Well-differentiated carcinoma (WDC) accounts for up to 90% of all thyroid cancers. The presence of a minor poorly differentiated (PD) component (mainly insular pattern) might represent an additional critical parameter for patients' prognosis and outcome. The role of both CXCR4 (a chemokine inducing cytoskeletal rearrangement and cell adhesion) and BRAF mutation have been studied in WDC (mainly papillary thyroid cancer and its variants), highlighting their critical role in tumor progression, local infiltration, and metastases. We discussed the clonal heterogeneity through the prognostic role of CXCR4 and BRAF mutation in WDC with a minor PD/insular component. Of our 16 WDC cases with a PD/insular component, up to 40% underwent surgery. The cases were subclassified according to the PD percentage as (1) <20% PD and (2) 20% to 40% PD, and were studied for CXCR4 expression and BRAF mutation. CXCR4 and molecular testing were distinctly performed on both components of each lesion. The majority of the cases (69%) showed an extrathyroid and metastatic dissemination. Regardless of the 2 categories, we had 8/16 (50%) patients with disease-free status. CXCR4 was negative in all 16 cases, whereas 3 of them (19%) had a mutated BRAF only in the WDC component of the lesion. WDCs with a minor PD pattern, even when <20%, showed more aggressive features than pure WDCs and should be entirely considered as PD carcinoma. The absence of CXCR4 expression and BRAF mutation in cancers with a minor PD component underlined different pathogenic and metastatic processes in comparison with WDCs.
Human immunodeficiency virus (HIV)-infected people exhibit a high incidence of vascular diseases. Since in the general population the high cardiovascular risk has been associated with an impaired endothelial cell function, we investigated circulating endothelial progenitor cells in HIV-positive patients.We evaluated circulating colony-forming unit-endothelial cell (CFU-EC) and endothelial colony-forming cell (ECFC) progenitors in 14 antiviral therapy-naive HIV-positive patients, in comparison with 15 normal controls.CFU-EC and ECFC derived from peripheral blood mononuclear cells from HIV-infected and HIV-uninfected individuals were recovered and evaluated for HIV genome presence by PCR. Vascular endothelial growth factor (VEGF) and apolipoprotein B mRNA-editing enzyme catalytic polypeptide like (APOBEC) subunits expression were evaluated in infected colonies by real-time PCR.We found that circulating CFU-EC but not ECFC were significantly reduced in HIV-positive patients and that proviral HIV DNA was detectable only in CFU-EC but not in ECFC. Furthermore, the expression of APOBEC subunits was significantly lower in CFU-EC than in circulating monocytes. Accordingly, the CFU-EC displayed a high content of proviral DNA copies, suggesting that these cells have a high sensitivity to the HIV infection.Although HIV does not affect the 'true endothelial progenitor' compartment, it infects and strongly depletes circulating endothelial progenitors with hematopoietic signature. We unravel a novel pathogenetic mechanism by which HIV infection might cause vascular diseases.
The occurrence of mitotic recombination of the short arm of chromosome 9 at the location of the JAK2 gene, determines that many patients with Polycythemia Vera (PV) and Primary Myelofibrosis (PM) harbour the JAK2V617F mutation in homozygous form (Scott et al, 2006). In contrast, those with essential thrombocythemia (ET) appear to be heterozygous in over 95% of cases (Scott et al, 2006). A more precise molecular quantification of the respective amounts of normal and mutant alleles highlighted that, either in PV or in ET, a high mutant allele burden is associated with a more symptomatic myeloproliferation, and probably includes an increased risk of vascular complication (Campbell et al, 2005; Vannucchi et al, 2007a; Vannucchi et al, 2007b). We recently observed that children with non-familial ET constitute a particular set of patients compared to their adult counterparts, with low incidence of the JAK2 mutation and thrombotic complications (Randi et al, 2006; Teofili et al, 2007). This study retrospectively investigated 13 children with ET and four with PV observed at the Departments of Paediatric Haematology of the University ‘La Sapienza’, Rome, and Paediatrics, Haemato-Oncology of the University of Padua between February 1988 and October 2008. All patients were diagnosed according to the PV Study Group or the World Health Organization criteria (Teofili et al, 2007). Among children with ET, seven were evaluated at diagnosis or during follow up, in the absence of any cytoreductive therapy (Theocharides et al, 2008), while six were on treatment at the time of the study (two with hydroxycarbamide, one with anagrelide; three patients experienced more than one line of treatment, including hydroxycarbamide, anagrelide and interferon). Among the children with PV, three were studied at diagnosis and one during therapy. Patient samples were obtained following informed consent from parents in accordance with the Declaration of Helsinki and with the approval from the local Institutional Review Boards. As a control group, we evaluated 32 adult patients with ET observed at the Haematology Department of the Catholic University of Rome between June 2004 and September 2008; 20 were investigated at diagnosis and 12 during follow-up in the absence of cytoreductive therapy. Table I shows the clinical and haematological features of the patients. Splenomegaly was defined by ultrasound scan reporting the longitudinal diameter of the spleen. The presence of the homozygous or heterozygous JAK2V617F mutation was investigated on the granulocyte cell fraction according to the method of Campbell et al, (2005); (Teofili et al, 2007) and the mutant allele burden was measured by a quantitative real time -polymerase chain reaction assay, according to the method of Vannucchi et al (2007b). All samples were analyzed in triplicate. Statistical analysis was performed with graphpad software by using the Mann–Whitney U-test for continuous variables and the Fisher’s exact test for categorical variables. P values <0·05 were considered statistically significant. As shown in Table I, clinical-haematological findings at diagnosis were similar in children and adult patients, except for a higher platelet count at diagnosis (P = 0·002) and a lower incidence of thrombosis (P = 0·048) in children. A mild tendency (statistically not significant) to a higher white blood cell (WBC) count in children than in adults was observed. This seemed to be in contrast to the more common presence of thrombotic complications in adult patients with higher WBC counts (Carobbio et al, 2008). All children and all adults but one with ET were heterozygous for the JAK2V617F mutation (data not shown). The median burden of mutated alleles in the seven children not receiving cytoreductive therapy was 20%, significantly lower than that of 33% observed in the group of adults (Fig 1A, P = 0·04). When the patients were grouped in quartiles according to the percentage of mutated alleles, the distribution of adults and children was significantly different (Fig 1B, P < 0·0001): the majority of children were in the 1st quartile, while 54% of the adults were distributed in the 2nd quartile; in addition no children had an allele burden higher than 50%, while 12% of the adult patient did. In children, the burden of mutated alleles at diagnosis did not correlate either with haematological parameters or with the presence of splenomegaly. Interestingly, the six children receiving cytoreduction showed a similar allele burden to those not receiving therapy (median value 31·5%, range 6–50, P = 0·78). Recent observations suggest that treatment can reduce the burden of JAK2V617F mutated alleles over time (Ricksten et al, 2008). It is possible that treated patients might, in reality, have a higher mutated allele burden at diagnosis, which is subsequently affected by the therapy needed to control the disease. An interesting observation emerging from the analysis of the medical history of this series of patients is that micro vessel disorders (in most cases headache), was the most frequent symptom requiring cytoreduction in childhood, contrary to adult patients, who are put on treatment mainly to prevent thrombosis or re-thrombosis. The four investigated children with PV exhibited a mutated allele burden of 48%, 43%, 44% and 39%, respectively, with the patient receiving antiproliferative therapy showing the lowest result. None of them exhibited the JAK2V617F mutation in the homozygous form. The values obtained in children with PV appeared significantly higher than that observed in children with ET (P = 0·012). Interestingly, no thrombotic events were recorded among the children with PV, with a median follow-up of 72 months. Cario et al (2008) have recently published clinical and haematological data in a series of eight children affected by PV. Among them, six had JAK2V617F mutation and two had exon 12 JAK2 mutation. The authors reported that two children suffered from Budd–Chiari syndrome (one patient at diagnosis and one during the follow-up). Unfortunately, no data about the burden of mutated alleles is given in these patients. Indeed, it could be hypothesized that the children suffering from thrombosis either had a higher burden than ours, or that they were homozygous for the mutation (Cario et al, 2008). (A) JAK2V617F allele burden in 32 adults and seven children with ET. Only patients not receiving cytoreductive treatment were included (boxes represent the interquartile range that contains 50% of the subjects, the horizontal line mean values; the bars show the upper and the lower range of values). (B) Distribution of adult (black blocks) and childhood (white blocks) patients in quartiles, according to the JAK2V617F mutant allele burden. In conclusion, this study showed that, similar to adults, children with ET exhibit lower amounts of mutant alleles than children with PV. Moreover, our findings indicate that ET in childhood is characterized by a lower mutant allelic burden than in adults, associated with a more pronounced thrombocytosis and a reduced incidence of thrombosis. This work was supported by Prin 2006, Ministero Università e Ricerca Scientifica (Rome and Padua, Italy) and by Fondi d’Ateneo, Progetti D1 2007–2008, Università Cattolica (Rome, Italy).
The past 20 years in cytopathology have demonstrated tremendous growth and evolution in the cytopathology of organs located below the diaphragm, with much of the progress centering on the pancreas.Cancer Cytopathology has been at the forefront of reporting advances in the field of pancreaticobiliary cytology, especially for the diagnosis of pancreatic cysts.
It has been generally demonstrated that the valine-to-glutamic acid substitution at position 600 (V600E) in the v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) gene is an effective diagnostic/prognostic marker mainly for papillary thyroid carcinoma (PTC). The detection of this mutation typically has been achieved using DNA-based techniques. The recently introduced monoclonal V600E antibody (clone VE1) is likely to be an alternative strategy for detecting this mutation in thyroid lesions. The authors investigated molecular and immunocytohistochemical BRAF analyses in a prospective series of samples from patients with PTC.Fifty-five prospective cytohistologic samples that were diagnosed as PTC were studied using both DNA BRAF testing and the VE1 antibody. The intensity of VE1 expression was graded from 0 (negative) to 3+ (strong).All diagnoses were histologically confirmed. In total, 37 samples with mutated BRAF and 18 samples with wild-type BRAF were reported with 100% cytohistologic concordance. Cytologic VE1 expression revealed 25 negative samples (including 16 with 0 expression and 9 with 1+ expression) and 30 positive samples (including 16 with 2+ expression and 14 with 3+ expression). On histology, there were 27 negative samples (20 with 0 expression and 7 with 1+ expression) and 28 positive samples (14 with 2+ expression and 14 with 3+ expression). Four specimens with the BRAF mutation had discrepancies in VE1 intensity between cytology and histology. Furthermore, 6 BRAF-mutated samples produced negative VE1 results.Although it has limitations, the VE1 antibody represents a feasible first-line approach for evaluating BRAF mutation status and may be a valid tool in the selection of samples for molecular analysis. The current report highlights the statistically significant difference between molecular and VE1 positivity in PTC (P < .0001). Nevertheless, in the authors' experience, BRAF mutations are more accurate for identifying VE1-negative cases.
