Dirofilariasis is a zoonosis caused by nematodes of the genus Dirofilaria.Dirofilaria immitis is cosmopolitan as regards its distribution in animals, being responsible for human pulmonary ...dirofilariasis in the New World. However, human infections by Dirofilaria immitis are exceptional in Europe, and the previously reported Italian cases of pulmonary dirofilariasis were due to Dirofilaria repens. We performed a systematic literature review of the Italian cases of human dirofilariasis due to Dirofilariaimmitis according to the PRISMA guidelines. We also report the first autochthonous case of human pulmonary dirofilariasis due to Dirofilariaimmitis, confirmed by polymerase chain reaction analysis. The patient was a 60-year-old man who lived in the Po river valley and had never traveled abroad; on histological examination, the 2-cm nodule found in his right upper lung was an infarct due to a parasitic thrombotic lesion. Only one other autochthonous (but conjunctival) case due to Dirofilariaimmitis (molecularly confirmed) was previously found in the same geographic area. Climatic changes, the increasing movements of animal reservoirs and vectors, and new competent carriers have expanded the geographic distribution of the Dirofilaria species, increasing the risk of human infections. Our report demonstrates that at least some pulmonary Italian cases of human dirofilariasis are due to Dirofilaria immitis, as in the New World.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Mutations of KRAS are detectable in 70–90% of pancreatic duct adenocarcinomas (PDAC), using direct sequencing. We used a highly sensitive molecular method in order to investigate: (a) the frequency ...and prognostic significance of different KRAS mutations and, (b) whether the presence of KRAS mutations in histologically-negative resection margins of PDAC could explain local tumor recurrence after surgery.
Twenty-seven patients with histologic diagnosis of PDAC, radical pancreaticoduodenectomy and histologically-negative margins were evaluated. KRAS mutations were searched for mutant-enriched PCR in tumor and negative resection margins.
KRAS mutations were detected in 85.2% of the cases; the most frequent mutation was G12D (48.1%). Shorter OS was found in patients with G12D (25 months; 95% CI, 20.5–29.5), vs patients with other mutations (31.5 months; 95% CI, 25.6–37.1) (N.S.). KRAS mutation in histologically-negative margins was detected in one patient who died of locoregional recurrence; six patients had tumor recurrence but no mutations in surgical margins.
The high frequency of KRAS mutations suggests a search for KRAS status to improve the diagnosis in suspected cases; the G12D mutation could be related to poor prognosis, but without statistical significance. No correlation was found between the frequency of cancer recurrence and KRAS mutations in surgical margins.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, SAZU, SBCE, SBJE, UL, UM, UPUK
We provide morphological, immunohistochemical and molecular characterization of the 3rd “intermediate-grade” orbital meningeal melanocytoma, testing for the first time Vysis Melanoma FISH Probe Kit. ...We reviewed the literature in order to discuss the main differential diagnoses and to provide a better molecular description of these unusual tumors of difficult diagnosis and controversial management.
Histochemical stains (Haematoxylin and Eosin, Perls, reticulin), immunohistochemistry (HMB45, p16, Melan-A, S100, EMA, Ki67, CD68), polymerase chain reaction amplification and sequence analysis (BRAF, exon 15; NRAS exons 2 and 3; c-KIT, exons 11, 13, 17, 18; GNAQ, exons 4 and 5; GNA11, exons 4 and 5) and fluorescent in situ hybridization (RREB1, 6p25; MYB, 6q23; CCND1, 11q13; CEP 6, 6p11.1-q11.1) were performed on paraffin-embedded, formalin-fixed material.
Histological diagnosis of “intermediate-grade” melanocytoma was supported by zonal necrosis and increased Ki67-index (12%). Immunophenotype: HMB45+(strong, >75%), Melan-A+(strong, >75%), p16+(∼20%), S100 −/+ (<5%), EMA −/+ (<5%), CD68 − (positive histiocytes). No gene mutations nor copy-number alterations were identified. The patient was asymptomatic and disease-free 3 years after total surgical excision.
Adequate sampling and accurate immunohistochemical characterization are important for a correct diagnosis. Molecular analysis could provide important additional information (especially for “intermediate-grade” tumors), but further data are needed.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, SAZU, SBCE, SBJE, UL, UM, UPUK, ZRSKP
Distinct human polyomavirus genotypes cause different diseases in patients with renal transplants: BK virus (BKV) causes tubulointerstitial nephritis and ureteral stenosis, whereas both JC virus ...(JCV) and BKV are responsible for hemorrhagic cystitis. These findings could result from a selective infection of kidney and urinary tract segments by JCV or BKV.
To verify this hypothesis, 10 complete, unselected, consecutive autopsies from 9 immunocompetent patients and 1 patient affected by acquired immunodeficiency syndrome were investigated.
Samples from kidneys (n = 80), renal pelvis (n = 20), ureter (n = 40), and urinary bladder (n = 30) obtained from 10 consecutive autopsies were investigated by means of multiplex nested polymerase chain reaction to detect polyomavirus DNA and to distinguish different species of the Polyomavirus genus. In situ hybridization and immunohistochemistry were also carried out to define the viral status of the infected tissues.
Polyomavirus DNA was detected in all of the subjects (positive samples ranging from 2 to 7 samples), for a total of 43 of 170 samples (25.3%), distributed as follows: urinary bladder (10/30, 33%), renal pelvis (6/20, 30%), ureter (10/40, 25%), and kidney tissue (17/80, 21%). We found that JCV was most frequently detected overall (23/43 samples, 53.5%) and was also detected most frequently within the kidney (8/17 positive samples, 47%), the renal pelvis (5/6 positive samples, 70%), and the ureter (7/10 positive samples, 70%), whereas BKV was found in 14 samples (32.5%), and it was the prevailing genotype in urinary bladder (6/10 positive samples, 60%). Coinfection of BKV-JCV was found in 6 samples (14%). Immunohistochemistry and in situ hybridization returned negative results.
The viruses JCV and BKV latently persist randomly in kidney and urinary tract. Distinct diseases induced by them could be related more closely to molecular viral rearrangements than to the topographic distribution of latent viruses.
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DOBA, IZUM, KILJ, NUK, OILJ, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK, VSZLJ
Malignant pleural mesothelioma (MPM) is often associated with a poor prognosis and options for the treatment of this disease are few. To date, the important role of the immune microenvironment in ...modifying the disease natural history is well established. The programmed cell death pathway (PD-1/PD-L1) limits the T lymphocyte activation in peripheral tissues when an inflammatory response occurs, and controls the tumour immune escape. PD-L1 is broadly expressed in several malignant tumours and associated with poor clinical outcomes. Thus, the aim of our study is to investigate the potential role of PD-L1 expression in MPM prognosis.
Biopsy samples from 198 patients diagnosed with MPM were examined by immunohistochemistry (IHC) and reverse transcription-polymerase chain reaction (RT-PCR) to evaluate PD-L1 protein and gene expression. For PD-L1 protein expression we consider at least 5% membranous staining as positive. Gene expression levels were calculated with ΔΔCt method. Positive expression of PD-L1 by IHC was correlated with worse overall survival (OS; p=0.0225) in MPM patients. PD-L1 positive status was correlated with worse OS in the subgroup of patients with ECOG score <2 (p=0.0004, n=129) and these data were confirmed by multivariate analysis. No significant correlation was found between PD-L1 gene expression and OS.
Our results show that PD-L1 evaluated by IHC assay may be a prognostic biomarker for MPM patients with good performance status.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) is the procedure of choice for the cytologic diagnosis of pancreatic masses. The specificity of EUS-FNA approaches 100%, but the ...sensitivity is still low, and the high rate of indeterminate (atypical and suspicious) and false-negative results needs improvement. KRAS gene is frequently mutated in pancreatic ductal adenocarcinoma (PDAC) (up to 90%), and mutation analysis of KRAS has been proposed as diagnostic biomarker of PDAC. In most laboratories, KRAS mutation testing is performed by Sanger sequencing or real time-quantitative polymerase chain reaction (RT-qPCR), but these methods may give false-negative results in routine samples, mainly due to low cellularity.
In order to increase the sensitivity of EUS-FNA, we propose a sequential approach for detecting KRAS mutations using mutant enriched-PCR (ME-PCR, sensitivity up to 0.1%) in cytologically indeterminate and negative samples tested wild-type by RT-qPCR.
EUS-FNA specimens from 107 patients with pancreatic masses (51 males, 56 females, mean age 67 years) were cytologically examined. According to the Papanicolaou Society of Cytopathology guidelines, 50 cases (47%) were classified malignant, 15 (14%) suspicious, 13 (12%) atypical and 10 (9%) negative for malignancy; 18 cases (17%) were non-diagnostic.
The overall specificity and sensitivity of cytological examination were 100% and 61%, respectively, when only negative and positive cases were considered; when atypical and suspicious were added to positive cases, the sensitivity increased to 95.1% and the specificity decreased to 85.7%.
In all the cases, DNA was extracted from the cell-block and KRAS mutations were investigated by RT-qPCR, followed by ME-PCR in non-amplifiable and negative cases. The overall sensitivity and specificity of KRAS mutation testing alone were 79.3% and 100%; when KRAS mutation testing was performed in indeterminate and negative cytology, the sensitivity increased to 90% with specificity to 100%.
Our data indicate that conventional cytology from EUS-FNA samples is highly specific for the diagnosis of pancreatic cancer. Indeterminate and negative cases need to be screened for KRAS mutations; this two-step approach may greatly improve the diagnostic accuracy of this method.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Epidermal growth factor receptor (EGFR) gene mutational analysis is critical for guiding the treatment of lung adenocarcinoma. In everyday clinical practice, EGFR testing is frequently centralized in ...referral laboratories that may receive paucicellular cytologic specimens, often fixed in various ways. We conducted a search for EGFR mutations in 108 cytologic samples of lung adenocarcinoma from different hospitals using the TheraScreen EGFR29 kit. These samples included 80 (74.1%) fine-needle aspirations, 13 (12%) pleural/ascitic fluids, 13 (12%) bronchial washings, and 2 bronchial brushings. The samples were fixed in ethanol (n = 79), Duboscq-Brasil (n = 18) or formalin (n = 10); 1 was unfixed. Ninety-two (85.2%) were amplified, 16 (14.8%) were not. Mutations were detected in 22 (23.9%) of 92 amplified samples, 9 containing less than 200 cancer cells, and 4 with less than 50% cancer cells. DNA was amplified in 12 of 18 Duboscq-Brasil-fixed samples. These findings indicate that cytologic specimens are adequate for EGFR testing when a highly sensitive assay is used, even if they are paucicellular or not optimally fixed.
Given the conflicting results of the few published studies, the aim of this retrospective molecular‐based study of 10 aborted fetuses that underwent complete autopsy and 10 placentas was carried out ...to determine whether BK polyomavirus (BKV) can be transmitted transplacentally. The interruption of pregnancy was due to a miscarriage (five cases) or a prenatal diagnosis of severe intrauterine malformations (five cases). Samples from the brain, heart, lung, thymus, liver, and kidney were taken from each fetus, and two samples were obtained from all of the placentas. The presence of BKV was investigated by means of PCR using primers specific for the transcription control region (TCR) and viral capsidic protein 1 (VP1) and DNA extracted from formalin‐fixed, paraffin‐embedded tissue. BKV genome was detected in 22 of 60 samples (36.6%) from seven fetuses (70%), regardless of the cause of abortion: VP1 was amplified in 12 samples (54%), TCR in seven (32%), and both in three (14%). VP1 was also detected in one placental sample. BKV sequences were most frequently detected in heart and lung (five cases), but sequence analyses of TCR and VP1 revealed a high degree of genomic variability among the samples taken from different organs and the placenta. These results indicate that BKV can cross the placenta during pregnancy and become latent in fetal organs other than the kidney and brain (previously considered the main targets of BKV latency). This may happen in early pregnancy and does not seem to be associated with an increased risk of abortion. J. Med. Virol. 82:2127-2132, 2010.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
JC virus (JCV) is a polyomavirus that asymptomatically infects up to 80% of the worldwide human population and establishes latency in the kidney. In the case of host immunodeficiency, it can cause ...progressive multifocal leukoencephalopathy (PML), which is a fatal demyelinating disease of the central nervous system. In an attempt to understand better PML pathogenesis and JCV infection, the presence of the JCV genome and expression of the early viral protein in the brain of deceased individuals, with and without HIV infection, was investigated. Sixty autopsy samples of brain tissues were collected from 15 HIV-positive PML patients, 15 HIV-positive patients with other neurological diseases, 15 HIV-positive patients without neurological disorders, and 15 HIV-negative individuals who died from diseases unrelated to the central nervous system. By means of specific Real Time Polymerase Chain Reaction, the JCV genome was detected in 14 of 15 PML brains, three of 15 HIV-positive brains (with and without neurological diseases), and 1 of 15 HIV-negative brains. JCV genotyping was also performed. Expression of the early JCV protein T Antigen was verified by a specific immunohistochemistry assay, and it was found in the brain tissues from 12 PML cases and one case with other neurological disease. The data obtained demonstrate that infection of the brain with JCV can also be observed in the brains of HIV-negative individuals, without neurological disorders. However, viral protein expression was limited to PML brains and to one brain from a patient with other neurological disease, suggesting that JCV can also be present in the brains of patients without PML. J. Med. Virol. 80:2147-2152, 2008.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
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