The expression of LINE1-MET chimeric transcript identifies a subgroup of aggressive breast cancers.

Demethylation of the long interspersed nuclear element (LINE-1; L1) antisense promoter can result in transcription of neighboring sequences as for the L1-MET transcript produced by the L1 placed in the second intron of MET. To define the role of L1-MET, we investigated the sequence and the transcription of L1-MET in vitro models and heterogeneous breast cancers, previously reported to show other L1-derived transcripts. L1-MET expressing cell lines were initially identified in silico and investigated for L1-MET promoter methylation, cDNA sequence and cell fraction mRNA. The transcriptional level of L1-MET and MET were then evaluated in breast specimens, including 9 cancer cell lines, 41 carcinomas of different subtypes, and 11 normal tissues. In addition to a L1-MET transcript ending at MET exon 21, six novel L1-MET splice variants were identified. Normal breast tissues were negative for the L1-MET expression, whereas the triple-negative breast cancer (TNBC) and the high-grade carcinomas were enriched with the L1-MET mRNA (p = 0.005 and p = 0.018, respectively). In cancer cells and tissues the L1-MET expression was associated with its promoter hypomethylation (ρ = -0.8 and -0.9, respectively). No correlation was found between L1-MET and MET mRNA although L1-MET expressing tumors with higher L1-MET/MET ratio were negative for the MET protein expression (p = 0.006). Besides providing the first identification and detailed description of L1-MET in breast cancer, we clearly demonstrate that higher levels of this transcript specifically recognize a subset of more aggressive carcinomas, mainly TNBC. We suggest the possible evaluation of L1-MET in the challenging diagnosis of early TNBCs.

Improved detection reveals active ß-papillomavirus infection in skin lesions from kidney transplant recipients.

The aim of this study was to determine whether detection of ß-HPV gene products, as defined in epidermodysplasia verruciformis skin cancer, could also be observed in lesions from kidney transplant recipients alongside the viral DNA. A total of 111 samples, corresponding to 79 skin lesions abscised from 17 kidney transplant recipients, have been analyzed. The initial PCR analysis demonstrated that ß-HPV-DNA was highly present in our tumor series (85%). Using a combination of antibodies raised against the E4 and L1 proteins of the ß-genotypes, we were able to visualize productive infection in 4 out of 19 actinic keratoses, and in the pathological borders of 1 out of 14 squamous cell carcinomas and 1 out of 31 basal cell carcinomas. Increased expression of the cellular proliferation marker minichromosome maintenance protein 7 (MCM7), that extended into the upper epithelial layers, was a common feature of all the E4-positive areas, indicating that cells were driven into the cell cycle in areas of productive viral infections. Although the present study does not directly demonstrate a causal role of these viruses, the detection of E4 and L1 positivity in actinic keratosis and the adjacent pathological epithelium of skin cancer, clearly shows that ß-HPV are actively replicating in the intraepidermal precursor lesions of kidney transplant recipients and can therefore cooperate with other carcinogenic agents, such as UVB, favoring skin cancer promotion.

α- and ß-papillomavirus infection in a young patient with an unclassified primary T-cell immunodeficiency and multiple mucosal and cutaneous lesions.

BACKGROUND: Correlating human papillomavirus (HPV) type with the clinical and histopathological features of skin lesions (from genital and nongenital sites) can present a diagnostic challenge. OBJECTIVE: In this study, HPV infection patterns were correlated with pathology and clinical presentation in lesional and nonlesional body sites from a young patient with a primary T-cell immunodeficiency. METHODS: HPV infection was evaluated at both DNA and protein levels by polymerase chain reaction and immunohistochemistry. RESULTS: The patient's genital lesions were caused exclusively by α-genotypes (high-risk type HPV-51 in the anal and low-risk type HPV-72 in the penile condylomas). The opposite was true for the skin lesions, which were infected by ß-genotypes alone (HPV-8 and HPV-24). HPV-24 was the predominant type in terms of viral load, and the only one found in productive areas of infection. The patient had already developed high-grade dysplasia in the anal condyloma-like lesions, and showed areas of early-stage dysplasia in the lesions caused by the ß-genotype HPV-24. LIMITATIONS: The basic origin of the immunodeficiency is not yet defined. CONCLUSION: These findings provide proof of principle that both α- and ß-genotypes can cause overt dysplastic lesions when immunosurveillance is lost, which is not restricted to epidermodysplasia verruciformis.

Quantifying mRNA in Highly Degraded Fixed Tissues by Nanostring Technology: A Comparative Study.

Archive tissues are the most available source of human tissues useful for molecular analysis in translational research. The main issues for those specimens are the modification and degradation of biomolecules, namely proteins, DNA, and RNA. In the last decade, several high-throughput analytical methods have been applied to archive tissues. Although histological tissues are fixed in neutral-buffered formalin nowadays, in the recent past, Bouin's solution was also used in tissue processing. The present study aims to investigate the feasibility of nCounter Nanostring hybridization in quantifying mRNA in highly degraded samples, such as Bouin's fixed and paraffin-embedded (BFPE) tissues, in comparison to the standard formalin-fixed and paraffin-embedded (FFPE) tissues as a source of RNA. A total of 16 paraffin-embedded tissue blocks from eight patients were analyzed (8 were FFPE and 8 were BEPE). Nanostring technology was applied to 300 ng of each RNA sample, whereas 360 ng of the same templates were retrotranscribed and submitted to qPCR and ddPCR. Our results show that the Nanostring technology outperforms the reference methods (ddPCR and qPCR) in detecting target mRNA in FFPE and BFPE samples. However, even Nanostring technology does not escape the limitation imposed by the degradation of the RNA templates, which could lead to misleading conclusions on the gene expression level.

The Tumor-Specific Expression of L1 Retrotransposons Independently Correlates with Time to Relapse in Hormone-Negative Breast Cancer Patients.

BACKGROUND: Long-Interspersed Nuclear Element (L1) retrotransposons are silenced in healthy tissues but unrepressed in cancer. Even if L1 reactivation has been associated with reduced overall survival in breast cancer (BC) patients, a comprehensive correlation with clinicopathological features is still missing. METHODS: Using quantitative, reverse-transcription PCR, we assessed L1 mRNA expression in 12 BC cells, 210 BC patients and in 47 normal mammary tissues. L1 expression was then correlated with molecular and clinicopathological data. RESULTS: We identified a tumor-exclusive expression of L1s, absent in normal mammary cells and tissues. A positive correlation between L1 expression and tumor dedifferentiation, lymph-node involvement and increased immune infiltration was detected. Molecular subtyping highlighted an enrichment of L1s in basal-like cells and cancers. By exploring disease-free survival, we identified L1 overexpression as an independent biomarker for patients with a high risk of recurrence in hormone-receptor-negative BCs. CONCLUSIONS: Overall, L1 reactivation identified BCs with aggressive features and patients with a worse clinical fate.

DNA damage response and repair genes in advanced bone and soft tissue sarcomas: An 8-gene signature as a candidate predictive biomarker of response to trabectedin and olaparib combination.

Background: Advanced and unresectable bone and soft tissue sarcomas (BSTS) still represent an unmet medical need. We demonstrated that the alkylating agent trabectedin and the PARP1-inhibitor olaparib display antitumor activity in BSTS preclinical models. Moreover, in a phase Ib clinical trial (NCT02398058), feasibility, tolerability and encouraging results have been observed and the treatment combination is currently under study in a phase II trial (NCT03838744). Methods: Differential expression of genes involved in DNA Damage Response and Repair was evaluated by Nanostring® technology, extracting RNA from pre-treatment tumor samples of 16 responder (≥6-month progression free survival) and 16 non-responder patients. Data validation was performed by quantitative real-time PCR, RNA in situ hybridization, and immunohistochemistry. The correlation between the identified candidate genes and both progression-free survival and overall survival was investigated in the publicly available dataset "Sarcoma (TCGA, The Cancer Genome Atlas)". Results: Differential RNA expression analysis revealed an 8-gene signature (CDKN2A, PIK3R1, SLFN11, ATM, APEX2, BLM, XRCC2, MAD2L2) defining patients with better outcome upon trabectedin+olaparib treatment. In responder vs. non-responder patients, a significant differential expression of these genes was further confirmed by RNA in situ hybridization and by qRT-PCR and immunohistochemistry in selected experiments. Correlation between survival outcomes and genetic alterations in the identified genes was shown in the TCGA sarcoma dataset. Conclusions: This work identified an 8-gene expression signature to improve prediction of response to trabectedin+olaparib combination in BSTS. The predictive role of these potential biomarkers warrants further investigation.

Serological evidence of vertical transmission of JC and BK polyomaviruses in humans.

Vertical transmission of JC virus and BK virus has been investigated by few authors, with conflicting results. We performed a combined serological and genomic study of 19 unselected pregnant women and their newborns. Blood and urine samples were collected during each gestational trimester from the pregnant women. Umbilical cord blood, peripheral blood, urine and nasopharyngeal secretion samples were taken from newborns at delivery and after 1 week and 1 month of life. Polyomavirus DNA was detected by nested PCR. Polyomavirus IgG-, IgM- and IgA-specific antibodies were measured in maternal and newborn serum samples using a virus-like-particle-based ELISA method. BKV and JCV DNA were detected in urine from 4 (21 %) and 5 (26 %) women, respectively. BKV and JCV seroprevalences in the pregnant women were 84 % and 42 %, respectively. Using a rise in the IgG level or the transient appearance of an IgA or IgM response as evidence of infection in the newborn, we detected BKV and JCV infections in four (21 %) and three (16 %) newborns, respectively. Three infants had serological evidence of infection with both BKV and JCV. In two of the four possible BKV-infected newborns, the mothers seroconverted during pregnancy, while another mother was viruric and IgA seropositive. The mother of one of the three possible JCV-infected newborns was viruric and IgA seropositive; another mother was viruric. These results suggest JC virus and BK virus can be transmitted from mother to newborn during pregnancy or soon after birth.

Mutations in the external loops of BK virus VP1 and urine viral load in renal transplant recipients.

Polyomavirus-associated nephropathy (PVAN) is a major complication that occurs after renal transplantation and is induced by reactivation of the human polyomavirus BK (BKV). The structure of the viral capsid protein 1 (VP1) is characterized by the presence of external loops, BC, DE, EF, GH, and HI, which are involved in receptor binding. The pathogenesis of PVAN is not well understood, but viral risk factors are thought to play a crucial role in the onset of this pathology. In an attempt to better understand PVAN pathogenesis, the BKV-VP1 coding region was amplified, cloned, and sequenced from the urine of kidney transplant recipients who did, and did not, develop the pathology. Urine viral loads were determined by using real time quantitative PCR (Q-PCR). Amino acid substitutions were detected in 6/8 patients, and 6/7 controls. The BC and EF loop regions were most frequently affected by mutations, while no mutations were found within the GH and HI loops of both patients and controls. Some mutations, that were exclusively detected in the urine of PVAN patients, overlapped with previously reported mutations, although a correlation between changes in amino acids and the development of PVAN was not found. Urine viral loads were higher than that of the proposed cut-off loads for identification of patients that are at a high risk of developing PVAN (10(7) copies/ml), both in the PVAN and control groups, thus confirming that urine viral load is not a useful predictive marker for the development of PVAN.

BK virus sequences in specimens from aborted fetuses.

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.

Viral infection in bone marrow transplants: is JC virus involved?

Hemorrhagic cystitis is characterized by hematuria due to inflammation of the bladder. In bone marrow transplants, this disease is linked to the infection by human polyomavirus BK, whereas the role of the human polyomavirus JC is unclear. The transcriptional control regions of both viruses contain important cellular transcription factor binding sites that undergo rearrangement process generating suitable variants that could be more active for viral replication and for the onset of hemorrhagic cystitis. In this study urine obtained from seven patients with bone marrow transplant were examined. Polyomavirus genomes were quantified by PCR and viral loads were compared. The transcriptional regions of both viruses were amplified and sequenced to determine the presence of variants. Subtypes of polyomaviruses were determined by amplification and sequencing of the viral protein 1 region. The results showed that four of seven patients were positive for BK DNA, two of seven patients had BK and JC DNA and one of seven had JC DNA. Positive samples were amplified and sequenced successively for transcriptional regions. The viral archetype was always found in both viruses. Finally, typing showed that BK virus subtype I infected patients with BK, whereas JC virus genotype IA and genotype 1B were found in patients infected with JC. The data suggest that new and different approaches are required to improve the morbidity and mortality caused by polyoma-associated hemorrhagic cystitis, since it known that BK virus is involved in the onset of hemorrhagic cystitis, whereas the role of JC virus should be investigated further.

Cold Formalin Fixation Guarantees DNA Integrity in Formalin Fixed Paraffin Embedded Tissues: Premises for a Better Quality of Diagnostic and Experimental Pathology With a Specific Impact on Breast Cancer.

Formalin fixation and paraffin embedding (FFPE) represent the standard method to preserve tissue specimens for diagnostic pathology, however formalin fixation induces severe fragmentation of nucleic acids. We investigated whether formalin fixation at 4°C could preserve DNA integrity in FFPE specimens. Paired samples from 38 specimens were formalin fixed at room temperature (stdFFPE) and at 4°C (coldFFPE), respectively. Two independent cohorts were prospectively collected, cohort A (collected 6 years prior to the study, n = 21), cohort B (collected at time of the study, n = 17). DNA was extracted and its integrity evaluated with a qPCR-based assay that produces a normalized integrity index, the QC score (ratio between the quantity of a long and a short amplicon of the same gene). We observed higher QC scores in coldFFPE compared to stdFFPE samples (mean values: 0.69 vs. 0.36, p < 0.0001) and stdFFPE breast cancer specimens showed the most detrimental effect overall. Comparable QC scores were obtained between coldFFPE tissues of both cohorts; conversely, DNA integrity of stdFFPE was significantly lower in cohort A compared to cohort B (p < 0.0001). Of note, QC scores of stdFFPE (but not of coldFFPE) samples were significantly reduced following 6 months of storage (p = 0.0001). Monitored formalin fixation at 4°C outperforms standard fixation in ensuring high-quality DNA, which is key to feasibility of downstream high-throughput molecular analyses. An important effect was observed over storage time, thus suggesting a likely better preservation of archival samples when this cold fixation protocol is used.

MiR-100 is a predictor of endocrine responsiveness and prognosis in patients with operable luminal breast cancer.

PURPOSE: Overexpression of miR-100 in stem cells derived from basal-like breast cancers causes loss of stemness, induction of luminal breast cancer markers and response to endocrine therapy. We, therefore, explored miR-100 as a novel biomarker in patients with luminal breast cancer. METHODS: miR-100 expression was studied in 90 patients with oestrogen-receptor-positive/human-epidermal growth factor receptor 2-negative breast cancer enrolled in a prospective study of endocrine therapy given either preoperatively, or for the treatment of de novo metastatic disease. Response was defined as a Ki67 ≤2.7% after 21±3 days of treatment. The prognostic role of miR-100 expression was evaluated in the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) and The Cancer Genome Atlas (TCGA) breast cancer datasets. Additionally, we explored the correlation between miR-100 and the expression its targets reported as being associated with endocrine resistance. Finally, we evaluated whether a signature based on miR-100 and its target genes could predict the luminal A molecular subtype. RESULTS: Baseline miR-100 was significantly anticorrelated with baseline and post-treatment Ki67 (p<0.001 and 0.004, respectively), and independently associated with response to treatment (OR 3.329, p=0.047). In the METABRIC dataset, high expression of miR-100 identified women with luminal A tumours treated with adjuvant endocrine therapy with improved overall survival (HR 0.55, p<0.001). miR-100 was negatively correlated with PLK1, FOXA1, mTOR and IGF1R expression, potentially explaining its prognostic effect. Finally, a miR-100-based signature developed in patients enrolled in the prospective study outperformed Ki67 alone in predicting the luminal A phenotype. CONCLUSIONS: Our findings suggest that miR-100 should be further explored as a biomarker in patients with luminal breast cancer.

Pazopanib and Trametinib as a Synergistic Strategy against Osteosarcoma: Preclinical Activity and Molecular Insights.

Receptor tyrosine kinases (RTKs) inhibitors' activity in advanced osteosarcoma is significant but short-lived. To prevent or at least delay drug resistance, we explored a vertical inhibition by combining drugs acting at different levels of the RTK pathways (pazopanib + trametinib). We studied pazopanib + trametinib antitumor activity both in vitro and in vivo (MNNG-HOS and KHOS xenografts in NOD/SCID mice) investigating the molecular mechanisms and potential escapes. The involvement of MAPK-PI3K pathways was validated by Nanostring technology, western blot and by silencing/overexpression experiments. Pazopanib targets were expressed on seven osteosarcoma cell lines and their pathways were activated. Pazopanib + trametinib exhibited synergistic antitumor activity by inducing apoptosis and inhibiting ERK1/2 and Akt. In vivo antitumor activity was shown in osteosarcoma-bearing mice. The drug combination significantly down-modulated RTK Ephrin Type-A Receptor 2 (EphA2) and Interleukin-7 Receptor (IL-7R), whereas induced mitogen-activated protein-kinase kinase (MAPKK) MEK6. EphA2 silencing significantly reduced osteosarcoma cell proliferation and migration, while impeding MEK6 up-regulation in the treated cells significantly increased the antitumor effect of the studied drugs. Moreover, the up-regulation of MEK6 reduced combination activity. Pazopanib + trametinib demonstrated synergistic antitumor effects in osteosarcoma models through ERK and Akt inhibition and EphA2 and IL-7R down-modulation. MEK6 up-regulation might evoke escaping mechanism.

Genomic mutations of viral protein 1 and BK virus nephropathy in kidney transplant recipients.

Genomic variability in the viral protein 1 region of BK polyomavirus (BKV) may change the ability of the virus to replicate. The significance of such changes was studied in clinical samples taken from kidney transplant patients with and without BKV nephropathy. A 94 base-pair fragment of viral protein 1 was amplified from 68 urine, 28 blood, and 12 renal biopsy samples from eight patients with BKV nephropathy, and from 100 urine samples, 17 blood and three renal biopsy samples from 41 of 218 controls. The DNA was sequenced and the amino acid changes were predicted by the Expert Protein Analysis System program (ExPASy, Swiss Institute of Bioinformatics, Geneva, Switzerland). Single base-pair mutations were detected more frequently in the samples from the BKV nephropathy patients than in the controls, and this was the only statistically significant finding of the study (P < 0.05), thus suggesting a greater genetic instability in BKV nephropathy associated strains. The amino acid changes were distributed at random in both BKV nephropathy patients and controls. However, one aspartic acid-to-asparagine substitution at residue 75 was detected in all samples of the one patient with BKV-associated nephropathy, who developed disease progression confirmed by histology, and not in any of the other patient or control samples. Whether this specific amino acid change plays a role in disease deserves further study.

Complications post renal transplantation: literature focus on BK virus nephropathy and diagnostic tools actually available.

Clinical diagnosis of kidney transplants related illnesses is not a simple task. Several studies were conducted to define diseases and complications after renal transplantation, but there are no comprehensive guidelines about diagnostic tools for their prevention and detection. The Authors of this review looked for the medical literature and pertinent publications in particular to understand the role of Human Polyomavirus BK (BKV) in renal failure and to recognize analytical techniques for BK virus associated nephropathy (BKVAN) detection.

Latent human polyomavirus infection in pregnancy: investigation of possible transplacental transmission.

AIMS: The purpose of the study was to investigate the transplacental transmission of the human polyomaviruses JCV and BKV. METHODS: Urine and blood samples from 300 pregnant women underwent cytological analysis to search for 'decoy cells', nested PCR to identify presence and genotype of isolated polyomaviruses, and sequence analysis of the transcription control region. Nested PCR was also used to study the umbilical cord blood of all their newborns. RESULTS: Decoy cells were identified in only one urine sample (1/300; 0.33%); polyomavirus DNA was detected in 80 urine samples (26.6%) corresponding to BKV alone in 28 samples (9.3%), JCV alone in 49 samples (16.3%) and both JCV-BKV in three samples (1%). Blood samples were positive in 17 cases (5.6%), corresponding to BKV alone in 10 (3.3%), and JCV alone in 7 (2.3%). Rearrangements of the transcription control region were found in only one urinary JCV strain, consisting of the insertion of 13 bp at D block, whereas point mutations were identified in 11 BKV and 11 JCV strains detected from urine. Sequence analysis of the BKV strains detected in blood samples revealed a 20 bp insertion of P block (P42-61) in human chromosomes 20 (five cases) and 14 (three cases); two JCV strains had single bp point mutations. The search for polyomavirus DNA in umbilical cord blood samples was always negative. CONCLUSIONS: Polyomavirus DNA was frequently detected in pregnancy, whereas genomic rearrangements were rare, and no evidence of transplacental transmission of polyomavirus was obtained.

Acid-free glyoxal as a substitute of formalin for structural and molecular preservation in tissue samples.

Tissue fixation in phosphate buffered formalin (PBF) remains the standard procedure in histopathology, since it results in an optimal structural, antigenic and molecular preservation that justifies the pivotal role presently played by diagnoses on PBF-fixed tissues in precision medicine. However, toxicity of formaldehyde causes an environmental concern and may demand substitution of this reagent. Having observed that the reported drawbacks of commercially available glyoxal substitutes of PBF (Prefer, Glyo-fix, Histo-Fix, Histo-CHOICE, and Safe-Fix II) are likely related to their acidity, we have devised a neutral fixative, obtained by removing acids from the dialdehyde glyoxal with an ion-exchange resin. The resulting glyoxal acid-free (GAF) fixative has been tested in a cohort of 30 specimens including colon (N = 25) and stomach (N = 5) cancers. Our results show that GAF fixation produces a tissue and cellular preservation similar to that produced by PBF. Comparable immuno-histochemical and molecular (DNA and RNA) analytical data were obtained. We observed a significant enrichment of longer DNA fragment size in GAF-fixed compared to PBF-fixed samples. Adoption of GAF as a non-toxic histological fixative of choice would require a process of validation, but the present data suggest that it represents a reliable candidate.

Frequency of O6-methylguanine-DNA methyltransferase promoter methylation in cytological samples from small cell lung cancer.

BACKGROUND: In a phase II study for patients with relapsed small cell lung cancer (SCLC), the administration of Temozolomide, an alkylating agent used in gliomas and anaplastic astrocytoma, showed a effective activity when O(6) -methylguanine-DNA methyltransferase (MGMT) gene promoter was methylated. METHODS: We tested the feasibility of MGMT promoter status evaluation in small biopsies and cytological specimens routinely processed for diagnostic purposes. We tested samples from 56 patients with SCLC: 30 tissue biopsies, 17 fine-needle aspiration biopsy, 8 bronchial washing, and 1 was a sputum. Biopsies and fine-needle aspiration biopsy were fixed in formalin, bronchial washing and sputum in Dubosq Brazil. DNA was extracted after macrodissection of the areas containing the maximum number of cancer cells. MGMT promoter methylation status was assessed by methylation specific PCR. RESULTS: Methylation analysis was obtained in 54 samples (54/56) and failed in two bronchial wash. MGMT promoter was methylated in 35.2% of the cases without any significant difference between histological and cytological samples (37.9% vs. 32%). CONCLUSION: MGMT promoter methylation is present in SCLC and cytological samples are perfectly adequate for methylation analysis, even if they were taken during routine diagnostic procedures, using different fixative and with low number and percentage of cancer cells.

Merkel cell carcinoma arising in inguinal lymph node in a patient with von Willebrand disease after multiple blood transfusions.

Merkel cell carcinoma (MCC) is an uncommon neuroendocrine tumour of the skin; rare cases have been reported within the lymph nodes without a primary site. The detection of Merkel cell polyomavirus (MCPyV) DNA, integrated within the genome of MCC, suggests its role for the onset of this tumour. We report a case of MCC in an inguinal lymph node of a patient with Von Willebrand disease (VWD), who underwent multiple blood transfusions following haemorrhoidectomy. The diagnosis was performed on the bases of morphology and immunohistochemistry; genomic sequences of LT and VP1 regions of MCPyV were amplified from MCC using a quantitative polymerase chain reaction (qPCR) assay. High levels of MCPyV antibodies were detected in the patient's serum by ELISA method. We discuss the role of MCPyV in the development of this tumour, the use of viral DNA detection for confirming the diagnosis of MCC in unusual sites and the possibility of MCPyV transmission from blood donors.

KRAS mutational analysis in ductal adenocarcinoma of the pancreas and its clinical significance.

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.