by therapy, and if the development of these antibodies carries any clinical significance on the response to treatment, as few studies in adults have been strictly designed to address the above hypothesis. METHODS: Samples at three time-points (baseline, end of treatment, end of follow-up) from 102 hepatitis C virus patients (39 sustained responders, 26 relapsers, 33 non-responders; four lost in follow-up) were studied for gastric parietal autoantibodies and/or non-organ-specific autoantibody by indirect immunofluorescence, commercial and in-house enzyme-linked immunosorbent assays. RESULTS: Sustained virological and biochemical response was associated with antinuclear antibody absence (end of treatment or end of follow-up), decrease of smooth-muscle antibody titres during therapy and gastric parietal autoantibodies negativity at baseline. However, after multivariate analysis only antinuclear antibody positivity at the end of treatment and increase of smooth-muscle antibody titres were associated with worst treatment response, independently of known factors of worst treatment outcome. CONCLUSIONS: We were able to demonstrate a negative correlation between the efficacy of anti-viral treatment for hepatitis C virus and the presence of antinuclear antibody and smooth-muscle antibody before treatment, or their increase during therapy.

Epidermal growth factor receptor expression in invasive thymoma.

PURPOSE: Epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein with intrinsic tyrosine kinase activity. Activation results in a variety of cellular responses including cell proliferation and differentiation. In clinical trials, anti-EGFR is showing promise in the treatment of solid tumors expressing EGFR. Thus, we assessed EGFR expression in a series of thymic epithelial tumors. METHODS: Tumors from 37 patients seen at Indiana University School of Medicine (IUMC) for treatment of thymoma (31 patients) or thymic carcinoma (six patients) were assessed for EGFR expression. Five-micron sections of formalin-fixed, paraffin-embedded tumor (28 invasive and/or metastatic thymomas, six thymic carcinomas, and three non-invasive thymomas) were stained with anti-EGFR. Any degree of cytoplasmic membrane staining of tumor cells was considered positive; furthermore, staining was scored 0 to 3+ using criteria as standardized for HER-2/neu assessment of breast carcinoma. Appropriate controls were performed. RESULTS: Positive staining of tumor was observed in 28 tumors (23 invasive and/or metastatic thymomas, two thymic carcinomas, and three non-invasive thymomas). CONCLUSIONS: EGFR is expressed in a high percentage of thymic epithelial tumors. EGFR is often strongly expressed and is a potential therapeutic target in patients with malignant thymic tumors. We are pursuing additional studies to assess anti-EGRF in the treatment of patients with advanced thymoma.

Cholangiocytic apoptosis in chronic ductopenic rejection.

The significance of cholangiocytic apoptosis as a mechanism of ductopenia in liver rejection remains controversial. In a previous study, the presence but not the extent of ductal apoptosis was assessed by electron microscopy. Other previously published studies using an in situ hybridization method (in situ end labeling) produced conflicting results (no apoptosis v massive apoptosis). We studied 47 liver needle biopsies from 8 patients with chronic ductopenic rejection confirmed by pathologic examination of the failed grafts. These biopsies were performed because of graft dysfunction, during a period of several months before retransplantation, and they showed cholangiocytic injury with progressive ductal paucity. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling (TUNEL) was used to detect apoptosis (tissue digestion with proteinase K 20 microg/mL for 20 minutes). The interlobular bile ducts did not show labeling, even in lymphocytic cholangitis with obvious epithelial injury. However, there was minimal staining of ductular nuclei. Lymphocytic nuclei were also labeled. Apoptosis was not detectable in the vanishing interlobular bile ducts, even when more representative samples were studied and a more sensitive method was used. Unless apoptosis of cholangiocytes is an exceptionally rapid process escaping detection by conventional methods, ductopenia results mainly from ordinary, nonprogrammed cholangiocytic death. Apoptosis could still be involved in the pathogenesis of ductopenia by depleting cholangiocytic precursors, generally presumed to reside in ductules. This is a possible mechanism suggested by the following: (1) the established role of apoptosis in the homeostatic control of immature/progenitor cells, (2) the paucity of ductular proliferation in chronic rejection, (3) the previously reported decrease of ductular bcl-2 expression in rejection, and (4) the sporadic ductular TUNEL labeling seen in this study.

Vitronectin in the cirrhotic liver: an immunomarker of mature fibrosis.

Vitronectin (Vn) is a multifunctional plasma glycoprotein produced by hepatocytes. Vn has been studied extensively as a cell adhesion molecule. However, its localization in the hepatic extracellular matrix has received relatively little attention. Cryosections of 5 normal liver samples and of 20 specimens showing posthepatitic cirrhosis were stained by the avidin-biotin complex method with a well-characterized monoclonal antibody to Vn. The extent and intensity of immunostaining were assessed semiquantitatively (0, no staining; 1+, weak focal staining; 2+, strong focal staining; 3+, strong diffuse staining). Paraffin sections from the same samples were stained with Masson trichrome (MT) and Shikata orcein (Or) methods. Frozen samples from selected cases were analyzed by Western blotting. In the normal liver, 3+ staining was limited to portal vessels. The portal tract connective tissue showed minimal staining (0 to 1+). Cirrhotic septa showed strong staining (2+). Septa lacking significant inflammation and composed of dense connective tissue, as indicated by MT and Or stains, showed the strongest Vn reactions (3+). Immunoblotting data strongly correlated with Vn increase in cirrhotic livers. Vn immunoreactivity is markedly increased in the cirrhotic liver matrix, regardless of the documented decrease in plasma Vn. Binding to collagen, elastin, and proteoglycans is the current favored mechanism of Vn deposition in tissues. Previous studies in cirrhotic patients showed increased affinity of plasma Vn to collagen. Vn is also increased in aged skin, associated with dermal elastic fibers. In other tissues, Vn deposition reflects chronicity of injury. Therefore, Vn immunoreactivity in liver can be considered a marker of fibrosis, especially of chronic/mature fibrosis, paralleling previous observations on enhanced orcein staining of cirrhotic septa. Immunolabeling of biopsy specimens with Vn and tenascin, a marker of ongoing remodeling or recently formed fibrous tissue, could be diagnostically helpful.

Enhanced tenascin expression correlates with inflammation in primary sclerosing cholangitis.

Tenascin (Tn) is an extracellular matrix (ECM) glycoprotein upregulated during development, repair and oncogenesis. In the normal adult liver, Tn is limited to vessels and, focally, to sinusoidal walls. In this study, samples were obtained from 12 livers removed during transplantation for primary sclerosing cholangitis (PSC). Paraffin sections were immunostained with monoclonal antibodies BC-4 which recognizes all isoforms of Tn and alpha-SMA-1 to alpha smooth muscle actin (alpha-SMA). Intense Tn reactions were noted in areas of ductular proliferation and inflammation at the parenchyma-stroma interface. In the absence of ductular proliferation, no selective Tn upregulation was noted. Staining was preferentially located adjacent to ductular basement membranes, with minimal extension into the surrounding ECM. Advanced histologic stages with micronodules rimmed by proliferating ductules showed the most florid Tn reactions, whereas fibrous septa and edematous perinodular haloes did not react. Increased periductal Tn was also seen associated with active inflammation, notably around large, dilated septal ducts, while fibro-obliterative ductal lesions and "onion skin fibrosis" did not stain. Focally enhanced Tn staining was noted in sinusoids neighboring ductular proliferation, and in dilated sinusoids within cirrhotic nodules. Reactions with alpha-SMA-1 highlighted myofibroblasts and activated Ito cells in topographic association with Tn reactions. We conclude that Tn is upregulated in PSC where it is preferentially localized in the remodeling matrix encompassing proliferating ductules and in altered periductal matrix. Our results suggest that Tn determinations in tissue or serum samples might be helpful in the clinical assessment of "activity" in PSC.

Immunolocalization of integrins in the normal lung and in pulmonary carcinomas.

Cryosections of normal adult lung (n = 7) and pulmonary epithelial tumors, including squamous (n = 8), adeno (n = 8), bronchioloalveolar (n = 5), and large cell (n = 4) carcinomas (SCC, ACC, BAC, LCC), carcinoids (Cd, n = 7), and neuroendocrine carcinomas (NEC) of variable grades (n = 14) were immunostained by the avidin-biotin peroxidase (ABC) method with monoclonal antibodies to the alpha1-6 and alpha(v) and the beta1-4 integrin subunits. Normal adult alveolar septae showed variably intense immunoreactivity for alpha1,3,6 and beta1, whereas reactions for alpha5 and alpha(v) were weaker and uneven; the remaining integrin subunits were not detected. Bronchial and bronchiolar epithelium showed variably intense staining for alpha2.3,6,v and beta1,4. Reactions were often, though not invariably, basally polarized. SCC, ADC, and LCC showed variably intense reactions for alpha2.3,6,v and beta1,4. BAC were strongly and uniformly stained for alpha1.3 and beta1. In Cd, alpha1,2,3,v and beta1 reactions were noted, whereas in NEC, weak alpha1,3 and beta1 staining was detected with only traces of alpha6 and alpha(v). We conclude that alveolar epithelial cells do not express the hemidesmosome-associated, laminin-binding integrin alpha6beta4 of the bronchial epithelium but rather the alpha1beta1 and alpha3beta1, collagen IV, and laminin receptors, respectively. SCC, ADC, and sampled LCC express an integrin repertory qualitatively similar to that of the bronchial epithelium. Distinct from the latter, the integrin repertory of BAC parallels that of the alveolar epithelium by its strong expression of the multipotential alpha1beta1 and alpha3beta1 integrins. NEC tumors do not display the laminin receptors alpha6beta4 and alpha6beta1 shown by SCC and ADC but express instead alpha1beta1, a collagen IV-laminin receptor rarely found in epithelial neoplasms except for BAC. In NEC tumors, integrins, especially alpha2, decrease with dedifferentiation. Notably distinct from epithelial mesotheliomas, the major fibronectin-binding integrin alpha5beta1 was not found in any type of lung carcinoma.

Pleural mesotheliomas have an integrin profile distinct from visceral carcinomas.

Cryosections of epithelial, sarcomatoid, and biphasic malignant mesotheliomas (EMM, n = 11; SMM, n = 5; BMM, n = 6) of the pleura were immunostained with monoclonal antibodies to integrin subunits alpha 1-6 and v, and beta 1-4. Localization patterns were compared with those known to occur in pulmonary and other adenocarcinomas (PADC, ADC). EMM and the epithelial component of BMM (ecBMM) expressed alpha 1,3,5,6, and v and beta 1 and 4. SMM and the sarcomatoid elements of BMM (scBMM) reacted variably for alpha 1,3,5,6 and v, and beta1. Reactions for alpha3, found in all tumors, were strongest in EMM, ecBMM, and PADC. Our findings indicate that EMM and ecBMM parallel PADC and most ADC in their expression of alpha6 beta4, underscoring that this laminin integrin receptor is intimately associated with these neoplastic epithelial phenotypes. Also, our observations on alpha3 beta1 suggest that this cell-cell adhesion-mediating integrin is related to the epithelial phenotype. Notably, all malignant mesotheliomas (MM), including those with distinct glandular structures, expressed the alpha5 beta1 fibronectin receptor, thus paralleling most sarcomas and differing from PADC and most other ADC. We conclude that irrespective of architectural and cytologic variants, transformed mesothelial cells possess an integrin repertory that differs significantly from that of most ADC, including those of the lung. These findings set mesothelium apart from epithelia and may prove helpful as adjunct tools for the differential diagnosis between EMM and AD.

Immunolocalization of cellular fibronectins in the normal liver, cirrhosis, and hepatocellular carcinoma.

Cellular (c) fibronectins (Fn) differ biochemically, immunologically, and functionally from plasma fibronectins (pFn). Most existing data on Fn distribution in the normal and diseased liver require revision because those studies were based on reagents that did not distinguish pFn from cFn and predated the development of specific cFn monoclonal antibodies (Mabs). We immunostained cryosections of normal adult livers (n = 5), cirrhotic livers (n = 20), and livers with hepatocellular carcinoma (HCC) (n = 10) by the avidin-biotin-complex method with specific Mabs to the extradomains A and B (EDA, EDB) and oncofetal (Onc) isoforms of cFn. Selected samples were stained with an antiserum to pFn; fetal livers served as controls. Normal and cirrhotic livers showed EDA-cFn staining in the portal, septal, and perisinusoidal matrix; its distribution was more restricted than that of pFn. In cirrhosis, EDA-cFn reactions were strongest at sites of piecemeal necrosis and around proliferating ductules in biliary cirrhosis. EDA-cFn reactions were consistently most intense in the matrix of HCC. Distinct from adult normal and cirrhotic livers, reactions for EDB- and Onc-cFn were noted exclusively in most cases of HCC. We conclude that the only cFn isoform indigenous to the normal adult liver matrix is EDA-cFn. Enhanced EDA-cFn in cirrhotic livers may serve as indicator of active stromal remodeling. The restriction of EDB- and Onc-cFn to a large subset of HCC and the putative role of cFn in modulating cell-matrix adhesive interactions would suggest that the emergence of these molecules may be related to the variably invasive and metastatic properties of these tumors.