Follicular Variant of Papillary Thyroid Carcinoma: Accuracy of FNA Diagnosis and Implications for Patient Management

Follicular variant of papillary thyroid carcinoma (FVPTC) creates a continuous diagnostic dilemma among pathologists because of the paucity of nuclear changes of papillary carcinoma and overlapping features with benign and other neoplastic follicular lesions. Current guidelines for the management of thyroid nodules recommend surgery for confirmed PTC, suspicious for PTC, and follicular neoplasm cases, while further immediate diagnostic studies or treatment are not routinely required if the nodule is benign on cytology. This study is designed to determine the accuracy of cytology in the diagnosis of FVPTC, based on the Bethesda classification system, and determine the implications for patient management based on the current recommendation. Based on a retrospective review of cytologic diagnoses between January 2008 and December 2011, thyroid fine needle aspiration (FNA) cytology specimens with subsequent surgical intervention and a final diagnosis of FVPTC were selected. The cytologic diagnoses were compared with the final diagnoses, and the percentage of cases contributing to the final diagnosis of FVPTC was calculated for each diagnostic category. Triage efficiency and diagnostic accuracy were calculated. One hundred and fifty-two cases with histologic confirmation of FVPTC were identified (representing 128 patients—101 female, 27 male). All patients had undergone either lobectomy with completion thyroidectomy or total thyroidectomy. The cytologic diagnosis of “positive for malignancy” accounted for only 27 % of the final histologic diagnosis of FVPTC, while suspicious for carcinoma, follicular neoplasm, follicular lesion of undetermined significance, and benign accounted for 11, 23, 23, and 16 % of the final diagnosis of FVPTC, respectively. Only 18 % of the 55 cases tested were positive for BRAF mutation. The subtle nuclear features of FVPTC pose challenges for an accurate diagnosis. Therefore, a better approach is to triage these cases for surgical intervention and/or further evaluation of the particular nodule. Our triage efficacy for FVPTC was 84 %; however, the diagnostic accuracy of PTC was 38 %. A negative diagnosis on FNA has diagnostic and management implications for up to 16 % of cases because they may have no further immediate diagnostic studies or treatment. BRAF mutation analysis provides minimal effect on diagnostic accuracy.     

Multifocal Fibrosing Thyroiditis and Its Association with Papillary Thyroid Carcinoma Using BRAF Pyrosequencing

Multifocal fibrosing thyroiditis (MFT) is characterized by numerous foci of fibrosis in a stellate configuration with fibroelastotic and fibroblastic centers entrapping epithelial structures. MFT has been proposed as a risk factor for papillary thyroid carcinoma (PTC) development. We attempted to identify whether MFT showed such molecular changes and could possibly be related to PTC. We identified seven cases of PTC with MFT in our institutional pathology database and personal consult service of one of the authors (VAL) for the years 1999 to 2012. Areas of PTC, MFT, and normal tissue were selected for BRAF analysis. Macro-dissection, DNA extraction and PCR amplification, and pyrosequencing were performed to detect BRAF mutations in codon 600. All of the MFT lesions and normal thyroid tissue were negative for BRAF mutations. Of the seven PTCs analyzed, five (71 %) were negative for BRAF mutations, while two cases were positive. In our study, none of the MFT lesions harbored BRAF mutations, whereas 29 % (two of seven) PTCs in the same gland were positive. Hence, in this small study, we found no evidence that the MFT lesion is a direct precursor to PTC. It is likely an incidental bystander in the process and a reflection of the background thyroiditis.     

Synchronous Occurrence of Medullary and Papillary Carcinoma of the Thyroid in a Patient with Cutaneous Melanoma: Determination of BRAFV600E in Peripheral Blood and Tissues. Report of a Case and Review of the Literature

The purpose of this study is to describe a case of concurrent medullary and papillary thyroid carcinoma (MTC and PTC) and cutaneous melanoma and to analyze BRAF^V600E mutation in plasma and tissues. We report the clinical history and the laboratory, imaging, and histopathological findings of a 47-year-old man affected by multinodular goiter. BRAF^V600E-mutated DNA was quantified in plasma samples and in cancer sections by quantitative real-time polymerase chain reaction (qPCR). At ultrasound examination, the dominant right nodule of the thyroid was weakly hyperechoic and hypervascularized, while the left one was hypoechoic without internal vascularization. Regional lymphadenomegalia was not detected. Basal plasma calcitonin was elevated, and the patient underwent total thyroidectomy and resection of central cervical lymph nodes. Histopathological examination identified two distinct foci of MTC and PTC and micrometastasis of well-differentiated carcinoma in one of the six resected lymph nodes. RET proto-oncogene germline mutations were not detected. Cutaneous melanoma of the thorax was subsequently diagnosed. BRAF^V600E tissue DNA was detected in PTC and melanoma but not in MTC. The cell-free plasma percentage of BRAF^V600E DNA was detected in pre-thyroidectomy peripheral blood and was drastically reduced after cancer treatments. This study confirms the occurrence of synchronous MTC and PTC and is the first evidence of the co-existence of melanoma and distinct thyroid cancers of different origin. BRAF^V600E allele was detected in PTC and melanoma but not in MTC tissues. BRAF^V600E molecular quantification in pre- and post-treatment blood supports our previous data, suggesting its possible role in diagnosis and follow-up of BRAF-positive tumors.     

Mutational profiling of the RAS,PI3K,MET and b-catenin pathways in cancer of unknown primary: a retrospective study of the Hellenic Cooperative Oncology Group

Cancer of unknown primary origin (CUP) had a poor prognosis, determined by clinico-histological characteristics, partly due to the lack of insights on its biology. We screened tumour DNA from 87 patients with CUP for CTNNB1 (coding exons 2,3,4,5), MET (coding exon 18), PIK3CA (coding exons 9,20), KRAS (coding exons 1,2), BRAF (coding exon 15) gene mutations by using dd-sequencing and evaluated their impact on prognosis. Mutated gene incidences in the 87 CUP cases were: KRAS 11 (12.6 %), BRAF 5 (5.7 %), PIK3CA 8 (9 %), MET 6 (6.7 %) and CTNNB1 18 (20.7 %). Several mutations in the KRAS gene were not the commonly encountered mutations in other solid tumours. Activating mutations were observed in 10.2 % in KRAS, 4.5 % in BRAF, 6.6 % in PIK3CA, 4.5 % in MET, and 19.5 % in CTNNB1. Activating mutations in PIK3CA coding exon 9 were inversely correlated with MET coding exon 18 activating mutations (p = 0.036). MET activating mutations were prognostic for poor Progression-Free Survival (median PFS 5 vs 9 months, p = 0.009) and Overall Survival (median OS 7 vs 20 months, p = 0.005). The complex profile of either CTNNB1 or MET mutations also had an adverse prognostic significance (median OS 11 vs 21 months, p = 0.015). No other gene mutation exhibited prognostic significance. In multivariate analysis, poor performance status, male gender, visceral disease and adenocarcinoma histology, but not gene mutations, were independently associated with poor patient outcome. CTNNB1 gene mutations are frequent, and along with MET mutations have an adverse prognostic effect in patients with CUP.     

Pulmonary adenocarcinoma with enteric differentiation: Dissecting oncogenic genes alterations with DNA sequencing and FISH analysis

Background

Pulmonary Adenocarcinoma with Enteric Differentiation (PAED) is a rare subtype of adenocarcinoma of emerging interest, recently introduced in the 2015 WHO classification. However, little is known about major molecular signatures of this class of adenocarcinomas and information about new biomarkers totally lack.

BRAF and RAS Mutations in Follicular Variants of Papillary Thyroid Carcinoma

Follicular variants of papillary thyroid carcinoma (FVPTC), particularly the encapsulated subtype, often cause a diagnostic dilemma. Therefore, many FVPTCs are interpreted as “indeterminate” in preoperative fine-needle aspiration (FNA). The aim of this study was to analyze the genotypic changes in BRAF codons 600 and 601, as well as the N, H, and KRAS codons 12, 13, and 61 in FVPTCs and investigate the usefulness of preoperative BRAF and RAS mutation analysis as an adjunct diagnostic tool along with routine FNA. Surgically resected thyroid nodules were reviewed to establish the histological diagnosis of FVPTC. All preoperative FNA diagnoses were categorized according to the Bethesda Reporting System. Mutations in BRAF codons 600 and 601, and N, H, KRAS codons 12, 13, and 61 were analyzed by pyrosequencing. Of 132 cases, 81 (61.4 %) had a point mutation in one of the BRAF V600E, BRAF K601E, or RAS oncogenes; BRAF V600E in 43(32.6 %), BRAF K601E in three (2.3 %), and RAS in 35 (26.5 %) cases. All mutations were mutually exclusive. Of 78 cases with an FNA indeterminate category diagnosis, 51 (65.4 %) were positive for mutations: 24 for BRAF V600E, 3 for BRAF K601E, and 24 for the RAS gene. The KRAS mutation was more frequently found than the HRAS mutation, comprising 22.9 % of the RAS mutations, and all KRAS mutations were located at codon 61. This study demonstrated that either BRAF or RAS mutations were present in two thirds of FVPTCs and these mutations were mutually exclusive.     

RET/PTC rearrangement is prevalent in follicular Hürthle cell carcinomas

de Vries M M, Celestino R, Castro P, Eloy C, Máximo V, van der Wal J E, Plukker J T M, Links T P, Hofstra R M W, Sobrinho‐Simões M & Soares P
(2012) Histopathology  61, 833–843 RET/PTC rearrangement is prevalent in follicular Hürthle cell carcinomas Aims: The molecular alterations underlying follicular Hürthle cell carcinomas (FHCCs) are largely unknown. In an attempt to clarify this issue, we analysed a series of Hürthle cell tumours for the presence of RET/PTC and PAX8/PPARG rearrangements and BRAF, HRAS and NRAS mutations. Methods and results: We investigated a series of 20 follicular Hürthle cell tumours [17 FHCCs and three follicular Hürthle cell adenomas (FHCAs)]. RET/PTC rearrangements were found in 33% of FHCAs and in 38% of FHCCs. All RET/PTC‐positive FHCCs had a solid pattern of growth. PAX8/PPARG rearrangement was present in 27% of the FHCCs which displayed, in most cases, a follicular architecture. NRAS mutation was detected in one FHCC. An FHCC with a solid/microfollicular growth pattern scored positive for both RET/PTC and PAX8/PPARG rearrangement. Conclusions: Our study has shown a significant association between RET/PTC rearrangements and FHCCs with a solid growth pattern, thus raising the possibility of using tyrosine kinase inhibitors for the treatment of patients with FHCCs, which are often refractory to radioiodine treatment.     

Thyroid carcinoma-associated genetic mutations also occur in thyroid lymphomas

Molecular testing for mutations activating the mitogen-associated protein kinase signaling pathway is being used to help diagnose thyroid carcinomas. However, the prevalence of these mutations in thyroid lymphomas has not been reported. Therefore, we studied the prevalence of BRAF, NRAS, HRAS, and KRAS mutations in 33 thyroid lymphomas and correlated the mutational status with the clinical, pathological, cytogenetic, and immunophenotypic findings. Eleven cases were also tested for PAX8/PPARγ translocations. The lymphomas included 25 diffuse large B-cell lymphomas, 6 extranodal marginal-zone lymphomas of mucosa-associated lymphoid tissue type, and 2 follicular lymphomas. Seventeen diffuse large B-cell lymphomas were germinal center type, six non-germinal center type, and two unclassifiable (Hans algorithm). None of the cases had an associated thyroid carcinoma. Mutations of the BRAF gene were identified in six (24%) diffuse large B-cell lymphomas (D594G in three germinal center diffuse large B-cell lymphomas, K601N in two germinal center diffuse large B-cell lymphomas, and V600E in one non-germinal center diffuse large B-cell lymphoma) and of the NRAS gene in two (8%) non-germinal center diffuse large B-cell lymphomas (Q61K and Q61H). BRAF and NRAS mutations were not found in any extranodal marginal-zone lymphomas of mucosa-associated lymphoid tissue type or follicular lymphomas. HRAS and KRAS mutations were not identified in any of the cases, nor were PAX8/PPARγ translocations found. Thus, interpretation of finding a BRAF or NRAS mutation in the thyroid, particularly in preoperative thyroid aspirates, must take into account the differential diagnosis of a lymphoma. In addition to the diagnostic importance, our data also demonstrate that alteration in the mitogen-associated protein kinase pathway may have a role in the pathogenesis of some large B-cell lymphomas of the thyroid with potential therapeutic implications.     

The preeminence of growth pattern and invasiveness and the limited influence of <Emphasis Type="Italic">BRAF</Emphasis> and <Emphasis Type="Italic">RAS</Emphasis> mutations in the occurrence of papillary thyroid carcinoma lymph node metastases

Prognostic factors indicative of papillary thyroid carcinoma (PTC) aggressive behaviour remain incompletely established partially due to the different composition of the series on record regarding the relative proportion of classic PTC (CPTC) and follicular variant PTC (FVPTC) subtypes. Several clinico-morphological features of PTC, together with the occurrence of BRAF mutations, are still not fully accepted as markers of aggressiveness. In the present clinico-pathological study of a series of 75 CPTC and FVPTC cases, we evaluated the relative contribution of the morphological features of the tumours and their BRAF and N-RAS status for the occurrence of nodal metastases. The morphological features most closely related to the occurrence of nodal metastases were extra-thyroid extension and poorly circumscribed growth pattern, in both CPTC and FVPTC. Additional features significantly associated to nodal metastases were multicentricity in the CPTC and vascular invasion in the FVPTC group. BRAF V600E mutation was detected in 29% of tumours, 41% of CPTC and 16% of FVPTC; N-RAS Q61R mutation was detected in 6% of tumours, 3% of CPTC and 10% of FVPTC. BRAF mutation was significantly more frequent in the CPTC group and in females, and it was detected only in patients older than 20 years, suggesting a late tumourigenic effect in the development of PTC. BRAF mutation was not significantly associated to any of the other studied features related to aggressiveness or nodal metastases. These results highlight the importance of infiltrative growth pattern and invasiveness over the presence of BRAF mutation in classic and follicular variant PTC for the development of nodal metastases.     

Analysis of <Emphasis Type="Italic">KRAS</Emphasis> and <Emphasis Type="Italic">BRAF</Emphasis> genes mutation in the central nervous system metastases of non-small cell lung cancer

KRAS mutations are associated with tumor resistance to EGFR TKIs (erlotinib, gefitinib) and to monoclonal antibody against EGFR (cetuximab). Targeted treatment of mutated RAS patients is still considered as a challenge. Inhibitors of c-Met (onartuzumab or tiwantinib) and MEK (selumetinib—a dual inhibitor of MEK1 and MEK2) signaling pathways showed activity in patients with mutations in KRAS that can became an effective approach in carriers of such disorders. BRAF mutation is very rare in patients with NSCLC, and its presence is associated with sensitivity of tumor cells to BRAF inhibitors (vemurafenib, dabrafenib). In the present study, the frequency and type of KRAS and BRAF mutation were assessed in 145 FFPE tissue samples from CNS metastases of NSCLC. In 30 patients, material from the primary tumor was simultaneously available. Real-time PCR technique with allele-specific molecular probe (KRAS/BRAF Mutation Analysis Kit, Entrogen, USA) was used for molecular tests. KRAS mutations were detected in 21.4 % of CNS metastatic lesions and in 23.3 % of corresponding primary tumors. Five mutations were identified both in primary and in metastatic lesions, while one mutation only in primary tumor and one mutation only in the metastatic tumor. Most of mutations were observed in codon 12 of KRAS; however, an individual patient had diagnosed a rare G13D and Q61R substitutions. KRAS mutations were significantly more frequent in adenocarcinoma patients and smokers. Additional analysis indicated one patient with rare coexistence of KRAS and DDR2 mutations. BRAF mutation was not detected in the examined materials. KRAS frequency appears to be similar in primary and CNS.     

Molecular profile and clinical-pathologic features of the follicular variant of papillary thyroid carcinoma. An unusually high prevalence of ras mutations

The follicular variant (FV) of papillary thyroid carcinoma is characterized by a follicular growth pattern and cytologic features of papillary carcinoma. ret/PTC rearrangements are common in classic papillary thyroid carcinoma (PTC) and PAX8-PPAR gamma and ras mutations in follicular thyroid carcinoma. Their prevalence in FV has not been established. We studied these genetic alterations and clinical-pathologic features in 30 FV cases and compared those with 46 non-FV papillary carcinomas. FV cases revealed 1 ret/PTC rearrangement (3%) and 13 ras mutations (43%). Non-FV cases harbored 13 ret/PTC (28%) (P = .006) and no ras mutations (P = .0002). No PAX8-PPAR gamma was found in either group. FV cases demonstrated a significantly higher prevalence of tumor encapsulation, angiovascular invasion, and poorly differentiated areas and a lower rate of lymph node metastases. These data indicate that the FV of papillary carcinoma has a distinct set of molecular alterations and is characterized by a high frequency of ras point mutations.     

Macrofollicular Variant of Papillary Thyroid Carcinoma with Extensive Lymph Node Metastases

The macrofollicular variant of papillary thyroid carcinoma is a rare subtype of the follicular variant of papillary thyroid carcinoma and is usually characterized by an indolent clinical course. The tumors are prone to be misdiagnosed as benign due to their macrofollicular architecture and bland cytologic features. We report a rare case of the macrofollicular variant of papillary thyroid carcinoma with extensive lymph node metastases. The patient was a 48-year-old female with a right thyroid nodule and multiple enlarged lymph nodes in the right neck. It was not possible to make a definitive diagnosis of malignancy on fine-needle aspiration cytology and intraoperative frozen section. She underwent total thyroidectomy with right modified radical neck dissection. The surgical specimen showed a 2.5?×?1.5?×?10 cm, well-circumscribed macrofollicular variant of papillary thyroid carcinoma in the right lobe and multiple central and right lateral neck lymph node metastases. Molecular testing for BRAF, NRAS, HRAS, and KRAS was all negative. We then reviewed the demographic and clinicopathologic characteristics of 71 patients with the macrofollicular variant of papillary thyroid carcinoma. The cytologic or histopathologic diagnosis of macrofollicular variant of papillary thyroid carcinoma can be difficult. Extensive lymph node metastases caused by the macrofollicular variant of papillary thyroid carcinoma may occur even in the absence of capsular or lymphovascular invasion. This review will help to better understand the nature of the macrofollicular variant of papillary thyroid carcinoma.     

BRAF V600E mutation detection by immunohistochemistry in colorectal carcinoma

The serine/threonine‐protein kinase B‐raf (BRAF) is an oncogene mutated in various neoplasms, including 5–15% of colorectal carcinomas. The T1799A point mutation, responsible for a large majority of these alterations, results in an amino acid substitution (V600E) causing the constitutive activation of a protein kinase cascade. BRAF V600E in MLH1 deficient tumors implicates somatic tumor‐only methylation of the MLH1 promoter region instead of a germline MLH1 mutation. BRAF V600E also predicts poor prognosis in microsatellite stable colorectal cancers and may be a marker of resistance to anti‐EGFR therapy in metastatic disease. Currently, only molecular methods are available for assessing BRAF mutational status. An immunohistochemical approach is evaluated here. Colon cancers from 2008 to 2012 tested by pyrosequencing for BRAF V600E mutation were selected. A total of 31 tumors with (n = 14) and without (n = 17) the BRAF V600E mutation were analyzed by immunohistochemistry using a commercially available antibody specific to the V600E‐mutated protein. All 14 colorectal carcinomas with the BRAF V600E mutation demonstrated cytoplasmic positivity in tumor cells with the anti‐BRAF antibody. In a minority of cases, staining intensity for the mutated tumor samples was weak (n = 2) or heterogeneous (n = 4); however, the majority of cases showed diffuse, strong cytoplasmic positivity (8 of 14 cases). None of the 17 BRAF wild‐type colorectal cancers showed immunoreactivity to the antibody. The overall sensitivity and specificity of the immunohistochemical BRAF V600E assay was 100%. Detection of the BRAF V600E mutation in colorectal cancer by immunohistochemistry is a viable alternative to molecular methods. © 2013 Wiley Periodicals, Inc.     

Frequency of KRAS, BRAF, and NRAS mutations in colorectal cancer

Mutational analysis of KRAS codons 12 and 13 is standard for patients with metastatic colorectal cancer since mutations in these codons predict lack of response to anti-EGFR therapies. However, even among patients whose tumors are wildtype for KRAS codons 12 and 13, only a subset respond to therapy. Since additional activating mutations downstream of EGFR may also play a role in treatment resistance, we sought to establish the frequency of these mutations. We evaluated 2121 colorectal tumors for mutations in codons 12 and 13 of the KRAS gene. A subset of these samples, comprised of 513 samples wildtype for KRAS codons 12 and 13, were tested for mutations in codons 61 and 146 of KRAS, codon 600 of BRAF, and codons 12, 13, and 61 of NRAS. Mutation status was determined by targeted pyrosequencing. Mutations in KRAS codon 12 or 13 were identified in 900/2121 (42.4%) samples. Of the 513 wildtype samples tested for additional mutations, 78 samples were mutant for BRAF, 19 for KRAS codon 61, 17 for KRAS codon 146, and 26 for NRAS. In total, 140/513 (27.3%) tumors wildtype for KRAS codons 12 and 13 harbored a mutation in another of the RAS pathway genes. While further study is needed to determine the full therapeutic implications of mutations in these codons, mutational testing of these codons may be useful for identifying a significant proportion of patients who may also be resistant to anti-EGFR therapies.     

Mutational status of KRAS,NRAS, and BRAF in primary clear cell ovarian carcinoma

Ovarian clear cell carcinoma (OCCC) is a subtype of epithelial ovarian cancer with characteristic biological features and aggressive clinical behavior. OCCCs show a pattern of gene mutations different from other type I ovarian malignancies, notably a higher frequency of PIK3CA mutations. In low grade serous ovarian cancer, KRAS and BRAF mutations are frequent, but little data are available on the mutational status of these genes in OCCCs. To clarify this issue, we designed a clinicopathological study with the aim to establish the incidence of KRAS, NRAS, and BRAF hot spot mutations in OCCC. Between December 2006 and June 2012, 22 patients with a proven diagnosis of OCCC were admitted to our Institutions. In all cases, final diagnosis was established according to FIGO and WHO criteria. All women received complete surgical staging. The PyroMark Q24 system (Qiagen GmbH, Hilden, Germany) was used for pyrosequencing analysis of KRAS, NRAS, and BRAF hot spot regions on 2.5-μm sections of formalin-fixed paraffin-embedded tissue from primary OCCC. Pyrosequencing analysis of KRAS, NRAS, and BRAF hot spot regions revealed the presence of mutations only at codon 12 in exon 2 of KRAS in 3 of 22 (14 %) cases. We found no mutations in the hot spot regions of NRAF (exons 2, 3, 4) or BRAF (exon 15). The median age of women with a KRAS mutated OCCC was 74 years. These OCCC were unilateral FIGO stage IA lesions in two cases associated with foci of endometriosis. We conclude that in 14 % of OCCCs, a KRAS mutation occurs in codon 2 exon 2. NRAS and BRAF mutations were not found.     

Clinicopathological Features of Rare BRAF Mutations in Korean Thyroid Cancer Patients

The most common BRAF mutation in thyroid cancer is c.1799T>A (p.Val600Glu), and other BRAF mutations are rarely reported. We investigated the clinicopathological features of thyroid cancer with rare BRAF mutations. A total of 2,763 patients with thyroid cancer underwent molecular testing by direct DNA sequencing for mutations in BRAF exon 15. Among them, 2,110 (76.4%) had BRAF mutations. The c.1799T>A mutation was found in 2,093 (76.9%) of 2,722 papillary carcinomas and in one of 7 medullary carcinomas. Sixteen cases (0.76%) harbored rare mutation types. Five cases had single-nucleotide substitutions, 5 cases had small in-frame deletion or insertion, and one harbored a two-nucleotide substitution. Of these mutations, 2 were novel (c.1797_1798insGAGACTACA, c.[1799T>A; 1801_1812del]). The c.1801A>C mutation was identified in 4 follicular variant papillary carcinomas and one follicular carcinoma. None of the patients with the c.1801A>C mutation showed extrathyroidal extension or lymph node metastasis. The prevalence of rare BRAF mutations was 0.76% of all BRAF-positive thyroid cancers, and the rare mutations were associated with less aggressive pathologic features. Although BRAF mutations are detected exclusively in papillary carcinoma, they are also found in medullary carcinoma and follicular carcinoma.

Graphical Abstract

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Mutations of FLT3, NRAS, KRAS, and PTPN11 are frequent and possibly mutually exclusive in high hyperdiploid childhood acute lymphoblastic leukemia

Although it has been suggested that mutations of the FLT3, NRAS, KRAS, and PTPN11 genes are particularly frequent in high hyperdiploid (>50 chromosomes) pediatric acute lymphoblastic leukemias (ALLs), this has as yet not been confirmed in a large patient cohort. Furthermore, it is unknown whether mutations of these genes coexist in hyperdiploid cases. We performed mutation analyses of FLT3, NRAS, KRAS, and PTPN11 in a consecutive series of 78 high hyperdiploid ALLs. Twenty-six (33%) of the cases harbored a mutation, comprising six activating point mutations and one internal tandem duplication of FLT3 (7/78 cases; 9.0%), eight codon 12, 13, or 61 NRAS mutations (8/78 cases; 10%), five codon 12 or 13 KRAS mutations (5/78 cases, 6.4%), and seven exon 3 or 13 PTPN11 mutations (7/78 cases; 9.0%). No association was seen between the presence of a mutation in FLT3, NRAS, KRAS, or PTPN11 and gender, age, white blood cell count, or relapse, suggesting that they do not confer a negative prognostic impact. Only one case harbored mutations in two different genes, suggesting that mutations of these four genes are generally mutually exclusive. In total, one third of the cases harbored a FLT3, NRAS, KRAS, or PTPN11 mutation, identifying the RTK-RAS signaling pathway as a potential target for novel therapies of high hyperdiploid pediatric ALLs.     

BRAF mutation in sporadic colorectal cancer and Lynch syndrome

The aim of the study was to detect mutations of BRAF oncogene in colorectal cancer and to use this information to identify Lynch syndrome patients. Consecutive cases of primary colorectal cancer (n?=?137) were analyzed for MLH1 protein expression using immunohistochemistry (IHC). BRAF V600E mutation was detected by IHC using a specific monoclonal antibody (VE1) and by qPCR. All MLH1 protein-negative cases were subjected to microsatellite instability analysis and MLH1 promoter methylation assay. MLH1 protein expression deficiency and high microsatellite instability (MSI-H) were detected in 18 of the 137 (13.1 %) consecutive colorectal cancer specimens. Detection of the BRAF V600E mutation by IHC was 100 % sensitive and specific as compared to qPCR, and this mutation was frequently present in the MSI-H group (77.8 %; 14/18) and less frequently in the microsatellite-stable group (7.6 %; 9/118). All BRAF V600E mutated cases of the MSI-H group presented with a MLH1 promoter methylation (14/14) as detected by methylation-specific multiplex ligation-dependent probe amplification. When BRAF was wild type in the MSI-H group, only one MLH1 promoter methylation was detected (1/4), and of the remaining three cases without MLH1 methylation, two were identified to harbor an MLH1 mutation consistent with Lynch syndrome. Finally, 11 previously confirmed Lynch syndrome cases were analyzed for BRAF V600E mutation, and all of them were wild type. In conclusion, detection of BRAF V600E in colorectal cancer specimens by IHC is sensitive and specific and may help to identify Lynch syndrome patients.     

Intratumoural lymph vessel density is related to presence of lymph node metastases and separates encapsulated from infiltrative papillary thyroid carcinoma

Papillary thyroid carcinoma (PTC) gives frequently rise to nodal metastases via lymphatic vessels while follicular thyroid carcinoma (FTC) metastasises mainly via blood vessels to lung and bones. The follicular variant of PTC (FVPTC) encompasses the infiltrative subtype (I-FVPTC), which shares most of the features of classic PTC (CPTC), and the encapsulated subtype (E-FVPTC), which appears to be related to minimally invasive FTC. In an attempt to contribute to the understanding of the aforementioned differences, we evaluated intratumoural and peritumoural lymph vessels density (LVD), using the immunomarker D2-40 in a series of E-FVPTC, I-FVPTC, and CPTC with known BRAF and RAS status. None of the E-FVPTC cases presented extra-thyroid extension, lymph vessel invasion or nodal metastases, at variance with I-FVPTC and CPTC cases. The BRAF V600E mutation was detected in 8.3% of E-FVPTC, 25.0% of I-FVPTC and in 40.7% of CPTC, while N-RAS Q61R mutation was detected only in 10.3% of FVPTC cases. Only one case of E-FVPTC (8.3%) had intratumoural D2-40-stained vessels in contrast to their presence in 76.5% of the cases of I-FVPTC. Intratumoural LVD determined by D2-40 expression correlated with the occurrence of extra-thyroid extension, lymph vessel invasion and lymph node metastases in PTC cases. At variance with intratumoural LVD, peritumoural LVD was not associated with any clinic-pathological or molecular feature, being similar in E-FVPTC, I-FVPTC and CPTC. Our study highlights the role of intratumoural lymph vessels in PTC nodal metastisation and reinforces the importance of distinguishing E-FVPTC from I-FVPTC regarding invasiveness, metastatic pattern and molecular profile.