A significant proportion of classic Hodgkin lymphoma recurrences represents clonally unrelated second primary lymphoma.

Despite high cure rates in classic Hodgkin lymphoma (cHL), relapses are observed. Whether relapsed cHL represents second primary lymphoma or an underlying T-cell lymphoma (TCL) mimicking cHL is underinvestigated. To analyze the nature of cHL recurrences, in-depth clonality testing of immunoglobulin (Ig) and T-cell receptor (TCR) rearrangements was performed in paired cHL diagnoses and recurrences among 60 patients, supported by targeted mutation analysis of lymphoma-associated genes. Clonal Ig rearrangements were detected by next-generation sequencing (NGS) in 69 of 120 (58%) diagnoses and recurrence samples. The clonal relationship could be established in 34 cases, identifying clonally related relapsed cHL in 24 of 34 patients (71%). Clonally unrelated cHL was observed in 10 of 34 patients (29%) as determined by IG-NGS clonality assessment and confirmed by the identification of predominantly mutually exclusive gene mutations in the paired cHL samples. In recurrences of >2 years, ∼60% of patients with cHL for whom the clonal relationship could be established showed a second primary cHL. Clonal TCR gene rearrangements were identified in 14 of 125 samples (11%), and TCL-associated gene mutations were detected in 7 of 14 samples. Retrospective pathology review with integration of the molecular findings were consistent with an underlying TCL in 5 patients aged >50 years. This study shows that cHL recurrences, especially after 2 years, sometimes represent a new primary cHL or TCL mimicking cHL, as uncovered by NGS-based Ig/TCR clonality testing and gene mutation analysis. Given the significant therapeutic consequences, molecular testing of a presumed relapse in cHL is crucial for subsequent appropriate treatment strategies adapted to the specific lymphoma presentation.

Reclassification of diffuse large B cell lymphoma to large B cell lymphoma with IRF4 rearrangement in an adult population.

AIMS: Large B cell lymphoma with IRF4 rearrangement (LBCL-IRF4) is a new entity in the 2017 revised World Health Organisation (WHO) classification that was initially mainly reported in children. After identification of a 79-year-old patient, we assessed how often IRF4 rearrangements can be detected in adult diffuse large B cell lymphomas (DLBCLs) which have to be reclassified to LBCL-IRF4 based on fluorescence in-situ hybridisation (FISH) for IRF4. METHODS AND RESULTS: With FISH, we studied the presence of IRF4 rearrangements in 238 lymphomas that were diagnosed as DLBCL according to the previous WHO classification of 2008. CONCLUSIONS: In addition to the index patient, an IRF4 rearrangement was detected in another five of 237 patients (2%). The immunohistochemical profile of these five IRF4 rearranged lymphomas was consistent with previous reports of LBCL-IRF4. One case was recognised to represent transformation of follicular lymphoma rather than de-novo LBCL-IRF4. BCL6 rearrangements were found in two cases of LBCL-IRF4; BCL2 and MYC rearrangements were excluded. Patients presented with limited stage disease with involvement of the head and neck in three patients, and involvement of the lung and thyroid in two others. This study shows that, although rare, LBCL-IRF4 should also be considered in older patients and at localisations other than the head and neck region.

Reassessment of Prostate Biopsy Specimens for Patients Referred for Robot-assisted Radical Prostatectomy Rarely Influences Surgical Planning.

BACKGROUND: The minimum volume standard is 100 robot-assisted radical prostatectomy (RARP) procedures per hospital in the Netherlands, so patients have to be referred to high-volume surgical centers for RARP. During preoperative work-up, prostate biopsies taken elsewhere are reassessed, with upgrading or downgrading of the initial Gleason grade group a possible consequence. OBJECTIVE: To determine if prostate biopsy reassessment leads to adjustment of the surgical plan regarding a nerve-sparing approach and extended pelvic lymph node dissection (ePLND) during RARP. DESIGN SETTING AND PARTICIPANTS: For 125 men who were referred to the Prosper prostate center at Canisius Wilhelmina Hospital (CWH) in the Netherlands between 2013 and 2016, results for the initial assessment of prostate biopsy by a local uropathologist were compared to results for biopsy reassessment by dedicated uropathologists at CWH. RESULTS AND LIMITATIONS: The pathologists reached agreement in 80% of the cases. In cases for which there was disagreement (n = 25), biopsy revision involved upgrading of the initial grade group in 68% and downgrading in 32%. Biopsy reassessment led to a change in surgical plan in ten cases (8%). As a result of upgrading, ePLND was performed in three patients (2%). ePLND was omitted in one patient (1%) because of downgrading. For three patients (2%) a non-nerve-sparing procedure was planned after upgrading of the initial grade group. For four patients (3%), a unilateral nerve-sparing procedure was performed after downgrading. CONCLUSIONS: This study shows that there is large interobserver agreement between uropathologists in the assessment of Gleason grade group in prostate biopsy specimens. Reassessment rarely leads to a change in surgical plan regarding the indication for a nerve-sparing approach and ePLND. Therefore, reassessment of prostate biopsy before radical prostatectomy can be omitted when the initial pathological assessment was performed by a dedicated uropathologist. PATIENT SUMMARY: Reassessment of the initial prostate biopsy specimen for patients referred to a specialist center for robot-assisted removal of the prostate rarely influences surgical planning and can be omitted.

Sequential immunohistochemistry: a promising new tool for the pathology laboratory.

AIMS: Current immunohistochemical methods to study the expression of multiple proteins in a single tissue section suffer from several limitations. In this article, we report on sequential immunohistochemistry (S-IHC), a novel, easy method that allows the study of numerous proteins in a single tissue section, while requiring very limited optimization. METHODS AND RESULTS: In S-IHC, a tissue section is stained for multiple antibodies, with intermediate scanning of the section and elution of chromogen and antibodies. Overlays are made of the digital images, allowing assessment of multiple proteins in the same tissue section. We used S-IHC to study nine nodular lymphocyte-predominant Hodgkin lymphomas (NLPHLs) and 10 T-cell-rich and histiocyte-rich diffuse large B-cell lymphomas (T/HRBCLs) for expression of cyclin D1, CD20, and CD68. We observed cyclin D1 expression in single tumour cells in 44% of NLPHLs and 60% of T/HRBCLs. Comparison of S-IHC with classic single immunohistochemical staining revealed discrepancies in eight cases (42%), demonstrating the difficulty of differentiating tumour cells from histiocytes on morphological grounds, and stressing the additional value of S-IHC. CONCLUSIONS: For research and diagnostic purposes, S-IHC is a promising technique that assesses the expression of numerous proteins in single tissue sections with complete architectural information, allowing phenotypic characterization of single cells.

Diagnostic value of histology compared with cytology in transbronchial aspiration samples obtained by histology needle.

OBJECTIVE: Results from endoscopic needle aspiration [transbronchial needle aspiration (TBNA), esophageal ultrasound-guided fine needle aspiration, real-time endobronchial ultrasound] mainly rely on cytology. We performed a retrospective study to evaluate the possible advantage of obtaining histologic samples during TBNA in the diagnostic assessment of mediastinal lymph node enlargement. MATERIALS AND METHODS: In a retrospective study 2 pathologists evaluated all TBNAs from patients with mediastinal lymph node enlargement in whom representative histologic and cytologic material was obtained, using only a histology needle. Cytology was reviewed before histology in a randomized, blinded fashion. Afterward, the results were related to the diagnosis made in the actual workup of the patient. RESULTS: A total of 50 TBNAs were reviewed. In 86% (43 of 50), both pathologists made the same diagnosis on both specimens, or a difference in cytology and/or histology specimens did not alter the eventual treatment. In 14% (7 of 50) of all TBNAs, histology revealed a diagnosis according to at least 1 pathologist, which altered patient treatment. CONCLUSIONS: Histologic material can reveal additional diagnostic information compared with sole cytologic examination in 14% of representative TBNA samples in patients with mediastinal lymph node enlargement. A discrepancy between cytologic and histologic TBNA results should prompt further investigation.

A panel of p16(INK4A), MIB1 and p53 proteins can distinguish between the 2 pathways leading to vulvar squamous cell carcinoma.

Two pathways leading to vulvar squamous cell carcinoma (SCC) exist. The expression of proliferation- and cell-cycle-related biomarkers and the presence of high-risk (hr) HPV might be helpful to distinguish the premalignancies in both pathways. Seventy-five differentiated vulvar intra-epithelial neoplasia (VIN)-lesions with adjacent SCC and 45 usual VIN-lesions (32 solitary and 13 with adjacent SCC) were selected, and tested for hr-HPV DNA, using a broad-spectrum HPV detection/genotyping assay (SPF(10)-LiPA), and the immunohistochemical expression of MIB1, p16(INK4A) and p53. All differentiated VIN-lesions were hr-HPV- and p16-negative and in 96% MIB1-expression was confined to the parabasal layers. Eighty-four percent exhibited high p53 labeling indices, sometimes with parabasal extension. Eighty percent of all usual VIN-lesions were hr-HPV-positive, p16-positive, MIB1-positive and p53-negative. Five (of seven) HPV-negative usual VIN lesions, had an expression pattern like the other HPV-positive usual VIN lesions. In conclusion, both pathways leading to vulvar SCC have their own immunohistochemical profile, which can be used to distinguish the 2 types of VIN, but cannot explain differences in malignant potential.

Discovery and validation of protein abundance differences between follicular thyroid neoplasms.

Distinguishing between benign follicular thyroid adenoma (FTA) and malignant follicular thyroid carcinoma (FTC) by cytologic features alone is not possible. Molecular markers may aid distinguishing FTA from FTC in patients with indeterminate cytology. The aim of this study is to define protein abundance differences between FTC from FTA through a discovery (proteomics) and validation (immunohistochemistry) approach. Difference gel electrophoresis (DIGE) and peptide mass fingerprinting were performed on protein extracts from five patients with FTC and compared with six patients with FTA. Individual gel comparisons (i.e., each FTC extract versus FTA pool) were also performed for the five FTC patients. Immunohistochemical validation studies were performed on three of the identified proteins. Based on DIGE images, 680 protein spots were matched on individual gels. Of these, 102 spots showed statistically significant differences in abundance between FTC and FTA in the individual gel analyses and were therefore studied further. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to identify 54 of these protein spots. Three candidates involved in protein folding (heat shock protein gp96, protein disulfide isomerase A3, and calreticulin) were studied by immunohistochemistry. Moderate calreticulin immunohistochemical staining was the best single marker with a high negative predictive value (88%); combining all three markers (any marker less than moderate staining) had the best positive predictive value (75%) while still retaining a good negative predictive value (68%). With DIGE, we identified 54 proteins differentially abundant between FTC and FTA. Three of these were validated by immunohistochemistry. These findings provide further insights into the diagnosis, prognosis, and pathophysiology of follicular-derived thyroid neoplasms.