Fine-needle Aspiration Biopsy Samples Research Articles

Comprehensive Cancer Panel Sequencing Defines Genetic Diversity and Changes in the Mutational Characteristics of Pancreatic Cancer Patients Receiving Neoadjuvant Treatment.

Background/AimsPancreatic ductal adenocarcinoma (PDA) is associated with an extremely poor prognosis. This study assessed the genetic diversity among patients with PDA and compared their mutational profiles before and after treatment.MethodsTumors and matched blood samples were obtained from 22 PDA patients treated with neoadjuvant chemoradiation therapy. The somatic mutations were analyzed with comprehensive cancer gene panel (CCP). In addition, the biopsy samples obtained at diagnosis and the surgically resected samples after treatment were compared for seven patients. The CCP provided formalin-fixed paraffin-embedded sample-compatible multiplexed target selection for 409 genes implicated in cancer.ResultsAssessments of the MLH1, MLH3, MSH2, and PMS2 genes showed that the four patients with the highest relative burdens of mutations harbored somatic mutations in at least three of these genes. Genes in the histone-lysine N-methyltransferase 2 (KMT2) family, such as KMT2D, KMT2A, and KMT2C, were frequently mutated in tumor samples. Survival was worse in patients with ARID1A gene mutations than those without ARID1A gene mutations. Mutation patterns were compared between tissue samples before and after neoadjuvant treatment in seven patients who underwent surgical resection. The allelic fraction of mutations in KRAS codon 12 was lower in the surgically resected samples than in the endoscopic ultrasonography-guided fine needle aspiration biopsy samples of six patients. The number of mutant alleles of the histone lysine methyltransferase gene WHSC1 also decreased after treatment.ConclusionsThese results indicate that tumor tissue from PDA patients is genetically diverse and suggest that ARID1A mutations may be a potential prognostic marker for PDA.

Cytology and the cell block method in diagnostic characterization of canine lymphadenopathy and in the immunophenotyping of nodal lymphoma.

Minimally invasive techniques used to evaluate canine peripheral lymphadenopathy (PLN), including fine needle aspiration biopsy with cytological evaluation (FNAB-C) and flow cytometry (FC), have benefits and limitations. The cell block (CB) method is an alternate processing technique in which fine needle aspirate biopsy samples are concentrated, fixed, and embedded in paraffin for routine histological processing/staining. Utilizing three observers, we determined the diagnostic value of the CB in evaluating canine PLN across six categories (non-diagnostic, reactive, inflammatory/infectious, probable lymphoma and lymphoma, metastatic neoplasia) and correlated findings to immunophenotypic and clonal antigen receptor rearrangement results in canine nodal lymphoma. Eighty-five paired FNAB-C and CB samples were evaluated from canine patients presenting to the University of Minnesota Veterinary Oncology or Internal Medicine services. Diagnostic quality samples were obtained in 55/85 (65%) CB and 81/85 (95%) FNAB-C samples, respectively, and nodal pathology impacted CB diagnostic yield. Overall percent agreement between diagnostic-quality FNAB-C and CB samples was 86%, but increased to 95% if the categories of lymphoma and probable lymphoma were combined. There was 100% agreement for both the diagnoses of metastatic neoplasia and reactive lymph nodes and 92% agreement for the diagnosis of lymphoma/probable lymphoma. Using immunohistochemistry (IHC), CB samples correctly immunophenotyped 22/23 (96%) cases of B-cell lymphoma, but only 1/6 (17%) cases of T-cell lymphoma. IHC was not completed on nine cases of lymphoproliferative disease because of insufficient cellularity. When the CB method (CBM) yielded diagnostic quality samples there was good to excellent agreement with FNAB-C samples and CB samples were suitable for some IHC tests.

Deciphering novel biomarkers of lymph node metastasis of thyroid papillary microcarcinoma using proteomic analysis of ultrasound-guided fine-needle aspiration biopsy samples

Thyroid papillary microcarcinoma is now a common clinical problem. Cervical lymph node metastasis is the main metastasis mode of PTMC. However, before operation, it is still difficult to determine exactly whether PTMC patient is suffering with cervical lymph node metastasis. To resolve this dilemma, for better selection of optimum treatment plans, it is necessary to investigate the overall changes in proteomes of PTMC, and evaluate the potential of biomarkers to predict lymph node metastasis. Tandem mass tags combined with multidimensional liquid chromatography and mass spectrometry analyses were used aiming to screen the proteomic profiles of fine-needle aspiration biopsy samples. Quantitative proteomic analysis, significant pathway and functional categories were investigated. In total, 3391 proteins of the 3793 protein groups identified were quantified. Bioinformatics analysis indicated that differentially expressed proteins were involved in multiple biological functions, metastasis-related pathways. Moreover, IFN-stimulated gene 15 proteins were found to be well distinguished between patients with lymph node metastatic and patients with nonmetastatic PTMC. Knocking down ISG15 with shRNA inhibited the xenografted tumor growth. This study provided a reference proteome map for lymph node metastatic PTMC. ISG15 probably is a prognosis marker of thyroid papillary microcarcinoma patients with lymph node metastasis. SignificanceNowadays, thyroid cancer has become a widespread epidemic. The rate of thyroid cancer incidence has been faster than any other cancers, reported by the American Cancer Society. Papillary thyroid microcarcinoma (PTMC) is a subset of PTC defined as PTC measuring≤1 cm in size, which comprises nearly one-half of all the cases of PTCs. Actually, the rapidly increasing global incidence of PTC is mainly attributed to the corresponding increase in the diagnosis of PTMC. Scholars have figuratively compared the increase of PTMC to the “tsunami”.The treatment scheme for PTMC is still not uniform, and the controversy is mainly focused on the necessity of surgery treatment. PTMCs often have an indolent course in the absence of evidence of metastatic cervical lymph nodes, distant metastases and extrathyroidal extension. Therefore, it is important for us to reliably differentiate the small number of PTMC patients developing significant metastases progression from the larger population of patients that harbor indolent PTMCs.The present study aimed to investigate the overall changes in proteomes of PTMC, and evaluate the potential of biomarkers to predict lymph node metastasis. Tandem mass tags (TMT) combined with multidimensional liquid chromatography and mass spectrometry analyses were used aiming to screen the proteomic profiles of fine-needle aspiration biopsy (FNAB) samples. Quantitative proteomic analysis, significant pathway and functional categories were investigated. Our results showed that some differential expression proteins were likely to be important resources for finding new diagnostic biomarkers.

Clinical assessment of the GNAS mutation status in patients with intraductal papillary mucinous neoplasm of the pancreas.

Intraductal papillary mucinous neoplasm (IPMN) of the pancreas is characterized by cystic dilation of the pancreatic duct, caused by mucin hypersecretion, with slow progression via the adenoma-carcinoma sequence mechanism. Mutation of GNAS at codon 201 is found exclusively in IPMNs, occurring at a rate of 41-75%. Recent advances in molecular biological techniques have demonstrated that GNAS mutation might play a role in the transformation of IPMNs after the appearance of neoplastic cells, rather than in the tumorigenesis of IPMNs. GNAS mutation is observed frequently in the intestinal subtype of IPMNs with MUC2 expression, and less frequently in IPMNs with concomitant pancreatic ductal adenocarcinoma (PDAC). Research has focused on assessing GNAS mutation status in clinical practice using various samples. In this review, we discuss the clinical application of GNAS mutation assessment to differentiate invasive IPMNs from concomitant PDAC, examine the clonality of recurrent IPMNs in the remnant pancreas using resected specimens, and differentiate pancreatic cystic lesions using cystic fluid collected by endoscopic ultrasound-guided fine needle aspiration (EUS-FNA), duodenal fluid, and serum liquid biopsy samples.

Precise Detection of Gene Mutations in Fine-Needle Aspiration Specimens of the Papillary Thyroid Microcarcinoma Using Next-Generation Sequencing.

Purpose To assess the feasibility of next-generation sequencing (NGS) to detect mutations in BRAF, RAS, TERT promoter, and TP53 genes in ultrasound-guided fine-needle aspiration (FNA) biopsy samples of the papillary thyroid microcarcinoma (PTMC). Methods A total of 135 FNA samples out of 135 patients with suspected PTMC were submitted for mutation testing using NGS. NGS was successfully performed in 114 specimens, while the remaining 21 samples were excluded due to insufficient amount/poor quality of DNA and sequencing failure. Of those 114 samples, 72 who were confirmed as having PTMC by postoperative histopathology were enrolled in our study, and the other 42 who had a follow-up with ultrasound were excluded. Mutations of genes including BRAF, NRAS, HRAS, KRAS, TERT promoter, and TP53 were evaluated using NGS. The associations of gene mutations and clinicopathological characteristics of PTMC were analyzed. Results BRAF mutation was observed in 59 (81.94%) of 72 specimens. This mutation detected in BRAF was p.V600E (c.1799T>A) in exon 15 of all 59 specimens. NRAS mutation was identified in 1 (1.39%) specimen classified as Bethesda III and pathologically confirmed as a follicular variant PTMC. There were no mutations found in TERT promoter or TP53. The tumor with a maximum diameter (Dmax) larger than 5 mm was shown to be significantly correlated with the BRAF mutation in a multivariate analysis (OR 5.52, 95% CI 1.51-26.42, P = 0.033). But the BRAF mutation was not found to be significantly associated with the gender or age of patients with PTMC (P > 0.05). Conclusions This study demonstrated that gene mutations in FNA specimens of PTMC could be successfully analyzed with a higher sensitivity using NGS compared to conventional methods for mutation detection. BRAF mutation of p.V600E was statistically associated with PTMC with a Dmax larger than 5 mm.

In Brief

In Brief

Teaching FNA techniques and ultrasound guided FNA

Teaching FNA techniques and ultrasound guided FNA

Efficacy of an Intensive, Ultrasound-Guided Fine-Needle Aspiration Biopsy Training Workshop in Tanzania.

BackgroundFine-needle aspiration biopsy (FNAB) is a minimally invasive, cost-effective diagnostic tool that can be used in low-resource settings. However, adequacy and accuracy of FNAB is highly dependent on the skills of the operator and requires specialized training. Poor technique can preclude definitive diagnoses because of insufficient quality or quantity of FNAB samples. We evaluated the efficacy of an intensive training experience in Tanzania on improving ultrasound-guided FNAB techniques.MethodsA 2-day workshop offered didactic lectures, demonstrations, and hands-on practicum on fundamentals of ultrasound imaging and FNAB technique. A prospective interventional study design was used with pre- and postintervention surveys and assessments to measure the effect of the workshop on specific skills related to slide smearing and ultrasound-guidance among participants.ResultsTwenty-six pathologists and radiologists, including trainees in each specialty, participated in the workshop. Pre- and postworkshop assessments demonstrated that most participants improved significantly in nearly all technical skills for slide smearing and ultrasound-guided FNAB. After the workshop, most participants demonstrated substantial improvements in ability to prepare the ultrasound equipment, measure the lesion in three dimensions by ultrasound, target lesions in one pass using both parallel and perpendicular approaches, and prepare high-quality aspirate smears.ConclusionAn in-country 2-day workshop in Tanzania was efficacious in transferring basic skills in FNAB smear preparation and ultrasound-guided FNAB, resulting in skills enhancement among participating pathologists and radiologists. Although mastery of skills was not the goal of this short workshop, participants demonstrated proficiency in most technical elements after workshop completion, and the workshop generated interest among select participants to pursue additional intensive training in cytopathology.

Training to Improve the Quality of Early Detection of Breast Cancer in Low-Resource Settings

Purpose Breast cancer (BC) is the most common cancer among women in Latin America, yet many countries lack the capacity to provide early detection and diagnostic services. In Peru, mammograms are not readily available in secondary cities, and 57% of women who are diagnosed with BC are diagnosed with stage III and IV disease. To bring services to women in a northern region of Peru, PATH collaborated with Peruvian health institutions at the local, regional, and national levels to design and implement a strategic algorithm with which to increase the early detection of BC in the absence of mammography. Here, we evaluate the impact of training on the quality of clinical breast exam (CBE), ultrasound triage, and fine-needle aspiration (FNA) sampling and the reading of FNA biopsy for triage-positive women. Methods Two hundred twenty-four midwives and 15 doctors were included in two-step training in Trujillo, Peru. CBE trainings were performed using anatomic models and patient exams in 2-day sessions. Team discussion with trainers was used to improve skills. Doctors’ trainings for FNA sampling were led by two international expert pathologists in 2014 and by a Peruvian expert pathologist in 2016. Then in 2017, 43 pretraining and 50 post-training FNA biopsy samples were evaluated for adequacy and quality of diagnosis. Results Since the trainings, 14,223 women have received CBE on an opportunistic basis. Midwives refer abnormal cases (n = 281) for follow-up. All doctors felt well trained for CBE and FNA, whereas five of 15 doctors perceived the need for additional ultrasound training. Quality assessment revealed that the adequacy of FNA samples improved from 26% pretraining to 37% post-training. Of the 43 FNA samples, a low concordance with reviewers was observed in the detection of suspicious/carcinoma (6.9% v 13.9%), whereas full concordance was observed after the training, although the numbers were small. Conclusion In Peru, CBE remains an acceptable and feasible approach if complemented with ultrasound triage and FNA biopsy. Additional efforts are needed to increase coverage through a structured program. Training and continuous monitoring are essential for quality assurance. AUTHORS' DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST The following represents disclosure information provided by authors of this manuscript. All relationships are considered compensated. Relationships are self-held unless noted. I = Immediate Family Member, Inst = My Institution. Relationships may not relate to the subject matter of this manuscript. For more information about ASCO's conflict of interest policy, please refer to www.asco.org/rwc or ascopubs.org/jco/site/ifc . Ronald Balassanian Stock or Other Ownership: Cerus Corp ($400.00)

Next-Generation Sequencing Identifies a Highly Accurate miRNA Panel That Distinguishes Well-Differentiated Thyroid Cancer from Benign Thyroid Nodules.

Background: Fine needle aspiration biopsy (FNAB) is the gold-standard procedure for diagnosing malignant thyroid nodules. Indeterminate cytology is identified in 10% to 40% of cases, and molecular testing may guide management in this setting. Current commercial options are expensive, and are either sensitive or specific. The aim of this study was to utilize next-generation sequencing (NGS) technology to identify informative diversities in the miRNA expression profile of benign versus malignant thyroid nodules.Methods:Ex vivo FNAB samples were obtained from thyroid specimens of patients who underwent thyroidectomy at a referral center. miRNA levels were determined using NGS and multiplexing technologies. Statistical analyses identified differences between normal and malignant samples and miRNA expression profiles that associate with malignancy were established. The accuracy of the miRNA signature in predicting histologic malignancy was validated using a group of patient specimens with indeterminate cytology results.Results: A total of 274 samples were obtained from 102 patients undergoing thyroidectomy. Of these samples, 71% were benign and 29% were malignant. Nineteen miRNAs were identified as statistically different between benign and malignant samples and were used to classify 35 additional nodules with indeterminate cytology (validation). The miRNA panel's sensitivity, specificity, negative and positive predictive values, and overall accuracy were 91%, 100%, 87%, 100%, and 94%, respectively.Conclusions: Using NGS technology, we identified a panel of 19 miRNAs that may be utilized to distinguish benign from malignant thyroid nodules with indeterminate cytology.Impact: Our panel may classify indeterminate thyroid nodules at higher accuracy than commercially available molecular tests. Cancer Epidemiol Biomarkers Prev; 27(8); 858-63. ©2018 AACR.

Clinical trial cytology: Use of on-site evaluation of small biopsy and FNA samples for clinical trials and biomarker research studies.

After increased requests for biopsies for clinical trials and biomarker research, the University of Pittsburgh Medical Center created a clinical trial research service that partnered pathology, radiology, and medicine to facilitate rapid on-site evaluation (ROSE) of fine-needle aspiration (FNA) and/or core needle biopsy (CNB) samples to confirm the presence of tumor in these studies. Clinical trial coordinators organized biopsies for patients needing tumor samples for trials, and informed the cytopathology and radiology team. ROSE was performed to confirm the presence of sufficient tumor in FNA specimens and/or touch preparations of CNB. A total of 79 cases from a total of 14 clinical trials were evaluated with ROSE, 77 of which (97%) were for research only. There were 53 cases (67%) from breast/ovarian cancer studies that were initiated between 2008 and 2009, whereas 26 cases (33%) included a variety of other tumors for studies that were started between 2011 and 2014. The majority required CNB samples (60 cases; 76%), 20% of which used an FNA for needle placement before obtaining CNB material and 56% of which had touch preparations of the CNB evaluated without a preceding FNA. The concordance rate for ROSE with final adequacy of the sample was 96% to 100%. The study institution has experienced an increase in the number of clinical trial studies requesting ROSE to confirm the presence of tumor in a variety of malignancies. Cytology laboratories can help with patient care by offering ROSE to determine the adequacy of clinical trial material to minimize the submission of unsatisfactory or nonrepresentative material. Developing a clinical research service enhances communication and the processing of novel research specimens for cancer patients. Cancer Cytopathol 2018. © 2018 American Cancer Society.

Model for Early Detection of Breast Cancer in Low-Resource Areas: The Experience in Peru.

PurposeLate-stage breast cancer detection should be something of the past; however, it is still all too common in low-resource areas, including Peru, where 57% of women diagnosed with cancer are diagnosed at stage III or IV disease. Early detection of breast cancer is feasible in low-resource semirural and rural areas where mammography is rarely accessible.MethodsPATH collaborated with Peruvian health institutions at local, regional, and national levels to design and implement a model of care for the early detection of breast cancer in Peru. The model includes training health promoters for community outreach, professional midwives in clinical breast exam, doctors to perform fine-needle aspiration biopsy sampling with ultrasound to triage, and patient navigators to ensure patients follow through with treatment.ResultsIn a northern region of Peru, 400 individuals, including health promoters, midwives, doctors, and volunteers, received early-detection training in two phases. In Peru, local health professionals continue to refine and improve methods and materials using locally available resources, and the Peruvian health information system now includes specific breast cancer detection categories. Despite challenges and limited resources, the model is effective, and partnership with government health administrations improves health systems and benefits the population.ConclusionGiven the absence of screening mammography, the public health challenge is to bring breast cancer early detection and diagnostic services closer to women’s homes and to ensure appropriate follow-up and care. The model is eminently transferable with appropriate adaptation and should now be tested in other settings within and outside of Peru.

Combined microRNA and mRNA microfluidic TaqMan array cards for the diagnosis of malignancy of multiple types of pancreatico-biliary tumors in fine-needle aspiration material.

Pancreatic ductal adenocarcinoma (PDAC) continues to carry the lowest survival rates among all solid tumors. A marked resistance against available therapies, late clinical presentation and insufficient means for early diagnosis contribute to the dismal prognosis. Novel biomarkers are thus required to aid treatment decisions and improve patient outcomes.We describe here a multi-omics molecular platform that allows for the first time to simultaneously analyze miRNA and mRNA expression patterns from minimal amounts of biopsy material on a single microfluidic TaqMan Array card. Expression profiles were generated from 113 prospectively collected fine needle aspiration biopsies (FNAB) from patients undergoing surgery for suspect masses in the pancreas. Molecular classifiers were constructed using support vector machines, and rigorously evaluated for diagnostic performance using 10×10fold cross validation. The final combined miRNA/mRNA classifier demonstrated a sensitivity of 91.7%, a specificity of 94.5%, and an overall diagnostic accuracy of 93.0% for the differentiation between PDAC and benign pancreatic masses, clearly outperfoming miRNA-only classifiers. The classification algorithm also performed very well in the diagnosis of other types of solid tumors (acinar cell carcinomas, ampullary cancer and distal bile duct carcinomas), but was less suited for the diagnostic analysis of cystic lesions.We thus demonstrate that simultaneous analysis of miRNA and mRNA biomarkers from FNAB samples using multi-omics TaqMan Array cards is suitable to differentiate suspect solid pancreatic masses with high precision.

TWENTY-FIVE-GAUGE CANNULA-ASSISTED FINE-NEEDLE ASPIRATION BIOPSY OF CHOROIDAL MELANOMA: Cytopathological Analysis.

To report cytopathological observations on the cells retrieved from the 25-G cannula used during prognostic transvitreal fine-needle aspiration biopsy of choroidal melanoma. Transvitreal fine-needle aspiration biopsy of choroidal melanoma was performed through a 25-G valved cannula. Twenty samples from 20 consecutive patients were obtained. Most tumors were treated with plaque radiation therapy (16/20, 80%) following standard clinical guidelines. Four enucleated globes (4/20, 20%) were subjected to a similar transvitreal biopsy before enucleation. Cytopathological analysis of the cells retrieved from the cannula revealed the absence of any cells in 4 of 20 samples (20%). In the remaining 16 samples, definite melanoma cells and atypical cells (probable melanoma cells) were observed in 2 samples each (total 4, 25%). Histiocytes (4/16, 25%) and lymphocytes (1/16, 6%) were also observed. Thirteen samples (13/16, 81%) contained conjunctival epithelial epithelium. Prognostication could be performed on all fine-needle aspiration biopsy samples (20, 100%). Use of a 25-G valved cannula offers potential advantages by isolating the needle tract and by allowing retrieval of the contaminating cells without affecting the prognostic yield of the fine-needle aspiration biopsy sample.

A 10-Gene Classifier for Indeterminate Thyroid Nodules: Development and Multicenter Accuracy Study.

Background: In most of the world, diagnostic surgery remains the most frequent approach for indeterminate thyroid cytology. Although several molecular tests are available for testing in centralized commercial laboratories in the United States, there are no available kits for local laboratory testing. The aim of this study was to develop a prototype in vitro diagnostic (IVD) gene classifier for the further characterization of nodules with an indeterminate thyroid cytology.Methods: In a first stage, the expression of 18 genes was determined by quantitative polymerase chain reaction (qPCR) in a broad histopathological spectrum of 114 fresh-tissue biopsies. Expression data were used to train several classifiers by supervised machine learning approaches. Classifiers were tested in an independent set of 139 samples. In a second stage, the best classifier was chosen as a model to develop a multiplexed-qPCR IVD prototype assay, which was tested in a prospective multicenter cohort of fine-needle aspiration biopsies.Results: In tissue biopsies, the best classifier, using only 10 genes, reached an optimal and consistent performance in the ninefold cross-validated testing set (sensitivity 93% and specificity 81%). In the multicenter cohort of fine-needle aspiration biopsy samples, the 10-gene signature, built into a multiplexed-qPCR IVD prototype, showed an area under the curve of 0.97, a positive predictive value of 78%, and a negative predictive value of 98%. By Bayes' theorem, the IVD prototype is expected to achieve a positive predictive value of 64–82% and a negative predictive value of 97–99% in patients with a cancer prevalence range of 20–40%.Conclusions: A new multiplexed-qPCR IVD prototype is reported that accurately classifies thyroid nodules and may provide a future solution suitable for local reference laboratory testing.

Concurrent fine needle aspirations and core needle biopsies: a comparative study of substrates for next-generation sequencing in solid organ malignancies

Minimally invasive procedures, such as fine needle aspiration and core needle biopsy, are commonly used for the diagnosis in solid organ malignancies. In the era of targeted therapy, it is crucial for molecular testing to be performed on these limited volume specimens. Although several recent studies have demonstrated the utility of small biopsy specimens for molecular testing, there remains debate as to whether core needle biopsy specimens are more reliable than fine needle aspiration for molecular studies. In this study, we reviewed concurrently acquired fine needle aspiration and core needle biopsy samples (n=24), and compared overall cellularity, tumor fraction, and the results of next-generation sequencing. All somatic mutations detected in core needle biopsy samples were also detected in fine needle aspiration samples. The estimated tumor fraction was significantly higher in fine needle aspiration smears than core needle biopsy samples (P=0.003), whereas the overall DNA yield from smears was significantly lower than that obtained from the core needle biopsy specimens (P=0.01). The normalized average amplicon coverage for the genes analyzed was significantly higher in cytology smears than paired core needle biopsy samples, with lower numbers of failed amplicons and higher overall mutation allelic frequencies seen in the former. We further evaluated 100 malignant fine needle aspiration and core needle biopsy samples, acquired concurrently, for overall cellularity and tumor fraction. Overall cellularity and tumor fraction of fine needle aspiration samples was significantly higher than concurrently acquired core needle biopsy samples (P<0.001). In conclusion, we show that fine needle aspiration samples frequently provide better cellularity, higher tumor fraction, and superior sequencing metrics than concurrently acquired core needle biopsy samples. Cytologic specimens, therefore, should be better integrated into routine molecular diagnostics workflow to maximize limited tissues for clinically relevant genomic testing.

Ultra-performance liquid chromatography tandem mass spectrometry for determination of Direct Acting Antiviral drugs in human liver fine needle aspirates.

An ultra-performance liquid chromatography with triple quadrupole mass spectrometry method was developed and validated for the determination of direct acting antiviral drug concentrations in human liver fine needle aspirates. Liver fine needle aspirate (FNA) biopsy samples were homogenized in acetonitrile to stabilize the analytes and precipitate protein. The acetonitrile supernatants were diluted with internal standards and mobile phase. Separation was achieved with a Waters Acquity BEH C18 column (50×2.1mm, 1.7um) with a gradient elution of 0.1% formic acid in water and acetonitrile. The total run time was 4.25min. Detection of analytes was achieved using electrospray ionization (positive mode) and triple quadrupole selected reaction monitoring. Standard curve concentrations ranged from 12.5 to 5000ng/mL for dasabuvir and the m1 metabolite of dasabuvir, 1.25 to 2500ng/mL for ombitasvir and ritonavir, and 5.00 to 5000ng/mL for paritaprevir. The intra- and inter-day accuracy and precision were less than 13.7% in low, medium, and high quality control samples. The validated method was applied to the analysis of a liver fine needle aspirate of a patient undergoing direct acting antiviral therapy for hepatitis C virus.

Early spermatogenesis changes in traumatic complete spinal cord-injured adult patients.

Prospective longitudinal study. To assess the impact of spinal cord injury (SCI) on the spermatogenesis of patients studied in the early subacute stage and ensuing months. National hospital for SCI patients. A prospective follow-up study was conducted on 28 male patients with complete SCI who were evaluated in the early subacute phase (~1 month), and 3 and 6 months after the injury. At each time point, fine needle aspiration biopsy samples were taken from the testes for cytological assessment, and serum levels of relevant hormones were measured. At the end of the study period, ejaculation was attempted for standard semen analyses. Cytological patterns indicative of defective spermatogenesis were found in 61%, 52% and 20% of the patients at 1, 3 and 6 months after SCI, respectively, suggesting an improvement over time. Serum hormone analyses showed a steady elevation of estradiol levels above the reference range, and increasing levels of testosterone, inhibin B and prolactin throughout the study period. Prolactin levels were above the reference range at all time points. Only 2 out of the 10 patients who were able to ejaculate at 6 months post injury showed normal sperm parameters. A majority of the patients showed impaired spermatogenesis soon after the injury, which in most cases recovered over time. That was accompanied by parallel increases in serum levels of inhibin B, testosterone and prolactin, possibly driving or reflecting the spermatogenesis recovery. Further studies are needed to elucidate the biological mechanisms underlying these changes.

DA-EPOCH-R Is an Effective Regimenin High Grade B-Cell Lymphoma Defined By Cell-of-Origin, Karyotype and BCL2/MYC/BCL6 Status and Expression

Background: MYC, BCL2 and BCL6 are known to be altered in high grade B-cell lymphoma (HGBL). Double/triple-hit lymphomas (D/THLs) are characterized by chromosomal rearrangementsof MYC, BCL2 and/or BCL6. D/THLs have been included in the updated 2016 WHO classification, as a new category of "High grade B-cell lymphoma with rearrangements" (HGBL-R) or Diffuse Large B-cell lymphoma (DLBCL) entity, depending on morphology/cytogenetic features. BCL2 protein is expressed in a much higher proportion of DLBCL and HGBL, not otherwise specified (HGBL,NOS), and is often associated with a concomitant expression of MYC and BCL6. Most of HGBLs do not carry BCL2/MYC/BCL6 rearrangements and are referred to as "double/triple-expressor lymphomas" (D/TELs). D/THLs patients usually progress rapidly, are resistant to R-CHOP immunochemotherapy, and have very poor prognosis. D/TELs also have a worse outcome than other DLBCL,NOS. BCL2 overexpression is observed in both germinal centre B-cell-like (GCB) and non-GCB HGBL. We have previously described a diagnostic algorithm for subtypes of HGBL based on flow-cytometry immunphenotyping (FCM) with CD38 overexpression, which correlates with MYC rearrangement assessed in fine needle aspiration biopsy (FNAB) samples. Here, we propose that patients with HGBL/DLBCL,NOS, with BCL2 overexpression, especially those with D/THLs and D/TELs, may benefit from DA-EPOCH-R (dose-adjusted cyclophosphamide, doxorubicin, etoposide, vincristine, prednisone with rituximab) treatment.Methods: 30 patients (male/female 18/12, median age/range 51/35-76/) diagnosed with DLBCL,NOS (13 pts), HGBL-R (11 pts), HGBL,NOS (2 pts), primary mediastinal B-cell lymphoma - PMBL (3 pts) and DLBCL,leg type (1 pt), based on 2016 WHO classification, were treated with DA-EPOCH-R as first-line therapybetween January 2015 - July 2016. Clinical stage III or IV and IPI 3 or more were found in 27 pts (90%) and 22 pts (73%), respectively. All cases were evaluated by histopathological/immunohistochemical/flow-cytometry examination (HP/IHC/FCM), with panels of antibodies, including also CD5/CD10/CD20/CD38/BCL2/BCL6/MYC. In most cases, MYC, BCL2, BCL6 and, in case of relapse, also TP53 status was evaluated by karyotyping (CC) and FISH.Results: Considering the cell-of-origin, there were 20 pts with GCB, 3 non-GCB, 4 CD5+ and 3 PMBL. In addition, 15 pts were DEL, 12 - TEL, 3 - one-expressor lymphoma, 8 - D/THL, 12 - one-hit lymphoma and 10 - non-hit lymphoma, mostly with BCL2 overexpression. Karyotype was successfully assessed in 70%, while BCL2 overexpression was found in 83% of HGBL pts. MYC, BCL2, and BCL6 rearrangement was found in 48%, 35% and 21%, respectively. In 50%, 46% and 54% of cases there was an increase in copy number/amplification of MYC, BCL2 and BCL6, respectively. In 15 evaluable pts., overall and complete response was 80% and 53%, respectively. Median follow-up of HGBL pts treated with DA-EPOCH-R was 5 months (range 1-17), and the probability of one year overall survival was 91%, 95% C.I. (80%,100%). Progression free survival at 1 year was 62%, 95C.I. (36%,90%). 3 of 4 patients with progressive disease had TP53 deletion. The main toxicity was pancytopenia with neutropenia grade 3 or more in 24 pts (80%). Treatment-related mortality due to septic shock occurred in 2 patients (7%).Conclusions: DA-EPOCH-R regimen shows a promising activity considering D/THL and D/TEL-associated drug resistance. DA-EPOCH-R regimen seems to overcome drug resistance associated with BCL2/MYC/BCL6 overexpression, but not with TP53 deletion. Combining HP/IHC with FNAB/FCM/CC/FISH is a reliable method for D/THL and D/TEL diagnosis but the most sensitive method for fast MYC/BCL2 rearrangement assessment in DHL is FNAB/FCM CD38 and BCL2 overexpression. DisclosuresRymkiewicz:Takeda: Other: travel, accommodation; Roche: Other: travel, accommodation. Romejko-Jarosinska:Celgene: Other: travel, accommodation; Servier: Other: travel, accommodation; Sanofi- Aventis: Other: travel, accommodation. Paszkiewicz-Kozik:Sandos: Other: fee; Hospira: Other: fee; Sanofi: Other: accommodation; Roche: Other: travel, accommodation. Domanska-Czyz:Roche: Other: travel, accommodation; Amgen: Other: travel, accommodation. Ostrowska:Roche: Other: travel, accommodation; Amgen: Other: travel, accommodation. Dabrowska-Iwanicka:Genzyme: Other: travel, accommodation; Roche: Other: travel, accommodation. Osowiecki:Sandoz: Other: travel, accommodation; Roche: Other: travel, accommodation. Sikorska-Mali:Roche: Other: travel, accommodation. Szymanski:Stada: Other: travel, accommodation. Swierkowska-Czeneszew:Stada: Other: travel, accommodation; Roche: Other: travel, accommodation; Amgen: Other: travel, accommodation. Prochorec-Sobieszek:Roche: Other: travel, accommodation. Walewski:Mundipharma: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Other: travel, accommodation, Research Funding; Takeda: Consultancy, Honoraria, Other: travel, accommodation; Roche: Consultancy, Honoraria, Other: travel, accommodation, Research Funding; Teva: Consultancy, Honoraria; Gilead: Consultancy, Honoraria, Other: travel, accommodation; Sanofi: Honoraria, Other: travel, accommodation; Janssen-Cilag: Consultancy; Boehringer Ingelheim: Consultancy; Karyopharm: Consultancy; Ariad: Consultancy; Servier: Consultancy; GSK/Novartis: Research Funding; Genetics: Other: travel, accommodation; Sanofi: Other: travel, accommodation.

APLP2, RRM2, and PRC1: New Putative Markers for the Differential Diagnosis of Thyroid Follicular Lesions.

Current methods based on fine-needle aspiration biopsy (FNAB) are not sufficient to distinguish among follicular thyroid lesions, follicular adenoma (FA), follicular thyroid carcinoma (FTC), and the follicular variant of papillary thyroid cancer (FVPTC). Furthermore, none of the immunohistochemical markers currently available are sensitive or specific enough to be used in the clinical setting, necessitating a diagnostic hemithyroidectomy. The aim of this study was to identify proteins of value for differential diagnosis between benign and malignant thyroid follicular lesions. This retrospective analysis is based on an assessment of the immunoexpression of 19 proteins on 81 benign thyroid lesions (FA) and 50 malignant tumors (FTC/FVPTC). The resulting expression profile allowed the design of a scoring system model to improve the differential diagnosis of benign and malignant thyroid lesions. The model was validated using an independent series of 69 FA and 40 FTC and an external series of 40 nodular hyperplasias, and was further tested in a series of 38 FNAB cell blocks. A model based on the nuclear and cytoplasmic expression of APLP2, RRM2, and PRC1 discriminated between benign and malignant lesions with 100% sensitivity in both main and validation groups, with specificities of 71.3% and 50.7%, respectively. For the nodular hyperplasia series, specificity reached 94.8%. Finally, in FNAB samples, the sensitivity was 100% and the specificity was 45% for discrimination between benign and malignant lesions. These findings suggest that the identified APLP2, RRM2, and PRC1 signature could be useful for distinguishing between benign (FA) and malignant (FTC and FVPTC) tumors of the thyroid follicular epithelium.