Mutation analysis of the EGFR gene and downstream signalling pathway in histologic samples of malignant pleural mesothelioma.

BACKGROUND: As epidermal growth factor receptor (EGFR) is involved in the pathogenesis of malignant pleural mesotheliomas (MPMs), the anti-EGFR drugs may be effective in treating MPM patients. Mutations of the EGFR gene or its downstream effectors may cause constitutive activation leading to cell proliferation, and the inhibition of apoptosis and metastases. Consequently, molecular profiling is essential for select patients with MPM who may respond to anti-EGFR therapies. METHODS: After manual macrodissection, genomic DNA was extracted from 77 histological samples of MPM: 59 epithelioid, 10 biphasic, and 8 sarcomatoid. Epidermal growth factor receptor gene mutations were sought by means of real-time polymerase chain reaction (PCR) and direct sequencing, KRAS gene mutations by mutant-enriched PCR, and PIK3CA and BRAF gene mutations by direct sequencing. RESULTS: Gene mutations were identified in nine cases (12%): five KRAS, three BRAF, and one PI3KCA mutation; no EGFR gene mutations were detected. There was no difference in disease-specific survival between the patients with or without gene mutations (P=0.552). CONCLUSIONS: Mutations in EGFR downstream pathways are not rare in MPM. Although none of those found in this study seemed to be prognostically significant, they may support a more specific selection of patients for future trials.

Severe crescentic BK virus nephropathy with favourable outcome in a transplanted patient treated with Leflunomide.

Herein reported is a severe case of BK virus nephropathy, probably caused by an intense/overimmunosuppression and identified 17 months after transplantation. The diagnostic biopsy showed extracapillary proliferation and typical cytopathic lesions, both in tubular epithelial cells and in those of the glomerular crescents. Severe inflammatory infiltrates, tubulitis, tubular atrophy and fibrosis were also observed. Immunohistochemistry and molecular biology disclosed the presence of an AS variant of the BK virus with a high viral load, both in renal tissue and urine. Immunosuppression was reduced and Leflunomide therapy administered for a month. Although this led to an improvement in the renal function, the therapy had to be suspended due to the onset of a skin rash. A second biopsy was performed 7 months later. The cellular crescents were no longer present and there was no evidence of either histologic or immunohistochemical findings consistent with an active BK virus infection. Tubular atrophy and interstitial fibrosis were still present. In addition, fibrotic crescents, which may be interpreted as late scarring changes of previous epithelial proliferation, were found. Although molecular investigation still showed the presence of the BK virus, the viral load in renal tissue, urine and serum was greatly reduced. Indeed, serum and urine viral load was still low at the last control, five months after the second biopsy. The morphologic and clinical evolution are reported and the possible role of the therapy is discussed.

Karyomegalic interstitial nephritis: report of 3 new cases and review of the literature.

Karyomegalic interstitial nephritis is a rare, but perhaps an "underdiagnosed" condition. Peculiar nuclear changes characterize it, involving mainly tubular cells along with glomeruli and blood vessels. Herein, 3 bioptically proven new cases of patients with chronic renal failure are discussed. The first case had a recently diagnosed karyomegalic nephritis which, to date, still does not require dialysis. The other 2 (brother and sister) required dialysis 4 and 1 years after diagnosis. Karyomegalic changes were found not only in the skin and duodenal biopsies of the male, in skin and liver biopsies of the female and in the urine cells of both patients, but also in several organs (brain, thyroid, lung, esophagus, arteries) as shown at the autopsy of the female. There was a fatal outcome for both patients. The data reported in this study emphasize the usefulness of pathologic investigation of both tissue and urine samples in the identification of this disease. Moreover, as karyomegalic interstitial nephritis is strongly suspected to have a genetic background, its identification may well not only be of clinical relevance, due to its ominous outcome, but may also bear eugenetic value.

GAS6, the ligand of Axl and Rse receptors, is expressed in hematopoietic tissue but lacks mitogenic activity.

GAS6, a gene previously identified as growth arrest specific, has been demonstrated to be the ligand of Axl, a novel tyrosine kinase receptor widely expressed in both normal and neoplastic hematopoietic tissue. We have observed previously that GAS6 mRNA was present in whole bone marrow. This preliminary finding prompted us to investigate the presence of GAS6 in hematopoietic tissue and the possible role of this molecule in controlling the proliferation of hematopoietic precursors. We report here that the protein GAS6 is diffusely present in hematopoietic tissue, both in stromal and in hematopoietic cells, and that, among these cells, positivity is observed in megakaryocytes and myelomonocytic precursors. Furthermore, our data suggest that GAS6 is not a growth factor for hematopoietic progenitors or stromal fibroblasts. Despite the fact that both the Axl receptor and its ligand, GAS6, are expressed in hematopoietic tissue, the biological role of their interactions remains to be determined.

Glomerulonephritis with organized deposits: a mesangiopathic, not immune complex-mediated disease? A pathologic study of two cases in the same family.

Similar glomerular changes (marked widening of the mesangial stalk, irregular basement membrane thickening, and presence of mesangial and subendothelial deposits) were observed by light microscopy in renal biopsy specimens from two patients (mother and daughter) affected by nephrotic syndrome. Electron microscopy disclosed huge glomerular electron-dense deposits containing 12-nm fibrils in both patients. Immunohistochemical investigations performed with antisera anti-immunoglobulin (Ig) and anti-complement fractions, anti-laminin, anti-collagen IV, and anti-fibronectin (FN) showed scant and focal Ig and complement deposits and strong deposits of FN in the mesangium and along glomerular basement membranes. Most glomerular FN was plasma-derived, as shown by immunohistochemical tests with monoclonal antibodies specific for both plasma and cell-derived FN (IST-4) and for cell-derived FN (IST-9). Electron-dense deposits with fibrillar component could hardly correspond to the Ig and complement deposits, whereas they could be related to FN deposits. Since it is known that in glomeruli FN binds to Ig and immune complexes, and the latter seem to be too scant to justify light and electron microscopic lesions and clinical findings, the hypothesis of a primary mesangiopathic glomerulonephritis in some way connected with abnormal plasma FN deposition within the glomeruli and subsequent non-specific immune reactant entrapment could be considered. We could be dealing with a peculiar form of fibrillary glomerulonephritis with rather indolent evolution, as shown by a slow decrease of glomerular function and the scarcely modified glomerular changes found in the second biopsy performed in the mother 8 years after the first investigation.

JC virus in human glial-derived tumors.

To investigate the presence and the role of polyomaviruses JC (JCV), BK (BKV), and the simian polyomavirus (SV40) in human brain tumors, samples from 25 glial-derived tumors (10 astrocytomas, 5 ependymomas, 5 oligodendrogliomas, and 5 glioblastomas) were examined by means of molecular biology and immunohistochemistry. Nested PCR of the large T (LT) region and its sequence analysis showed JCV in 6 cases (4 astrocytomas, 1 oligodendroglioma, and 1 ependymoma), while the transcriptional control region (TCR) was amplified only in 1 astrocytoma, the oligodendroglioma, and the ependymoma, one of which (astrocytoma) also stained positively by immunohistochemistry (JCV LT). TCR sequence analysis of the oligodendroglioma showed a JCV rearranged structure not related to a known viral strain, while the astrocytoma and the ependymoma disclosed a JCV Mad-4 strain that is known to induce brain tumors in animals. We suggest that JCV could have played a role in the pathogenesis of these brain tumors.

Molecular characterization and sequence analysis of polyomavirus BKV-strain in a renal-allograft recipient.

The significance of polyomavirus (PV) infection was investigated in a 53-year-old patient who underwent renal transplantation and was treated with triple immunosuppressive therapy (tacrolimus, prednisone, and azathioprine). A renal biopsy taken because of the suspicion of acute rejection showed focal inflammatory interstitial infiltration, tubulitis, and tubular cell nuclear changes consistent with the hypothesis of viral infection. Both the tubular and decoy cells identified by means of urinalysis positively stained for anti-SV40 antibody. Polymerase chain reaction performed on the DNA extracted from renal tissue and isolated from urine showed the presence of an antigenic variant (AS) of the BKV archetype after sequence analysis of the transcription control region (TCR). On the basis of the diagnosis of BKV infection, immunosuppressive therapy was reduced. The patient's renal function improved and was still stable 8 months later when urinalysis showed only a few decoy cells, which were found to be infected by JC but not BK virus. These data suggest that only the BKV, probably favoured by immunosuppressive therapy (tacrolimus), causes renal damage. It is worth underlining that even small and sporadic viral genome mutations may lead to pathologic effects.

Immunohistochemical distinction between amyloidosis and fibrillar glomerulopathy.

Six patients with glomerulonephritis and glomerular proteinaceous deposits constituted by fibrillar ultrastructures similar to those of amyloid but lacking the Congo red tinctorial affinity characterizing amyloid were studied. Clinically, these patients had proteinuria and hematuria; in addition, three patients had hypertension and one renal failure. Protein deposits in their kidney biopsy sections were evaluated by immunofluorescence, immunoperoxidase, and immunoelectron microscopic (protein A-gold) techniques, using antibodies against IgG, IgA, IgM, C3, C1q, fibrinogen, immunoglobulin kappa and lambda light chains, and against amyloid fibril proteins of different types, including AA, A lambda, A kappa, and AF. By immunofluorescence and immunoperoxidase, in all cases the deposits stained intensely with antibodies against IgG, C3, and kappa and lambda light chains; one case also showed C1q immunoreactivity. By contrast, none stained with antibodies against various amyloid fibril proteins. Immunoelectron microscopic findings corroborated this data, indicating that the nonamyloid fibrillar deposits studied are antigenically distinct from known amyloid deposits and that they contain IgG-derived material.

Molecular characterization and sequence analysis of polyomavirus strains isolated from needle biopsy specimens of kidney allograft recipients.

We retrospectively examined 29 renal allograft biopsy specimens from 42 kidney transplant recipients by means of molecular biologic techniques (nested polymerase chain reaction), immunohistochemical analysis (anti-SV40 antibody), and histologic examination to evaluate the presence of polyomaviruses (PVs), viral genotypes, genomic mutations, and their pathologic significance. PV genomes were found in six cases (21%); restriction fragment length polymorphism analysis characterized 4 as JC virus (JCV) and 2 as BK virus (BKV). The latter also were positively stained immunohistochemically and showed histologically typical intranuclear viral inclusions; JCV cases were negative. DNA sequence analysis revealed only minor changes in the 4 JCV cases (3 archetypes and 1 JCV type 3, not associated with a known pathogenic genotype) but identified 2 specific variants in the BKV isolates (AS and WW strains). Given the different histologic findings (mixed inflammatory infiltration in the AS and no inflammation in the WW strain), we speculate that different BKV strains may cause differential damage in transplanted kidneys. Finally, the negative histologic and immunohistochemical JCV results, as well as the absence of viral mutations, indicate that JCV renal infection is latent in transplant recipients.

Lymphoepithelioma-like carcinoma of the breast. An unusual pattern of infiltrating lobular carcinoma.

A case of breast carcinoma, showing both lymphoepithelioma-like and lobular infiltrating carcinoma, is described, which must be distinguished from the medullary carcinoma with which it shares some features, such as the strong lymphocytic infiltration, but not sharp circumscription, syncytial growth pattern, nuclear pleomorphism, and high mitotic rate. Unlike the lymphoepithelial carcinoma of the nasopharynx and some lympho-epithelioma-like carcinomas of the lung, stomach, salivary glands, and thymus, it does not seem to be connected with Epstein-Barr virus (EBV) infection, as shown by negative results of both in situ hybridization and polymerase chain reaction. This neoplasia may be defined as a peculiar form of lobular carcinoma, therefore, more representative of an unusual microscopic pattern than a distinctive clinicopathologic entity in itself.

Hepatic histology of patients with HIV infection and chronic hepatitis C treated with interferon.

AIMS: To evaluate the histological changes seen in liver biopsies after interferon (IFN) treatment in patients with chronic hepatitis C and human immunodeficiency virus (HIV) infection. METHODS: Twenty four intravenous drug users with chronic hepatitis C were investigated histologically before beginning a 12 month course of IFN treatment and 18 months later. Twelve were HIV positive, without opportunistic or other viral infections (group A), and 12 were HIV negative (group B). RESULTS: According to alanine amino-transferase concentrations, four sustained responders and eight non-responders were found in group A; six sustained responders, five relapsers, and one non-responder were found in group B. HCV RNA became negative in one sustained responder of group A and in the six sustained responders of group B. When histological findings of biopsies performed before therapy and 18 months later were compared, no significant changes in the mean value of Knodell's index and subindices were found in group A, whereas in group B Knodell's index, piecemeal necrosis, and focal hepatocellular necrosis decreased significantly. CONCLUSIONS: In chronic hepatitis C, coinfection with HIV showed a tendency towards a lower response to IFN, although this did not reach statistical significance; however, none of the HIV positive patients developed cirrhosis during the follow up and this should be considered in clinical management of such patients.

Acute hepatic and renal failure caused by Pneumocystis carinii in patients with AIDS.

Clinical and pathological findings are described in two AIDS patients with Pneumocystis carinii infection who received prophylactic treatment with nebulised pentamidine and developed unusual hepatic and renal failure. Histological examination showed clumps of P carinii massively obstructing hepatic sinuses and portal vessels in the first patient, and merular and intertubular capillaries in the second. These findings could explain the unusual clinical features, characterised by acute hepatic and renal failure.

Renal Encephalitozoon (Septata) intestinalis infection in a patient with AIDS. Post-mortem identification by means of transmission electron microscopy and PCR.

We describe the occurrence of renal Encephalitozoon (Septata) intestinalis infection in a 35-year-old AIDS patient who died with disseminated tuberculosis. The patient did not complain of specific symptoms involving the kidney or lower urinary tract during life, but at autopsy, light microscopic examination of the kidney revealed numerous small round or oval bodies in the tubules and tubular cell cytoplasm that were interpreted as intracellular protozoa. Transmission electron microscopy of tissue retrieved from paraffin-embedded samples identified these organisms as microsporidia belonging to the Encephalitozoonidae family, but did not allow definitive identification of the species of infecting parasite. This was made possible only by means of Southern blot hybridization after the polymerase chain reaction, which recognized the micro-organism as E. intestinalis.

Early sarcolemmal dysfunction in skeletal muscle amyloidosis.

Weakness and stiffness appeared in a 65-year-old man affected by multiple myeloma. Muscle fibre conduction velocity was recorded in situ in the biceps brachii and found to be significantly decreased. Muscle biopsy, performed in the same muscle, showed amyloid deposition and moderate atrophy of muscle fibres, which was not sufficient to explain the reduction in muscle fibre conduction velocity. The results of the study suggest that amyloid interferes with conduction along the sarcolemma and that this plays a pathogenetic role mainly in the early stages of the disease.

Mitochondrial abnormalities of late motor neuron degeneration following poliomyelitis and other neurogenic muscular atrophies.

A case of late motor neuron degeneration following poliomyelitis with abnormal mitochondria in muscle fibers is presented with two additional cases of systemic neurogenic muscular atrophy (Charcot-Marie-Tooth disease). Muscle biopsy revealed a neurogenic pattern of variable severity in all cases. Subsarcolemmal zones of hyperactivity and hyperpositive intermyofibrillar collections of granular material present in a variable proportion of type I fibers were demonstrated by oxidative enzymes. Ultrastructurally they corresponded to abnormal mitochondria, with paracrystalline inclusions in one case. The finding is discussed in the light of the previous literature on mitochondrial myopathies. Mitochondrial alterations are not specific and their significance in neurogenic conditions is debated.

Histochemistry and electron microscopy of muscle fibres in a case of congenital paramyotonia.

In a case of congenital paramyotonia a muscle biopsy was performed and studied morphologically, histochemically and ultrastructurally. A clearcut pattern of changes has been observed with ATPase and oxidative enzymes. On electron microscopy special changes known as "tubular aggregates" were found. The relationship between the two findings, as well as the significance of such alterations in the range of periodic paralyses and myotonic phenomena, are discussed.

Central core disease: histochemical and ultrastructural study of muscle biopsies of father and daughter.

Two cases of central core disease, father and daughter, of a family with dominant autosomal inheritance, are presented, one with bilateral congenital dislocation of the hip. Muscle biopsy was performed in both cases. Oxidative enzymes evidenced only type I fibers, most of them presenting a central core and not uncommonly more than one. On electron microscopy the cores generally appeared well demarcated from the surrounding fibrils and were characterized by lack of mitochondria and abnormalities of the Z line. Transitional aspects from normal fibers to completely unstructured cores were observed, as well as from well structured and unstructured cores. These findings are discussed in the light of the previous literature and particular attention is paid to the problem of differentiation between central core and multicore disease. The pathogenesis of the muscular alteration is also discussed in relation with the possibility of their neurogenic origin. Eventually, the histochemical and ultrastructural similarities between central cores and target fibers are focused.

Simulation of right atrial cardiac myxoma by silent hepatocellular carcinoma.

A clinically silent hepatocellular carcinoma presenting as a mixoma of the right atrium is described. Intra-atrial growth has been reported in advanced, clinically manifested cases of liver carcinomas in African and Japanese subjects, but very occasionally in Caucasian people. Our case further suggests that this occurrence should also be considered in Western Countries.

Light microscopic and immunohistochemical evaluation of vascular and neural invasion in colorectal cancer.

One hundred and sixty consecutive surgically removed colorectal carcinomas were investigated on histological sections stained with Hematoxylin and Eosin (H & E). Vascular and neural neoplastic invasion was found in 49 (30.6%) and 50 (31.3%) patients. In addition, immunohistochemical investigation was performed on step sections to those stained with H & E in the first 50 patients, using anti-human Factor VIII related antigen, anti-actin and anti-protein S 100 antisera. The percentages of positive cases for vascular invasion in this group of 50 patients were 20% on H & E sections and 62% on those stained with anti-Factor VIII and anti-actin antisera. Neural infiltration was identified in 14% of cases on H & E sections and in 70% of cases on anti-protein S 100 treated sections. Since vascular and neural infiltration are known to be ominous prognostic factors, their identification has great clinical relevance. The use of these simple immunohistochemical stains, using readily available antisera on formalin-fixed material, is recommended as routine procedure in surgical pathology laboratories.

The use of Mallory's phosphotungstic acid-hematoxilin (PTAH) stain in renal pathology.

High quality histology is needed in renal biopsy examination, especially when material for immunofluorescence and/or electron microscopy is not available. For these purposes Mallory PTAH stain was tried on 34 renal biopsies and the results were compared with the immunofluorescent and ultrastructural findings (used as a control of the reliability of the method). PTAH, besides showing extracellular structures, cytological details and rather subtle abnormalities, was able to detect the presence and the site of deposits. PTAH stains the material which appears as electron dense deposits in electron microscopy and as granular deposits in immunofluorescence, whereas it fails to stain linear deposits in immunofluorescence (which are not electron dense). Moreover, a good correspondence between PTAH and electron microscopic data was detected as far as the location of deposits is concerned.