PET/MRI for staging patients with Hodgkin lymphoma: equivalent results with PET/CT in a prospective trial.

To compare FDG-PET/unenhanced MRI and FDG-PET/diagnostic CT in detecting infiltration in patients with newly diagnosed Hodgkin lymphoma (HL). The endpoint was equivalence between PET/MRI and PET/CT in correctly defining the revised Ann Arbor staging system. Seventy consecutive patients with classical-HL were prospectively investigated for nodal and extra-nodal involvement during pretreatment staging with same-day PET/CT and PET/MRI. Findings indicative of malignancy with the imaging procedures were regarded as lymphoma infiltration; in case of discrepancy, positive-biopsy and/or response to treatment were evidenced as lymphoma. Sixty of the 70 (86%) patients were evaluable having completed the staging program. Disease staging based on either PET/MRI or PET/CT was correct for 54 of the 60 patients (90% vs. 90%), with difference between proportions of 0.0 (95% CI, -9 to 9%; P=0.034 for the equivalence test). As compared with reference standard, invasion of lymph nodes was identified with PET/MRI in 100% and with PET/CT in 100%, of the spleen with PET/MRI in 66% and PET/CT in 55%, of the lung with PET/MRI in 60% and PET/CT in 100%, of the liver with PET/MRI in 67% and PET/CT in 100%, and of the bone with PET/MRI in 100% and PET/CT in 50%. The only statistically significant difference between PET/MRI and PET/CT was observed in bony infiltration detection rates. For PET/CT, iodinate contrast medium infusions' average was 86 mL, and exposure to ionizing radiation was estimated to be 4-fold higher than PET/MRI. PET/MRI is a promising safe new alternative in the care of patients with HL.

2-deoxy-2[F-18] fluoro-D-glucose positron emission tomography Deauville scale and core-needle biopsy to determine successful management after six doxorubicin, bleomycin, vinblastine and dacarbazine cycles in advanced-stage Hodgkin lymphoma.

BACKGROUND: The clinical impact of the positivity of the Deauville scale (DS) of positron emission tomography (PET) performed at the end of doxorubicin, bleomycin, vinblastine and dacarbazine (ABVD) in patients with advanced Hodgkin lymphoma (HL), in terms of providing rationale to shift poor responders onto a more intensive regimen, remain to be validated by histopathology. PATIENTS AND METHODS: This prospective trial involved patients with stage IIB/IV HL who after six ABVD cycles underwent PET (PET6) and core-needle cutting biopsy (CNCB) of 2-deoxy-2[F-18] fluoro-d-glucose (FDG)-avid lymph nodes. Patients received high-dose chemotherapy/autologous haematopoietic stem cell rescue (HDCT/AHSCR) if CNCB was positive for HL, alternatively, if CNCB or PET was negative, received observation or consolidation radiotherapy (cRT) on residual nodal masses, as initially planned. The end-point was 5-year progression-free survival (PFS). RESULTS: In all, 43 of the 169 (25%) evaluable patients were PET6 positive (DS 4, 32; DS 5, 11). Among them, histology showed malignancy (HL) in 100% of DS 5 scores and in 12.5% of DS 4 scores. Fifteen patients with positive biopsy received HDCT/AHSCR, whereas 28 patients with negative biopsy, as well as 126 patients with negative PET6, continued the original plan (cRT, 78 patients; observation, 76 patients). The 5-year PFS in the negative PET6 group, negative biopsy group and positive biopsy group was 95.4%, 100% and 52.5%, respectively. CONCLUSION: DS positivity of end-of-ABVD PET in advanced HL carried a certain number of CNCB-proven non-malignant FDG-uptakes. The DS 4 scores which were found to have negative histology appeared to benefit from continuing the original non-intensive therapeutic plane as indicated by the successful outcome in more than 95% of them by obtaining similar 5-year PFS to the PET6-negative group. By contrast, the DS 5 score had consistently positive histology and was associated with unsuccessful conventional therapy, promptly requiring treatment intensification or innovative therapeutic approaches.

Intra-institutional second opinion diagnosis can reduce unnecessary surgery for indeterminate thyroid FNA: A preliminary report on 34 cases.

INTRODUCTION: Indeterminate diagnoses are rendered on 15%-30% of thyroid fine-needle aspirates (FNA). Thus, a second diagnostic opinion given by an outside expert pathologist is a common practice that facilitates a more appropriate clinical management. Conversely, the role of an intra-institutional second opinion diagnosis (iSOD), which is usually informally performed in-house, has not been well established. METHODS: To assess the contribution of iSOD, a retrospective series of 34 thyroid FNA diagnosed as follicular neoplasm/suspicious follicular neoplasm (FN/SFN) with matched histological follow-up and a malignancy rate of 17.6% was selected and independently reviewed by two cytopathologists (CYT1 and 2). Cases with discrepant diagnoses were referred to a third in-house senior cytopathologist for the iSOD. The malignancy rates (MR) obtained after single independent reviews and iSOD were compared. RESULTS: MR obtained after CYT1 and CYT2 re-screening was similar (14.28% and 19.04%, respectively) and did not improve the original MR (17.64%). Conversely, after the iSOD of discrepant diagnoses, the overall malignancy rate increased up to the 27.27%, potentially sparing unnecessary surgical procedures. CONCLUSIONS: Intra-institutional second opinion practice for "indeterminate" thyroid FNA avoids unnecessary surgeries and maximises the detection of malignant cases diagnosed as FN/SFN.

Cytopathologists can reliably perform ultrasound-guided thyroid fine needle aspiration: a 1-year audit on 3715 consecutive cases.

OBJECTIVE: In our Pathology Department, fine needle aspiration (FNA) of palpable thyroid nodules is performed by cytopathologists who ensure correct sample management and rapid on-site evaluation (ROSE). Conversely, ultrasound (US)-guided FNAs have traditionally been carried out by endocrinologists and radiologists in outside clinics, where the presence of a cytopathologist is not always feasible. To overcome this limitation, cytopathologists have started to perform US-guided FNAs themselves. This study retrospectively evaluates 1 year of this novel practice. METHODS: A total of 2225 US-guided FNAs were performed in our clinic by cytopathologists, whereas 1490 aspirates were taken by a group of non-cytopathologists. Among these, 756 FNAs were taken by a single experienced endocrinologist. The distribution of the Bethesda classification categories was evaluated in each of these groups. RESULTS: FNAs performed by cytopathologists were more often diagnostic and better prepared than those taken by non-cytopathologists, including those taken by the experienced endocrinologist (P < 0.01). The latter operator yielded a higher rate of suspicious and malignant FNAs, reflecting a more appropriate clinical triage of worrisome nodules. CONCLUSION: Although the endocrinologist's evaluation is crucial to select clinically relevant thyroid nodules, cytopathologists can reliably perform US guidance in addition to their traditional expertise in sampling, specimen preparation and ROSE.

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.

Outsourcing cytological samples to a referral laboratory for EGFR testing in non-small cell lung cancer: does theory meet practice?

OBJECTIVE: Guidelines from the College of American Pathologists (CAP), the International Association for the Study of Lung Cancer (IASLC) and the Association for Molecular Pathology (AMP) consider cytology suitable for testing epidermal growth factor receptor (EGFR) mutations in lung adenocarcinoma. The guidelines recommend that cytopathologists first discuss the possibility of testing squamous cell carcinomas (SqCC) in multidisciplinary meetings. Second, cell blocks should be analysed rather than smear preparations and, third, specimens should be sent to external molecular laboratories within three working days of receiving requests. This study monitored how these recommendations are met in practice. METHODS: Our laboratory received 596 requests from cytologists from 13 different institutions. For each case, the cytological diagnosis, cytopreparation type, and time between the request and sample mailing were compared with the recommendations. RESULTS: Of the 596 samples, 32 (5.4%) had been reported as SqCC. Three of these (9.4%) showed EGFR mutation. Cytological slides, either ThinPrep(™) (51.2%) or direct smears (43.2%), were more frequently received than cell blocks (5.7%). The mean time between the oncologist's request and specimen dispatching was 5.8 working days. CONCLUSIONS: The occurrence of mutations in samples reported as SqCC was higher than expected. This questions the reliability of the original diagnosis, which reinforced the recommendation to evaluate the opportunity for testing non-adenocarcinoma cytology on a case-by-case basis. In spite of CAP/IASLC/AMP recommendations, cell blocks were underutilized for EGFR testing, but cytological slides were suitable for DNA analyses. Significant efforts are needed to avoid delays in outsourcing cytological samples for EGFR testing.

CD10 is useful to identify gastrointestinal contamination in endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) cytology from pancreatic ductal adenocarcinoma.

OBJECTIVE: Endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) cytology is an effective tool to diagnose pancreatic ductal adenocarcinoma (PDA). Standard morphological criteria are usually reliable. When contaminating gastrointestinal (GI) epithelial cells are prevalent among neoplastic cells, these can be highlighted by carcinoembryonic antigen (CEA) staining. CD10 is a cell-surface metallopeptidase normally expressed by the GI epithelial apical border, whose expression is decreased or lost in PDA. We included CD10 in a panel, together with CEA, to discriminate the GI contaminant cells from PDA cells on cell blocks. METHODS: Eight cases of EUS-FNA of PDA, featuring both contaminating GI cells and neoplastic cells, whose corresponding cell blocks were available for immunostaining, were selected. CD10 and CEA were stained on cell blocks by standard methods. RESULTS: CD10 strongly labelled only the GI cells, with a well-defined apical membrane signal; conversely, GI cells did not show CEA staining; benign duodenal cells were faintly labelled in only one case. Malignant cells were positive for CEA and negative for CD10, with the exception of one case with labelled neoplastic cells with weak diffuse cytoplasmic positivity. CD10 apical membrane staining was a feature only seen in benign GI cells. CONCLUSIONS: As a loss of CD10 is a consistent feature of PDA, this marker can be useful, together with CEA, to aid the cytopathologist to identify neoplastic cells in a background rich in GI contaminant cells.

Performance of EGFR mutant-specific antibodies in different cytological preparations: a validation study.

OBJECTIVES: Molecular testing for epidermal growth factor receptor (EGFR) mutations is required to select the most appropriate treatment for advanced-stage non-small cell lung cancer (NSCLC). In routine practice, cytological samples are often the only specimens available for testing. When the number of neoplastic cells is large, DNA-based assays are the gold standard. When cytological samples contain only a few neoplastic cells, immunocytochemistry (ICC) using anti-EGFR mutant-specific antibodies may be more effective. We aim to assess the specificity and sensitivity of IHC staining in cytological specimens using mutated cell lines subjected to different cytopreparations and staining methods. METHODS: HCC827 (exon 19 p.E746-A750 del) and H3255 (exon 21 p.L858R) cell lines were subjected to different fixation (air dried, alcohol or CytoLyt(®)), staining (Diff-Quik(®) or Papanicolaou) and preparation (smears or cell blocks) methods before ICC. In a second set of experiments, mutated cells were mixed with EGFR wild-type cells to obtain low-level (10%) mutated cytological samples. The intensity and percentage of cells stained were evaluated against validated molecular techniques. Moreover, the cell lines were subjected to poor growing conditions to simulate routine specimens that are less optimal than in vitro samples. RESULTS: The cytological preparations showing the most intense staining were formalin-fixed cell blocks and samples fixed with CytoLyt or alcohol, including Papanicolaou-destained samples. Conversely, air-dried slides showed the least intense staining. Mutant antibodies allowed the detection of mutated cells, even when representing only 10% of the total population. Although, in necrotic specimens, an aspecific background signal appeared, the viable cells still retained anti-mutant EGFR positivity. CONCLUSIONS: All cytological preparations are suitable for ICC using anti-EGFR mutant-specific antibodies, in particular formalin-fixed cell blocks and alcohol- or CitoLyt-fixed samples. the method is also validated to detect even a few mutant cells in less than optimal samples.

Fine needle aspiration cytology of lymphoproliferative lesions of the oral cavity.

OBJECTIVE: 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. METHODS: The results of FNA cytology on 16 consecutive lymphoproliferative lesions of the oral cavity collected over an 8-year period in three institutions were retrieved. Sampled lesions were submucosal masses of different sizes bulging into the oral cavity. Rapid on-site evaluation (ROSE) and routine cytological staining were performed. Immunocytochemistry (ICC), flow cytometry (FC) and polymerase chain reaction (PCR) of the IGH (immunoglobulin heavy) locus were performed on additional passes according to ROSE. RESULTS: Fourteen OCLs, one myeloma and one florid reactive lymphoid hyperplasia (FRLH) were diagnosed by FNA. OCLs were diagnosed as large B-cell (eight cases) and small B-cell (six cases) lymphomas. Histology revealed eight diffuse large B-cell lymphomas (DLBCL), four lymphomas of mucosa-associated lymphoid tissue (MALT), two follicular lymphomas and one FRLH; no false-negative or false-positive results were diagnosed, but accurate subclassification was obtained in four cases only. CONCLUSIONS: FNA diagnosis of OCLs may be hampered by the rare incidence, anatomical context and difficulties in obtaining a sufficient amount of cells. Ancillary techniques should be used according to ROSE; a pre-operative FNA cytology diagnosis can avoid unnecessary extensive surgery and speed up the institution of therapeutic procedures.

Non lymphomatous clonal B-Cell populations in enlarged lymph nodes in acquired immunodeficiency syndrome.

Clonal B-cell populations in non-lymphomatous processes have been sporadically reported in enlarged reactive lymph nodes and mucosa-associated lymphoid cell populations. These generally small clones are considered non-malignant proliferations of B-lymphocytes determined by an abnormal response to bacterial or viral antigen stimulation. In cases reported in literature, clonality was detected by light chain assessment and or by polymerase chain reaction (PCR) analysis of immunoglobulin heavy chain (IgH) gene in histologically and clinically proven non lymphomatous processes. In this study the clinical, cytological, phenotypical and pathological features of three HIV patients in which non-lymphomatous clonal B-cell populations detected in enlarged lymph nodes are reported. All the patients complained for later cervical lymph nodes enlargement, positive at the FDG-positron emission tomography scan. Fine needle cytology, coupled with flow cytometry showed atypical lymphoid cell proliferations and kappa (2 cases) or lambda (1 case) light chain restriction. Reactive, non lymphomatous nature of these processes were then proven by histological control in two cases and by clinical follow-up in the last one; corresponding clinical and pathological aspects are discussed. Clonal B-cell populations in non-lymphomatous processes can sporadically occur in enlarged reactive lymph nodes in immunodeficiency as well as in autoimmune processes. Awareness of the phenomenon and attention should be paid in the evaluation of corresponding pathological features and in the clinical management of corresponding patients.

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

OBJECTIVE: To evaluate the diagnostic efficiency of fine needle aspiration cytology/flow cytometry (FNAC/FC) in the diagnosis and classification of non-Hodgkin lymphoma (NHL) in a series of 446 cases and to compare the results with those of previous experiences to evaluate whether there had been an improvement in FNAC/FC diagnostic accuracy. METHODS: FNAC/FC was used to analyse 446 cases of benign reactive hyperplasia (BRH), NHL and NHL relapse (rNHL) in 362 lymph nodes and 84 extranodal lesions. When a diagnosis of NHL was reached, a classification was attempted combining FC data and cytological features. Sensitivity, specificity and positive and negative predictive values (PPV and NPV) of FNAC/FC in the diagnosis and classification of NHL were calculated and compared with those available in the literature. RESULTS: FNAC/FC provided a diagnosis of NHL and rNHL in 245 cases and of BRH in 188 cases. In nine cases, the diagnosis was 'suggestive of NHL' (sNHL) and in four cases was inadequate. Histology and clinical follow-up confirmed 102 cases of NHL and detected one false positive. In 18 cases of BRH diagnosed by FNAC/FC, histological examination revealed 14 BRH and four NHL (false negatives). All nine cases diagnosed as sNHL were confirmed by histology. Including sNHL cases as false negatives, statistical analysis showed 94.9% sensitivity, 99.4% specificity, 99.6% PPV and 93.4% NPV in the diagnosis of NHL. A specific subtype was diagnosed in 125 cases and confirmed in 67 of 70 cases that had histological biopsies. Statistical analysis did not demonstrate significant improvements between the present series and previous studies either in diagnosis or in classification of NHL. CONCLUSIONS: FNAC/FC is a fundamental tool in the diagnosis and classification of NHL but the exiguity of diagnostic material and other technical and clinical limitations will probably continue to limit further improvement of the technique.