Update regarding the role of PD-L1 in oncocytic thyroid lesions on cytological samples.

AIMS: Several papers have shown that programmed death-ligand 1 (PD-L1) expression is a relevant predictive biomarker in anti-PD-L1 cancer immunotherapy. While its role in several human cancers is correlated with poor prognosis and resistance to anticancer therapies, in thyroid cancers the role of PD-L1 remains questionable. Few articles have studied PD-L1 in thyroid fine-needle aspiration cytology (FNAC), demonstrating a possible correlation with papillary thyroid carcinoma. However, its role in oncocytic thyroid lesions remains controversial. We accordingly examine the performance of PD-L1 immunostaining in liquid based cytology (LBC) from oncocytic lesions. METHODS: From January 2019 to March 2021, 114 thyroid lesions diagnosed by FNAC from lesions with a predominant oncocytic component, were enrolled for evaluation by PD-L1 immunostaining on both LBC and corresponding histology samples. RESULTS: The FNAC cohort included 51 benign (B, negative controls), 4 atypia of undetermined significance/follicular lesions of undetermined significance (AUS/FLUS), 57 follicular lesions (follicular neoplasm/suspicious for FN, FN/SFN) and 2 suspicious for malignancy (SFM) cases. Fifty-four cases (11B, 2 AUS/FLUS, 39 FN/SFN and 2 SFM) had histological follow-up including: 1B case resulted as a hyperplastic oxyphilic nodule in Hashimoto thyroiditis (HT), 10B as goitre, 2 AUS/FLUS cases as oncocytic adenomas (OAs); 39 FN/SFN included 27 OAs, 4 FA and 8 oncocytic follicular carcinoma (OFC). The two SFM cases were diagnosed on histopathology as OAs. Increased plasma membrane and cytoplasmic PD-L1 expression were found in 47 cases of the LBC cases (41.2%). Among the histological series, 67.3% of OAs and 75% of OFC had PD-L1 expression, while negative PD-L1 was found in hyperplastic oncocytic cells in HT. A positivity in more than 30% of the neoplastic cells was found in 72.9% of the cases including six OFC. CONCLUSIONS: These data suggest that PD-L1 expression is expressed in oncocytic thyroid lesions. While weak PD-L1 expression failed to discriminate benign from malignant lesions, OFC demonstrated more intense cytoplasmic and membranous expression.

The Role of Cytology in the Diagnosis of Subcentimeter Thyroid Lesions.

Thyroid nodules are common and typically detected by palpation and/or ultrasound (US). Guidelines have defined the management of large nodules, but controversy exists regarding nodules ≤ 1 cm. We evaluated a cohort of patients with subcentimeter nodules to determine their rate of malignancy (ROM). A total of 475 thyroid FNAs of lesions ≤ 1 cm with available follow-up were identified from January 2015-December 2019. For comparative analysis, we added a control series of 606 thyroid lesions larger than 1 cm from the same reference period. All aspirates were processed with liquid-based cytology and classified according to The Bethesda System for Reporting Thyroid Cytopathology (TBSRTC). Subcentimeter nodules were stratified as 35 category I-non-diagnostic cases (ND; 7.3%), 144 category II-benign lesions (BL; 30.3%), 12 category III-atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS; 2.5%), 12 category IV-follicular neoplasm/suspicious for follicular neoplasm (FN/SFN; 2.5%), 124 category V-suspicious for malignancy (SM; 26.1%), and 148 category VI-positive for malignancy (PM; 31.1%). A total of 307 cases (64.6%) underwent subsequent surgery. Only one ND and three BLs had a malignant outcome. ROM for indeterminate lesions (III + IV) was 3.2%; with 1.6% for category III and 3.2% for category IV. ROM for the malignant categories (V + VI) was 88.2%. The control cohort of lesions demonstrated a higher number of benign histological diagnoses (67.3%). We documented that 57.2% of suspected subcentimeter lesions were malignant, with a minor proportion that belonged in indeterminate categories. There were very few ND samples, suggesting that aspirates of subcentimeter lesions yield satisfactory results. Suspected US features in subcentimeter lesions should be evaluated and followed by an interdisciplinary team for appropriate patient management.

How limited molecular testing can also offer diagnostic and prognostic evaluation of thyroid nodules processed with liquid-based cytology: Role of TERT promoter and BRAF V600E mutation analysis.

BACKGROUND: Mutational analysis contributes to the diagnosis and prognosis of thyroid nodules analyzed with fine-needle aspiration cytology (FNAC). Although several advanced molecular tests based on multiple molecular markers are available for clinical use and have increased their impact on clinical management of patients, they are not widely available. Among them is BRAF V600E, one of the most studied mutations. Other genetic alterations, such as TERT promoter mutations, may coexist in thyroid carcinomas. Previous studies have demonstrated that this duet might be involved in the aggressiveness of thyroid cancer, although its prognostic value related to mortality remains undefined. The detection of such genetic alterations in thyroid liquid-based cytology (LBC) thus may assist with patient management. METHODS: From January 2013 to June 2014, 356 thyroid FNAC samples were processed by LBC, including 174 surgical follow-up samples. BRAF V600E and TERT mutation analyses were performed on both LBC and histopathology. RESULTS: The study included 119 samples categorized as atypia of undetermined significance, 42 categorized as follicular neoplasms, 61 categorized as suspicious for malignancy, and 34 categorized as positive for malignancy. BRAF V600E mutation was detected in 10.4% of all cases, whereas TERT promoter mutations were identified in 1.1%. TERT-mutated cases belonged to the positive for malignancy category, with a histologic diagnosis of tall cell variant of papillary thyroid carcinoma. These genetic alterations correlated with lymph node metastases (P = .0349) and higher disease stage. CONCLUSIONS: BRAF V600E and TERT analysis can be performed on LBC. TERT mutations are rarely identified in well differentiated thyroid carcinoma but are associated with higher stage. Although a larger molecular panel may offer more information, analyzing these few point mutations is still likely to be useful for managing potentially more aggressive thyroid carcinomas.

The combination cytology/epichek test in non muscle invasive bladder carcinoma follow-up: Effective tool or useless expence?

OBJECTIVE: To identify in which cases after cytological diagnosis, the Bladder EpiCheck test could represent an effective tool in non-muscle invasive bladder carcinoma or an useless expence. MATERIALS AND METHODS: 375 patients diagnosed with non-muscle invasive bladder cancer, 269 with high grade urothelial carcinoma and 106 with carcinoma in situ, were treated and followed for 1 year. The treatment was an intravesical instillation of Bacillus Calmette-Guerin in 305 patients and Mitomycin-C in 70 patients. During the follow-up patients were evaluated by voided urine cytology and white-light cystoscopy, according to the European Association of Urology Guidelines. Bladder EpiCheck test was performed together with cytology in all cases. RESULTS: Analyzing Bladder Epicheck results for each category defined by the Paris System for Reporting Urinary Cytology, we found that the Episcore >60 correlates with histological diagnosis of high grade urothelial carcinoma (HGUC) in atypical urothelial cells and Suspicious for High Grade Urothelial Carcinoma (P = 0.0002 Odds Ratio 0.05926 95% Confidence Interval from 0.01127 to 0.3116 and P = 0.0009 Odds Ratio 0.03155 95% Confidence Interval from 0.001683 to 0.5914, Fisher's exact test, respectively), while in Negative for high grade urothelial carcinoma and HGUC patients Episcore is not helpful to identify cases with histological diagnosis of HGUC (P = 0.101 and P = 0.58 Fisher's exact test, respectively). Considering an Episcore ≥ 90 in the HGUC cytological group, this seems not to be correlated with a histological diagnosis of HGUC (P = 0.090 Fisher's exact test). CONCLUSIONS: Cytology and Bladder EpiCheck test in combination may have the potential to reduce cystoscopies in the follow-up of non-muscle invasive bladder cancer only for cytological diagnoses of atypical urothelial cells and Suspicious for High Grade Urothelial Carcinoma . Moreover, in patients with a cytological diagnosis of Negative for high grade urothelial carcinoma or HGUC, cytology alone seems to be safe and cost-effective.

PD-L1 and thyroid cytology: A possible diagnostic and prognostic marker.

BACKGROUND: Programmed death-ligand 1 (PD-L1) expression is emerging as an important predictive biomarker in anti-PD-L1 cancer immunotherapy. Its role has been clearly defined in various human cancers and is linked to a poor prognosis and resistance to anticancer therapies. The role of PD-L1 in thyroid cancers has not been well defined in fine-needle aspiration cytology (FNAC). The authors examined the performance of PD-L1 immunostaining in liquid-based cytology (LBC) to determine whether it could be a biomarker of malignancy or aggressive disease. METHODS: From January 2018 to March 2019, 236 thyroid lesions, which had been diagnosed by FNAC as indeterminate lesions, suspicious for malignancy (SFM), and malignant, were enrolled. PD-L1 immunostaining was performed on both LBC and corresponding histology samples. RESULTS: The FNAC cohort included 50 benign negative controls, 42 samples of atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS), 33 samples of follicular neoplasm/suspicious for follicular neoplasm (FN/SFN), 53 samples that were suspicious for malignancy (SFM), and 58 malignant samples. AUS/FLUS samples included 3 goiters, 32 follicular adenomas (FAs), 1 noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP), 5 invasive follicular variants of papillary thyroid carcinoma (I-FVPTCs), and 1 follicular carcinoma; whereas FN/SFN samples included 24 FAs and 9 malignancies (4 I-FVPTCs, 1 NIFTP, 3 papillary thyroid carcinomas [PTCs], and 1 oncocytic follicular carcinoma). The 53 SFM samples were diagnosed on histopathology as 2 FAs, 5 NIFTPs, 15 I-FVPTCs, and 31 PTCs; whereas the 58 malignant specimens included 5 NIFTPs, 5 I-FVPTCs, and 48 PTCs. Increased plasma membrane and cytoplasmic PD-L1 expression was found in 79 cases (38.5%), including 61 PTCs (conventional and variants). Negative PD-L1 expression was found in NIFTPs and FAs. A BRAF V600E mutation was identified in 15% of PD-L1-positive malignancies. CONCLUSIONS: The current data suggest that PD-L1 expression in the thyroid gland might represent a marker of malignancy that correlates with PTC, but not with NIFTP. Thyroid neoplasms with PD-L1 expression also ae enriched with BRAF V600E mutations, suggesting that they are associated with more aggressive behavior.

A large series of hyalinizing trabecular tumors: Cytomorphology and ancillary techniques on fine needle aspiration.

BACKGROUND: Hyalinizing trabecular tumors (HTTs) are rare, essentially benign, follicular cell-derived thyroid neoplasms characterized by a trabecular growth pattern and nuclear pseudoinclusions. Their cytological findings are misleading, because these tumors are often misinterpreted on fine needle aspirate cytology as malignant lesions, such as papillary thyroid cancer and/or medullary thyroid cancer, leading to unnecessary total thyroidectomy. The aim of this study was to analyze the cytomorphological features and application of ancillary techniques in a series of HTTs. METHODS: Of 26 histological cases of HTT collected from September 2001 to December 2018, 18 cases had concomitant cytopathology. Cytological cases were processed with liquid-based cytology (LBC). Immunocytochemistry for HBME-1 and galectine-3 as well as molecular testing for BRAFV600E mutation were performed on both LBC and histological specimens. RESULTS: The 18 lesions with fine needle aspirate cytology ranged in size from 5 to 45 mm. Cytological diagnoses included: 1 benign lesion favoring goiter (5.5%), 4 atypia of undetermined significance (22.2%), 6 follicular neoplasms (33.3%), 5 suspicious for malignancy favoring papillary thyroid cancer (28%), and 2 malignant (11%). Hence, 89% HTT had a negative concordant immunopanel, and they were 100% wild-type BRAFV600E . CONCLUSION: The majority of our HTTs (83.3%) were diagnosed in the indeterminate Bethesda categories, suggesting that their cytomorphological features pose issues for reaching a conclusive cytological diagnosis. The ancillary test results in our series support the fact that HTT is a benign neoplasm.

To Obtain More With Less: Cytologic Samples With Ancillary Molecular Techniques-The Useful Role of Liquid-Based Cytology.

CONTEXT: - Fine-needle aspiration cytology has been increasingly used as the first tool in the evaluation of several diseases. Although cytology has a relevant role in the discrimination between benign and malignant lesions, conventional slides cannot lead to 100% conclusive results. It was hoped that the introduction of liquid-based cytology (LBC) would improve the efficacy of cytology through standardization, quality improvement, and the possibility of carrying out ancillary techniques on the residual stored material. In recent decades, the application of genomic alterations has been studied on cytologic samples with feasible and reliable results. The molecular analysis offers a powerful aid to define the best clinical or surgical approaches and follow-up for patients. In recent years, the application of different ancillary techniques has been carried out on conventional slides even though LBC represents a useful additional and alternative method for molecular testing. OBJECTIVE: - To demonstrate the relevance of LBC as a valid aid to overcoming the difficulties encountered in the application of ancillary techniques on conventional slides. DATA SOURCES: - We examined and reviewed our experience with the application of ancillary techniques on LBC performed on different body sites. CONCLUSIONS: - We emphasize that LBC achieves significant and accurate results. It represents a valid method for cytologic evaluation and it provides highly reproducible and informative molecular yields.

The role of thyroid FNA cytology in pediatric malignant lesions: An overview of the literature.

When one is dealing with pediatric thyroid lesions, fine-needle aspiration is the first diagnostic tool for the correct characterization of these nodules. Despite the apparent infrequency of thyroid cancers in children, recent data from the National Cancer Institute prove that the incidence has been increasing, especially in adolescents. With the same data, a higher prevalence of well-differentiated cancers can be estimated, with 90% diagnosed as papillary thyroid cancer. Nonetheless, some publications have demonstrated that some specific malignant variants are more frequent in children and have a more aggressive behavior that justifies the increased number of surgical procedures. For this reason, the American Thyroid Association recommends the performance of neck ultrasonography and fine-needle aspiration cytology (FNAC) for the evaluation of pediatric thyroid nodules. Accordingly, as reported in adult thyroid series, several authors have documented the high sensitivity and diagnostic accuracy of FNAC in pediatric series; they have also shared the same problematic issues encountered in adult populations, mostly in the diagnosis of indeterminate lesions. To provide precise clinical and/or surgical management, the correct cytological identification of specific malignant histotypes/entities should be mandatory because lymph nodes, distant metastases, and extrathyroidal infiltration are more frequent within specific histotypes. A perusal of the literature shows that their identification has not been extensively studied and investigated in cytological samples. This review focuses on the analysis of data from the literature on the evaluation of malignancies and specific morphological features in pediatric thyroid lesions. Cancer Cytopathol 2017;125:594-603. © 2017 American Cancer Society.

The role of miRNAs in the evaluation of follicular thyroid neoplasms: an overview of literature.

MicroRNA (miRNA) deregulation has been frequently associated with different human cancers. Not only have miRNAs been involved in almost every cellular function but they have also been linked with a significant number of cancers including thyroid carcinomas. Specifically, thyroid tumors encompass several different miRNA profiles based on the histotypes. Furthermore, thyroid lesions with their broad spectrum of neoplasms (from benign to malignant entities) offer the possibility of studying and recognizing specific subsets of different up-and downregulated miRNAs in each different entity. To date, the majority of authors completed their evaluation mostly by including histologic samples of thyroid tumors. Nonetheless, in the last years, a few studies are focusing on the role of miRNA expression in thyroid fine-needle aspiration cytology (FNAC) regardless of the cytologic preparation, including liquid-based cytology. This growing interest is driven by the possible role of miRNAs in the malignant risk stratification, especially for the indeterminate categories of follicular neoplasms (FNs). In this review we overview the reliability of analyzing miRNAs on thyroid lesions, including those diagnosed as FNs, to identify whether their profiles are likely to distinguish benign from malignant lesions, providing a predictive molecular diagnosis on FNAC.

The expression of monocarboxylate transporters in thyroid carcinoma can be associated with the morphological features of BRAF V600E mutation.

BRAF V600E mutation, usually performed by DNA techniques, is one of the most common diagnostic markers in papillary thyroid carcinoma. Few papers have demonstrated that plump cells (eosinophilic cytoplasms and papillary thyroid carcinoma nuclei) and peculiar sickle-shaped nuclei represent morphological features of BRAF V600E on papillary thyroid carcinomas. These features seem to be linked to glycolytic phenotype whereby monocarboxylate transporters 1-4 are hypothesized to have a dominant role as lactate transporters. We investigated the association between these morphological features and monocarboxylate transporters 1 and 4 in 48 cyto-histological samples diagnosed as "positive for malignancy-favoring papillary thyroid carcinoma". These cases were processed with liquid-based cytology and underwent BRAF V600E mutational analysis (pyrosequencing) on liquid-based cytology and monocarboxylate transporters immunostaining on histology. The expression of monocarboxylate transporter 1, monocarboxylate transporter 4, glucose trasporter-1 and carbonic anhidrase were scored semi-quantitatively with expression from 0 to 3+ (strong positivity). The 33 mutated and 15 wild type cases showed 100 % cyto-histological concordance. The cytological evaluation revealed plump cells and sickle nuclear shape in 100 % mutated cases. Monocarboxylate transporter 1 yielded 76 % positivity in the mutated cases especially in both the plump cells and sickle-shaped nuclei, whereas the wild types showed 13.3 % positive monocarboxylate transporter 1 (p = 0.00013). Monocarboxylate transporter 4 resulted in 100 % positivity in mutated and 40 % in wild types (p < 0.005). Furthermore, 20 % of the wild types showed weak monocarboxylate transporter 1 nuclear expression associated to a less aggressive behavior. The analysis of glucose trasporter-1 and carbonic anhidrase did not highlight any statistical significance (p > 0.05). This is the first report analyzing the association between monocarboxylate transporter expression and the morphological features of BRAF V600E mutated papillary thyroid carcinomas suggesting the possible involvement of lactate in the morphological features.

Young investigator challenge: The morphologic analysis of noninvasive follicular thyroid neoplasm with papillary-like nuclear features on liquid-based cytology: Some insights into their identification.

BACKGROUND: Noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) represents a challenge for the diagnosis and management of thyroid carcinoma. Some authors have proposed histological criteria that are able to distinguish NIFTPs from invasive follicular variant of papillary thyroid carcinoma (I-FVPTC). Hence, NIFTPs may have repercussions in the diagnostic categories on fine-needle aspiration. In the current study, the authors evaluated the criteria for NIFTPs on liquid-based cytology samples. METHODS: The authors recorded all 61 liquid-based cytology samples proved to be histological FVPTC between January 2013 and March 2016 and analyzed the architectural, cytoplasmic, and nuclear parameters. They compared them with a cohort of 40 PTC cases and 20 follicular adenoma cases. RESULTS: The authors reported 37 NIFTP cases and 24 I-FVPTC cases at histology. The cytological diagnoses of follicular nodules in the NIFTP cases were twice those found in the I-FVPTC cases (54.1% vs 29.2%). The number of positive for malignancy cases among the NIFTPs were approximately half those of I-FVPTC cases. When compared with I-FVPTCs, 70% of the NIFTP cases demonstrated a nuclear size <20 µm (P = .025) and rarely exhibited grooves (13% vs 42%; P = .009). The authors found 100% of cases with wild-type BRAF gene in NIFTP cases versus 38.4% in mutated I-FVPTC cases (P = .046). The cytoplasmic features might help to discriminate NIFTPs from follicular adenomas but not from I-FVPTCs (P>.05). A predominant microfollicular pattern was recognized in both NIFTPs and I-FVPTCs (97.3% vs 100%). CONCLUSIONS: The majority of NIFTPs appear to be devoid of nuclear pseudoinclusions and papillary structures, thereby allowing the inclusion in the follicular nodule cases. Nuclear size and microfollicular clusters may suggest the discrimination between NIFTPs and I-FVPCs. Cancer Cytopathol 2016;124:699-710. © 2016 American Cancer Society.

Human cord blood endothelial progenitors promote post-ischemic angiogenesis in immunocompetent mouse model.

BACKGROUND: Human cord blood (CB) endothelial colony forming cells (ECFCs) are endowed with high vascular regenerative ability in immunodeficient mice, but their immunogenicity and susceptibility to rejection in immunocompetent models has yet to be explored. METHODS: We injected CB ECFCs in non-immuno-suppressed C57BL/6J mice after having induced the hindlimb ischemia and we investigated their contribution to the recovery from the ischemic injury. Human ECFCs (hECFCs) were administered by intramuscular injection and hindlimb blood perfusion was measured by laser Doppler analysis at 7-day intervals for 28days after treatment. Mice were sacrificed after 7 and 28days and immunohistochemistry for specific human (CD31) and mouse (von Willebrand factor) endothelial antigens was carried out. Before euthanasia, blood samples to assess cytokines and angiogenic growth factor levels were collected. RESULTS: Mice injected with hECFCs showed a prompter and greater recovery of blood flow than controls. Several endothelial cells of human origin were detected at day7 after injection and their number declined progressively. Likewise, a progressive increase of mouse-derived vascular structures were observed, paralleled by the amplified endogenous production of various soluble mediators of angiogenesis, including Vascular Endothelial Growth Factor and Fibroblast Growth Factor. CONCLUSIONS: Overall, our findings are consistent with the hypothesis that human ECFCs might expand the endogenous vascular repair potential of recipients and support their possible HLA-independent unconventional use.

The evaluation of miRNAs on thyroid FNAC: the promising role of miR-375 in follicular neoplasms.

Fine needle aspiration cytology (FNAC) plays an essential role in the evaluation of thyroid nodules especially for the category of follicular neoplasms (FN) representing 25 % of all thyroid cases including different neoplastic entities. Hence, one of the most promising areas is the application of molecular tests to FNAC. Among them, microRNAs (miRNA),identified as negative (post-transcriptional) gene expression regulators involved in tumor development, are likely to discriminate among FNs. Limited data explored the use of miRNAs on FNAC as well as their role in the malignant risk stratification. We aimed to define whether liquid-based cytology (LBC) is a valid method for miRNA evaluation. From June 2014 to March 2015, we enrolled 27FNs with histological follow-up. In the same reference period, 13 benign nodules (BN) and 20 positive for malignancy (PM) were selected as controls. Histologically, FNs resulted in 14 malignancies (3 papillary thyroid carcinoma-PTC and 11 follicular variant of PTC-FVPC) and 13 follicular adenomas (FA). The 20 PMs included two FVPC, 16 PTC and two medullary thyroid carcinoma (MTC). Five miRNAs (10b, 92a, 221/222 cluster, and 375) were studied on LBC and quantified by real-time PCR. Only miR-375 was over-expressed in the FNs diagnosed as carcinomas and in the PMs. A cut-off of 12 miR-375/U6 relative ratio recognized all BNs and 95 % PMs. Specifically, in each category, FVPCs and PTCs did not show any difference while MTCs had the highest value. miR-375 shows 97.1 % sensitivity, 100 % specificity, 96.3 % negative predictive value (NPV), 100 % positive predictive value (PPV), and 98.3 % diagnostic accuracy. LBC is suitable for miRNAs evaluation. miR-375 resulted over-expressed in all malignant FNs and 95 % PMs. It may represent a valid aid in ruling out BNs and supporting PTCs and/or FVPCs.

KRAS Exon 2 Mutations as Prognostic Indicators in Advanced Colorectal Cancer in Clinical Practice: A Mono-Institutional Study.

BACKGROUND: Kirsten-Ras (KRAS) mutations are widely accepted negative predictive factors for anti-EGFR therapies in metastatic colorectal cancer (mCRC), while their prognostic significance is still under discussion. OBJECTIVE: This mono-institutional retrospective study aims to investigate the real-life impact of exon 2 codon 12 and 13 mutations in mCRC. METHODS: All mCRC patients treated at our institution between 2008 and 2014 carrying KRAS exon 2 mutations were included. The primary endpoint was to determine any significant difference in overall survival (OS) between codon 12 and 13 mutations. Secondary endpoints included progression-free survival (PFS), OS in both populations according to antiangiogenic treatment, and OS in liver-limited disease (LLD). RESULTS: Of 620 mCRC patients, 218 carried KRAS exon 2 mutations (35.1%): 162 (26.1%) at codon 12 and 56 (9.0 %) at codon 13. Median OS results were similar: 32.0 months (codon 12) and 31.0 months (codon 13). PFS was also comparable, reaching 10.8 months in both populations. The addition of bevacizumab to chemotherapy conferred a trend toward survival advantage in codon 12 but not codon 13 mutation (p = 0.058). A high proportion of LLD patients underwent hepatic surgery with radical purpose (62.3%): in these patients, median OS has not yet been reached, while OS in non-LLD patients was 30.2 months. CONCLUSIONS: No difference in OS between KRAS codon 12/13 mutated disease was found. This analysis showed a very prolonged OS for KRAS-mutated patients, even when LLD patients were excluded; OS of our real-life series favorably compares with OS of all-RAS wild-type patients in recent randomized studies.

In vitro cardiomyocyte differentiation of umbilical cord blood cells: crucial role for c-kit(+) cells.

BACKGROUND AIMS: Although bone marrow c-kit(+) progenitor cells support myocardial regeneration, the cardiomyocyte differentiation potential of umbilical cord blood (UCB) c-kit(+) cells is unknown. METHODS: UCB mononuclear cells (MNCs) and c-kit(+) cells purified by use of immunomagnetic beads were used. Cardiomyocyte differentiation was induced with (i) α-minimum essential medium (MEM) with cyclosporine A, (ii) α-MEM with bone morphogenic protein 4 (BMP-4) and transforming growth factor-ß (TGF-ß) or (iii) MEM with dexamethasone. The expression of cardiac markers (GATA4, GATA6, ß-myosin heavy chain, α-sarcomeric actin and cardiac Troponin T) was investigated, and whole-cell current and voltage-clamp recordings were performed. RESULTS: Although c-kit(+) cells revealed an immature gene profile, with high expression of CD34, CD133, aldehyde dehydrogenase-A1 and c-myc RNAs, purified c-kit(+) cells did not succeed in differentiating into cardiomyocyte-like cells in culture. In contrast, MNCs (either in α-MEM plus cyclosporine A or in α-MEM plus BMP-4 and TGF-ß) produced large, adherent cells expressing several cardiac genes and exhibiting an excitable phenotype. Cardiomyocyte-like cell formation was prevented by removing the c-kit(+) cell fraction from MNCs. Furthermore, after co-culturing carboxyfluorescein diacetate succynimidyl ester (CFSE)-tracked c-kit(+) cells together with c-kit(-) cells, we found that cardiac Troponin T--expressing cells were also CFSE(+). CONCLUSIONS: We show that UCB contains progenitors endowed with differentiation potential into cardiomyocytes: these cells reside in the c-kit(+) fraction and require the presence of abundant accessory cells to accomplish the differentiation. These preliminary observations provide the basis for consider the storage of autologous UCB in patients with prenatal diagnosis of congenital heart diseases potentially amenable by myocardial regenerative approaches.

Uncommon BRAF mutations in the follicular variant of thyroid papillary carcinoma: New insights.

BACKGROUND: Mutational analysis is reshaping the practice of fine-needle aspiration cytology for the diagnosis of thyroid nodules. The v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) valine (V) to glutamic acid (E) substitution at codon 600 (BRAF(V600E)) is the most effective diagnostic/prognostic marker and is used mainly for papillary thyroid carcinomas (PTCs). Although BRAF(V600E) represents 95% of all BRAF mutations, uncommon BRAF mutations have been identified in thyroid carcinomas. For the current study, the authors evaluated morphologic (plump pink cells and sickle-shaped nuclei) anti-BRAF(V600E) antibody (VE1) immunocytochemical and molecular findings of BRAF mutations in PTCs and in the follicular variant of PTC (FVPC). METHODS: Between January 2013 and June 2014, there were 150 cytologic samples with surgical follow-up at the authors' institution. BRAF mutations, which were identified using liquid-based cytology, were classified into wild-type BRAF, BRAF(V600E), and uncommon BRAF mutations. All clinicopathologic correlations between BRAF and FVPCs were analyzed. RESULTS: Forty-four of 150 samples were identified as benign histologic lesions, and the authors focused on the 106 cytologic samples from patients who had malignant outcomes (60 PTCs and 46 FVPCs). The series included 16 follicular neoplasms, 36 samples diagnosed as suspicious of malignancy, and 54 samples diagnosed as positive for malignancy. The BRAF(V600E) mutation was detected in 17.4% of FVPCs and in 66.6% of PTCs, whereas uncommon BRAF mutations were detected only in FVPCs. Plump pink cells and VE1 expression were not identified in samples that had uncommon BRAF mutations. VE1 immunocytochemistry yielded positive results in all 36 samples that had the BRAF(V600E) mutation. CONCLUSIONS: Uncommon BRAF mutations were observed only in FVPCs and were linked to less aggressive behavior. Negative/weak VE1 expression was observed in both wild-type and uncommon BRAF mutations. The current investigation did not reveal any plump cells or morphologic BRAF findings in samples that had uncommon BRAF mutations. In the authors' experience, BRAF mutations detected by DNA methods were more accurate in identifying FVPCs.

Endothelial progenitor cell dysfunction in myelodysplastic syndromes: possible contribution of a defective vascular niche to myelodysplasia.

We set a model to replicate the vascular bone marrow niche by using endothelial colony forming cells (ECFCs), and we used it to explore the vascular niche function in patients with low-risk myelodysplastic syndromes (MDS). Overall, we investigated 56 patients and we observed higher levels of ECFCs in MDS than in healthy controls; moreover, MDS ECFCs were found variably hypermethylated for p15INK4b DAPK1, CDH1, or SOCS1. MDS ECFCs exhibited a marked adhesive capacity to normal mononuclear cells. When normal CD34+ cells were co-cultured with MDS ECFCs, they generated significant lower amounts of CD11b+ and CD41+ cells than in co-culture with normal ECFCs. At gene expression profile, several genes involved in cell adhesion were upregulated in MDS ECFCs, while several members of the Wingless and int (Wnt) pathways were underexpressed. Furthermore, at miRNA expression profile, MDS ECFCs hypo-expressed various miRNAs involved in Wnt pathway regulation. The addition of Wnt3A reduced the expression of intercellular cell adhesion molecule-1 on MDS ECFCs and restored the defective expression of markers of differentiation. Overall, our data demonstrate that in low-risk MDS, ECFCs exhibit various primary abnormalities, including putative MDS signatures, and suggest the possible contribution of the vascular niche dysfunction to myelodysplasia.

Well-differentiated thyroid cancer with a minor poorly differentiated component: clonal heterogeneity through the prognostic role of CXCR4 and BRAF analysis.

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.

Morphological parameters able to predict BRAF(V600E) -mutated malignancies on thyroid fine-needle aspiration cytology: Our institutional experience.

BACKGROUND: The BRAF(V600E) mutation is the most common diagnostic/prognostic marker in papillary thyroid carcinoma (PTC). Its evaluation is typically performed with DNA-based techniques; nonetheless, a few articles have recently proposed the morphological prediction of BRAF(V600E) in histological PTCs. We investigated this morphological parameter in our cytological series. METHODS: We re-analyzed all 72 cytohistological samples diagnosed as positive for malignancy (favoring PTC) on fine-needle aspiration cytology from January 2012 to December 2013. We included 22 male patients and 50 female patients. The cytological cases were processed with liquid-based cytology. We performed molecular analysis and immunocytochemistry for the VE1 BRAF(V600E) antibody. RESULTS: We reported 47 mutated cases and 25 wild-type (WT) cases with 100% cytohistological concordance. The cytological evaluations revealed plump cells (abundant eosinophilic cytoplasm and PTC nuclei) in all 47 mutated cases, with only 6 having a focal plump cell component (≤20% cells). Furthermore, 5 of the 25 WT cases showed focal plump cells. A distinctive sickle nuclear shape was found only in the mutated cases. VE1 yielded 100% positivity for all 24 mutated cases that were tested, including 3 cases with focal plump cells. CONCLUSIONS: We demonstrated that the BRAF(V600E) mutation might be predicted in cytological samples on the basis of some specific morphological features. Although the detection of plump cells (mainly focal) was also observed in WT cases, the detection of sickle-shaped nuclei provided the highest specificity and sensitivity as a predictive mutational parameter. These morphological features might be a valid tool for selecting cases for molecular analysis.