Elena Vigliar

Fine needle aspiration cytology and flow cytometry immunophenotyping of non-Hodgkin lymphoma: can we do better?

P. Zeppa, E. Vigliar, I. Cozzolino, G. Troncone, M. Picardi, A. De Renzo, F. Grimaldi, F. Pane, A. Vetrani and L. Palombini
Fine needle aspiration cytology and flow cytometry immunophenotyping of non‐Hodgkin lymphoma: can we do better?

Ion Torrent next-generation sequencing for routine identification of clinically relevant mutations in colorectal cancer patients.

Aims To evaluate the accuracy, consumable cost and time around testing (TAT) of a next-generation sequencing (NGS) assay, the Ion Torrent AmpliSeq Colon and Lung Cancer Panel, as an alternative to Sanger sequencing to genotype KRAS, NRAS and BRAF in colorectal cancer patients. Methods The Ion Torrent panel was first verified on cell lines and on control samples and then prospectively applied to routine specimens (n=114), with Sanger sequencing as reference. Results The Ion Torrent panel detected mutant alleles at the 5% level on cell lines and correctly classified all control tissues. The Ion Torrent assay was successfully carried out on most (95.6%) routine diagnostic samples. Of these, 12 (11%) harboured mutations in the BRAF gene and 47 (43%) in either of the two RAS genes, in two cases with a low abundance of RAS mutant allele which was missed by Sanger sequencing. The mean TAT, from sample receipt to reporting, was 10.4 (Sanger) and 13.0 (Ion Torrent) working days. The consumable cost for genotyping KRAS, NRAS and BRAF was €196 (Sanger) and €187 (Ion Torrent). Conclusions Ion Torrent AmpliSeq Colon and Lung Cancer Panel sequencing is as robust as Sanger sequencing in routine diagnostics to select patients for anti-epidermal growth factor receptor (EGFR) therapy for metastatic colorectal cancer.

Challenges and opportunities of next-generation sequencing: a cytopathologist's perspective

Molecular cytopathology has gene sequencing as its core technology. Until recently, cytological samples were only tested by sequential single‐gene mutational tests. Today, with the better understanding of the molecular events involved in malignancy and the mechanisms of pharmacotherapy, larger gene panels are more informative than a single biomarker. Next‐generation sequencing (NGS), matched with the multiplex capture of targeted gene regions and analysed by sophisticated bioinformatics tools, enables the simultaneous detection of multiple mutations in multiple genes. With the development of miniaturised technology and benchtop sequencers, it is not unlikely that NGS will soon be adopted for routine molecular diagnostics, including cytological samples. This review addresses (1) the most relevant methodological and technical aspects of the NGS analysis workflow and the diverse platforms available; (2) the issues related to daily practice implementation, namely, the cytological sample requirement and the validation procedures; and (3) the opportunities that NGS offers in different fields of cytopathology, to increase mutation detection sensitivity in paucicellular smears and to extend the analysis to a larger number of gene regions. Cytopathologists involvement and coordination in this rapidly evolving field is crucial for the effective implementation of NGS in the present and future cytological practice.

Lymph node fine needle Cytology in the staging and follow-up of Cutaneous Lymphomas

BackgroundLymph nodal involvement is an important clinical-pathological sign in primary cutaneous lymphoma (PCL), as it marks the transformation/evolution of the disease from localized to systemic; therefore the surveillance of lymph nodes is important in the staging and follow up of PCL. Fine needle cytology (FNC) is widely used in the diagnosis of lymphadenopathies but has rarely been reported in PCL staging and follow-up. In this study an experience on reactive and neoplastic lymphadenopathies arisen in PCL and investigated by FNC, combined to ancillary techniques, is reported.MethodsTwenty-one lymph node FNC from as many PCL patients were retrieved; 17 patients had mycosis fungoides (MF) and 4 a primary cutaneous B-cell lymphoma (PBL). In all cases, rapid on site evaluation (ROSE) was performed and additional passes were used to perform flow cytometry (FC), immunocytochemistry (ICC) and/or polymerase chain reaction (PCR) to assess or rule out a possible clonality of the corresponding cell populations.ResultsFNC combined with FC, ICC, and PCR identified 12 cases of reactive, non specific, hyperplasia (BRH), 4 dermatopathic lymphadenopathy (DL), 4 lymph nodal involvement by MF and 1 lymph nodal involvement by cutaneous B-cell lymphoma.ConclusionsFNC coupled with ancillary techniques is an effective tool to evaluate lymph node status in PCL patients, provided that ROSE and a rational usage of ancillary techniques is performed according to the clinical context and the available material. The method can be reasonably used as first line procedure in PCL staging and follow up, avoiding expensive and often ill tolerated biopsies when not strictly needed.

Immunoglobulin heavy-chain fluorescence in situ hybridization-chromogenic in situ hybridization DNA probe split signal in the clonality assessment of lymphoproliferative processes on cytological samples.

The human immunoglobulin heavy‐chain (IGH) locus at chromosome 14q32 is frequently involved in different translocations of non‐Hodgkin lymphoma (NHL), and the detection of any breakage involving the IGH locus should identify a B‐cell NHL. The split‐signal IGH fluorescence in situ hybridization‐chromogenic in situ hybridization (FISH‐CISH) DNA probe is a mixture of 2 fluorochrome‐labeled DNAs: a green one that binds the telomeric segment and a red one that binds the centromeric segment, both on the IGH breakpoint. In the current study, the authors tested the capability of the IGH FISH‐CISH DNA probe to detect IGH translocations and diagnose B‐cell lymphoproliferative processes on cytological samples.

Less frequently mutated genes in colorectal cancer: evidences from next-generation sequencing of 653 routine cases.

Aims The incidence of RAS/RAF/PI3KA and TP53 gene mutations in colorectal cancer (CRC) is well established. Less information, however, is available on other components of the CRC genomic landscape, which are potential CRC prognostic/predictive markers. Methods Following a previous validation study, ion-semiconductor next-generation sequencing (NGS) was employed to process 653 routine CRC samples by a multiplex PCR targeting 91 hotspot regions in 22 CRC significant genes. Results A total of 796 somatic mutations in 499 (76.4%) tumours were detected. Besides RAS/RAF/PI3KA and TP53, other 12 genes showed at least one mutation including FBXW7 (6%), PTEN (2.8%), SMAD4 (2.1%), EGFR (1.2%), CTNNB1 (1.1%), AKT1 (0.9%), STK11 (0.8%), ERBB2 (0.6%), ERBB4 (0.6%), ALK (0.2%), MAP2K1 (0.2%) and NOTCH1 (0.2%). Conclusions In a routine diagnostic setting, NGS had the potential to generate robust and comprehensive genetic information also including less frequently mutated genes potentially relevant for prognostic assessments or for actionable treatments.

Fine needle aspiration cytology of lymphoproliferative lesions of the oral cavity

Oral cavity non‐Hodgkin lymphoma (OCL) is a rare condition that may be clinically and radiologically indistinguishable from other pathologies of the mouth. A complete excision or adequate biopsy of the OCL may be difficult. Fine needle aspiration (FNA) cytology has been successfully utilized in the pre‐operative diagnosis of oral masses and in lymphoma involving other anatomical areas. Our experience with FNA pre‐operative cytological diagnosis of 16 OCLs is reported herein.

Epidermal Growth Factor Receptor Test Performed on Liquid-Based Cytology Lung Samples: Experience of an Academic Referral Center

Objectives: In this study we reviewed our practice of lung cancer epidermal growth factor receptor (EGFR) mutational testing in an academic centralized laboratory setting, where direct smears and liquid-based cytology (LBC) slides represent the most frequent cytological specimens received. The aim was to assess the differences, if any, between these sample types in terms of DNA yield, adequacy rates and overall EGFR testing performance. Study Design: A total of 362 cases were retrieved - received from January 2012 to January 2014 for EGFR testing - including 204 LBC specimens and 158 smears. Exon 19 deletions and the L858R point mutation in exon 21, detected by fragment assay and TaqMan assay, respectively, were confirmed by direct sequencing or by high-resolution melting. Results: Although the direct smears showed a higher DNA yield (60.94 vs. 23.07 ng/µl) and were more frequently cell-rich (54%) than the LBC slides (31.4%), the differences in adequacy (direct smears: 97.4%; LBCs: 94.1%) and in mutant rate (direct smears: 10.3%; LBCs: 14.0%) between the two sample types did not reach statistical significance. Conclusions: Not only direct smears but also LBC slides represent an effective preparation and storage medium for cytological material to be used for EGFR molecular testing.

Cytological diagnosis of thyroid nodules in Hashimoto thyroiditis in elderly patients

BackgroundLong standing Hashimoto Thyroiditis (HT) causes shrinking and atrophy of the thyroid, but may also lead to diffuse enlargement of the gland and/or formation of nodules. These nodules should be differentiated from papillary thyroid carcinoma (PTC) and primary thyroidal non-Hodgkin lymphoma (PTL), which are possible complications of HT, and require pre-surgical diagnoses and different treatments.This study focuses on the role of fine-needle cytology (FNC) in the clinical surveillance and pre-surgical diagnosis of HT with diffuse and nodular enlargement of the gland in elderly patients.MethodsThirty-four elderly patients (≥ 65 yrs) with HT and diffuse or nodular enlargement of the thyroid underwent ultrasound (US)-guided FNC. Smears were routinely stained and evaluated; additional passes were used for flow cytometry (FC) assessment of lymphoid infiltrate in 6 cases.ResultsThe cytological diagnosis was HT in 12 cases with prevalence of Hurtle cells in 2 cases, PTC in 1 case and PTL in 2 cases. FC assessed the reactive, non-lymphomatous nature of the lymphoid infiltrate in 5 cases and demonstrated light chain restriction, hence the lymphomatous nature of the lymphoid infiltrate in 2 cases of PTL.ConclusionsFNC plays a key role in the clinical surveillance and pre-surgical diagnosis of diffuse enlargement and nodular presentation of HT in elderly patients. FNC can correctly diagnose HT, PTC and PTL indicating the need for surgery and its extension in suspicious or neoplastic cases, leaving other cases to the medical treatment and clinical surveillance.

Early cytological diagnosis of extranodal stage I, primary thyroid Non-Hodgkin lymphoma in elderly patients. Report of two cases and review of the literature

BackgroundPrimary thyroid lymphomas (PTLs) account for 5% of thyroid malignant tumors and often develop in patients with Hashimoto Thyroiditis (HT). Fine-needle cytology (FNC) is widely used in the diagnosis of thyroid nodules, including those arising in HT. Two PTL cases in HT elderly patients are here described and discussed.MethodsFNC was performed in rapidly enlarged thyroid nodules of 2 elderly patients under ultrasound (US) control. FNC was used to prepare conventional cytologic smears, immunocytochemistry (ICC) and flow cytometry (FC) assessment of cell populations.ResultsThe above cases were diagnosed as well differentiated, small B-cell and diffuse large B-cell thyroid lymphomas, respectively, by means of FNC. The histological diagnoses were mucosa-associated non Hodgkin lymphoma (MALT) and diffuse large B-cell lymphoma (DLBCL), confirming FNC diagnoses, and patients were treated accordingly.ConclusionsFNC diagnosis of PTL is reliable and accurate; it may be conveniently used in the clinical practice since it provides indications for appropriate therapeutic procedures or diagnostic surgery, and avoids to treat benign nodules.