False-negative programmed death-ligand 1 immunostaining in ethanol-fixed endobronchial ultrasound-guided transbronchial needle aspiration specimens of non-small-cell lung cancer patients.

AIMS: Programmed death-ligand 1 (PD-L1) immunostaining is used to predict which non-small-cell lung cancer (NSCLC) patients will respond best to treatment with programmed cell death protein 1/PD-L1 inhibitors. PD-L1 immunostaining is sometimes performed on alcohol-fixed cytological specimens instead of on formalin-fixed paraffin-embedded (FFPE) biopsies or resections. We studied whether ethanol prefixation of clots from endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) results in diminished PD-L1 immunostaining as compared with formalin fixation. METHODS AND RESULTS: FFPE cell blocks from EBUS-TBNA specimens of 54 NSCLC patients were identified. For each case, paired samples were available, consisting of clots directly immersed in formalin and clots prefixed in Fixcyt (50% ethanol). Serial sections were immunostained for PD-L1 by use of the standardised SP263 assay and the 22C3 antibody as a laboratory-developed test (LDT). PD-L1 positivity was determined with two cut-offs (1% and 50%). Concordance of PD-L1 positivity between the formalin-fixed (gold standard) and ethanol-prefixed material was assessed. When the 22C3 LDT was used, 30% and 36% of the ethanol-prefixed specimens showed false-negative results at the 1% and 50% cut-offs, respectively (kappa 0.64 and 0.68). When SP263 was used, 22% of the ethanol-prefixed specimens showed false-negative results at the 1% cut-off (kappa 0.67). At the 50% cut-off, concordance was higher (kappa 0.91), with 12% of the ethanol-prefixed specimens showing false-negative results. CONCLUSION: Ethanol fixation of EBUS-TBNA specimens prior to formalin fixation can result in a considerable number of false-negative PD-L1 immunostaining results when a 1% cut-off is used and immunostaining is performed with SP263 or the 22C3 LDT. The same applies to use of the 50% cut-off when immunostaining is performed with the 22C3 LDT.

Challenges in Diagnosing Myelodysplastic Syndromes in the Era of Genetic Testing: Proceedings of the 13th Workshop of the European Bone Marrow Working Group.

The 13th workshop of the European Bone Marrow Working Group in Utrecht, The Netherlands, was devoted to studying myelodysplastic syndromes (MDS) and their boundaries. The panel received 44 cases submitted to the 3 invited categories, which included: reactive cytopenias with dysplasia, idiopathic cytopenia of undetermined significance, clonal haematopoiesis of indeterminate potential, idiopathic dysplasia of uncertain significance and overt MDS. For this summary, we have selected 17 cases that highlight difficulties in separating true MDS from other causes of cytopenia and the intricate relationship between clonal haematopoiesis and true MDS. In addition, cases of overt MDS with challenging features were also selected. All cases were stained for p53 expression. Using instructive submitted cases we discuss the following: (1) cytopenia with clonal haematopoiesis not fulfilling MDS criteria, (2) cytopenia and/or dysplasia with germline mutations and/or familial history suggesting an underlying gene defect, (3) MDS based on a recurrent chromosomal abnormality and (4) overt MDS with diagnostic difficulties due to concurrent treatment or disease. The lively discussion in the open forum of the workshop illustrated the need for better integrative understanding of the evolution of acquired genetic abnormalities in haematopoiesis, and the challenge of diagnosing true MDS in cytopenic patients with genetic abnormalities, either germline or acquired.

Role of columnar cell lesions in breast carcinogenesis: analysis of chromosome 16 copy number changes by multiplex ligation-dependent probe amplification.

Columnar cell lesions have been proposed as precursor lesions of low-grade breast cancer. The molecular characteristic of low-grade breast neoplasia is whole-arm loss of chromosome 16q. Copy number changes of 6 genes on 16p and 20 genes on 16q were analysed by multiplex ligation-dependent probe amplification in 165 lesions of 103 patients. Twenty-three columnar cell lesions and 19 atypical ducal hyperplasia lesions arising in columnar cell lesions were included, as well as cases of usual ductal hyperplasia, blunt duct adenosis, ductal carcinoma in situ, lobular neoplasia and invasive carcinoma. Usual ductal hyperplasia and blunt duct adenosis lacked whole-arm losses of 16q. In contrast, columnar cell lesions without atypia, columnar cell lesions with atypia, atypical ductal hyperplasia, low-grade ductal carcinoma in situ and low-grade invasive carcinomas increasingly harboured whole-arm losses of 16q (17%, 27%, 47% and 57%, respectively). However, no recurrent losses in specific genes could be identified. In several patients, columnar cell lesions and atypical ductal hyperplasia harboured similar losses as related ductal carcinoma in situ or invasive carcinomas within the same breast. There were indications for 16q breakpoints near the centromere. Whole-arm gains on 16p were relatively scarce and there was no relation between whole-arm gains of 16p and progression of lesions of the low-grade breast neoplasia family. In conclusion, columnar cell lesions (with and without atypia) often harbour whole-arm losses of 16q, which underlines their role as precursors in low-grade breast carcinogenesis, in contrast with usual ductal hyperplasia and blunt duct adenosis. However, no recurrent losses in specific genes could be identified, pointing to minor events in multiple tumour suppressor genes rather than major events in a single 16q gene contributing to low-grade breast carcinogenesis.

The use of droplet digital PCR in liquid biopsies: A highly sensitive technique for MYD88 p.(L265P) detection in cerebrospinal fluid.

The gold standard for diagnosis of central nervous system lymphomas still regards a stereotactic brain biopsy, with the risk of major complications for the patient. As tumor cells can be detected in cerebrospinal fluid (CSF), CSF analysis can be used as an alternative. In this respect, mutation analysis in CSF can be of added value to other diagnostic parameters such a cytomorphology and clonality analysis. A well-known example of targeted mutation analysis entails MYD88 p.(L265P) detection, which is present in the majority of Bing Neel syndrome and primary central nervous system lymphoma (PCNSL) patients. Unfortunately, tumor yield in CSF can be very low. Therefore, use of the highly sensitive droplet digital PCR (ddPCR) might be a suitable analysis strategy for targeted mutation detection. We analyzed 26 formalin fixed paraffin embedded (FFPE) samples (8 positive and 18 negative for MYD88 p.(L265P) mutation) by ddPCR, of which the results were compared with next generation sequencing (NGS). Subsequently, 32 CSF samples were analyzed by ddPCR. ddPCR and NGS results on FFPE material showed 100% concordance. Among the 32 CSF samples, 9 belonged to patients with lymphoplasmacytic lymphoma (LPL) and clinical suspicion of Bing Neel syndrome, and 3 belonged to patients with PCNSL. Nine of these samples tested positive for MYD88 p.(L265P) (8 LPL and 1 PCNSL). This study shows that sensitive MYD88 mutation analysis by ddPCR in CSF is highly reliable and can be applied even when DNA input is low. Therefore, ddPCR is of added value to current diagnostic parameters, especially when the available amount of DNA is limited.

Prediction of ultrasound guided fine needle aspiration cytology results by FDG PET-CT for lymph node metastases in head and neck squamous cell carcinoma patients.

INTRODUCTION: Accurate assessment of cervical lymph node status is essential in patients with head and neck squamous cell carcinoma (HNSCC) as it influences prognosis and treatment decisions. During patient workup, lymph node status is often examined by ultrasound guided fine needle aspiration cytology (USgFNAC). 18F-Fluorodeoxyglucose positron emission tomography combined with computed tomography (FDG PET-CT) is frequently used to assess primary tumor and distant metastases but provides information on lymph node status as well. It is possible that FDG PET-CT (if already made for abovementioned indications) can predict the results of USgFNAC in subgroups of lymph nodes based on FDG-uptake and size. The objective of this study is to identify maximum standardized uptake (SUVmax) and short axis diameter cutoff values of lymph nodes at which FDG PET-CT can reliably predict USgFNAC results. METHODS: One hundred and seventeen patients with HNSCC were retrospectively analyzed. Patients were included when FDG PET-CT and USgFNAC were available. SUVmax measurements were performed and compared to the USgFNAC results. RESULTS: Using USgFNAC as a reference standard, the area under the curve of the receiver operating curve was 0.91. At an SUVmax cutoff value of 4.9, the accuracy of FDG PET-CT was the highest (85%). Lymph nodes with short axis diameter ≥1.0 cm and SUVmax ≥4.9 were in 91% positive on USgFNAC. If SUVmax was below 2.2, no nodes were positive on USgFNAC. Of all lymph nodes 52% either had a short axis diameter ≥1.0 cm and SUVmax ≥4.9 or an SUVmax <2.2. FDG PET-CT and USgFNAC results were very similar in these nodes. CONCLUSIONS: By measuring SUVmax values and minimal axial diameters of lymph nodes and using appropriate cutoff values, FDG PET-CT can predict the results of USgFNAC examinations in half of the examined lymph nodes. This information may lead to a reduction of USgFNAC examinations in HNSCC patients if FDG PET-CT is already performed for other indications.

Dimorphic cells: a common feature throughout the low nuclear grade breast neoplasia spectrum.

Columnar cell lesions (CCLs) are recognized precursor lesions of the low nuclear grade breast neoplasia family. CCLs are cystic enlarged terminal duct lobular units with monotonous (monoclonal) columnar-type luminal cells. CCLs without atypia are regarded as benign and CCLs with atypia as true precursor lesions with clonal molecular changes, a certain progression risk, and an association with more advanced lesions. However, reproducibility of designating atypia in CCL is not optimal, and no objective markers of atypia have been identified, although 16q loss seems to be associated with atypical CCLs. Dimorphic ("pale") cell populations have been described in low nuclear grade ductal carcinoma in situ (DCIS) but not in CCLs and atypical ductal hyperplasia (ADH). Therefore, we searched for pale cells in CCL (N = 60), ADH (N = 41), and DCIS grade 1 (N = 84). Diagnostic criteria were derived from the WHO, and atypia was designated according to the Schnitt criteria. Pale cells occurred in 0% (0/30), 73% (22/30), 56% (23/41), and 76% (64/84) of CCLs without atypia, CCLs with atypia, ADH, and DCIS grade 1, respectively. Pale cells expressed ERα, E-cadherin and p120 and variably cyclin D1, and lacked expression of CK5 and p63. In conclusion, dimorphic "pale" cells occur throughout the low nuclear grade progression spectrum, increasing in frequency with progression. Interestingly, CCL lesions without atypia do not seem to bear showed pale cells, indicating that the presence of pale cells may serve as a diagnostic morphological feature of atypia in CCLs.

Papillomatous breast lesions with atypical columnar cell features.

AIMS: Columnar cell lesions (CCLs) are recognised breast cancer precursor lesions. Intraductal papillomas are usually lined by benign (polyclonal) cells. Although papillomas with monoclonal lesions (atypical ductal hyperplasia (ADH)/ductal carcinoma in situ (DCIS)) have been described, CCLs have not been described in papillomas. METHODS: We present two papillary breast lesions lined by a single layer of luminal cells resembling atypical CCL/flat epithelial atypia (FEA). We compared these two lesions with 13 benign intraductal papillomas, and 2 papillomas with ADH/DCIS grade 1 features as controls were immunohistochemically stained for the oestrogen receptor alpha (oestrogen receptor) and progesterone receptors (PR), cytokeratin 5 (CK5) and cyclin D1. RESULTS: Oestrogen receptor/PR expression was variable, with areas with ≥85% hormone receptor positivity in both morphologically normal papillomas and papillomas with ADH. In ADH areas, CK5 expression was seen in ≤5% of cells while cyclin D1 expression was high (>60%). The two papillary lesions with FEA were 100% oestrogen receptor and 90% cyclin D1 positive, and low on PR/CK5. There was only one morphologically normal papilloma with similar areas of low CK5 (5%) and high cyclin D1 expression; in all other morphologically benign papillomas CK5 expression varied between 10% and 50% and cyclin D1 expression was ≤50%. The papillary lesion with FEA that could be tested showed 16q losses, the hallmark genetic change in low nuclear grade breast neoplasias, in contrast to nine morphologically benign papillomas that could be tested. CONCLUSION: We present two papillomatous breast lesions with atypical CCL morphology and 16q loss, for which we propose the term papillary FEA.

Nationwide differences in cytology fixation and processing methods and their impact on interlaboratory variation in PD-L1 positivity.

Programmed death ligand-1 (PD-L1) immunostaining, which aids clinicians in decision-making on immunotherapy for non-small cell lung cancer (NSCLC) patients, is sometimes performed on cytological specimens. In this study, differences in cytology fixation and cell block (CB) processing between pathology laboratories were assessed, and the influence of these differences on interlaboratory variation in PD-L1 positivity was investigated. Questionnaires on cytology processing were sent to all Dutch laboratories. Information gathered from the responses was added to data on all Dutch NSCLC patients with a mention of PD-L1 testing in their cytopathology report from July 2017 to December 2018, retrieved from PALGA (the nationwide network and registry of histo- and cytopathology in the Netherlands). Case mix-adjusted PD-L1 positivity rates were determined for laboratories with known fixation and CB method. The influence of differences in cytology processing on interlaboratory variation in PD-L1 positivity was assessed by comparing positivity rates adjusted for differences in the variables fixative and CB method with positivity rates not adjusted for differences in these variables. Twenty-eight laboratories responded to the survey and reported 19 different combinations of fixation and CB method. Interlaboratory variation in PD-L1 positivity was assessed in 19 laboratories. Correcting for differences in the fixative and CB method resulted in a reduction (from eight (42.1%) to five (26.3%)) in the number of laboratories that differed significantly from the mean in PD-L1 positivity. Substantial variation in cytology fixation and CB processing methods was observed between Dutch pathology laboratories, which partially explains the existing considerable interlaboratory variation in PD-L1 positivity.

Blunt duct adenosis: a separate entity from columnar cell lesions?

Blunt duct adenosis (BDA) is a breast lesion first described by Foote and Stewart in 1945 as a proliferative benign lesion of the terminal duct lobular unit. Throughout recent decades, further literature descriptions of BDA have been confusing. Some consider BDA to be a separate entity, some a growth pattern of columnar cell changes. The WHO 2012 considered BDA and columnar cell changes to be synonyms, while columnar cell lesions, especially those with atypia, are part of a spectrum of early precursors of the low nuclear grade breast neoplasia family. In the updated WHO 2019 version, BDA is mentioned as 'not recommended' terminology for columnar cell lesions without further discussing it, leaving the question open if BDA should be considered a separate entity.Good diagnostic criteria for BDA have however largely been lacking, and its biological background has not yet been unravelled. In this paper, we point out that BDA is mainly associated with benign breast lesions and not with other recognised precursor lesions. Further, 16q loss, which is the hallmark molecular event in the low nuclear grade breast neoplasia family, is lacking in BDA. We therefore hypothesise that BDA may not be a true precursor lesion but a benign polyclonal lesion, and propose morphological diagnostic criteria to better differentiate it from columnar cell lesions.

First Report of IgG4 Related Disease Primary Presenting as Vertebral Bone Marrow Lesions.

IgG4-related disease is a fibro-inflammatory disorder characterized by swelling of tissues and affected organs accompanied by the development of scar tissue (fibrosis) and infiltration by IgG4 positive plasma cells. Almost any organ can be affected, including, but rarely, bone marrowinvolvement. Here we present a case of a 76-year-old male with IgG4-related disease presenting primarily with vertebral bone marrow lesions. Histopathology showed the typical features of storiform fibrosis, and increased IgG4 positive plasma cells. Treatment with corticosteroids significantly improved wellbeing and resolved lesion size on MRI.

Lymphoblastic lymphoma with a triple-hit profile: a rare but distinct and relevant entity.

Follicular lymphoma with progression to a high-grade lymphoma bears a poor prognosis. We describe a case of a 60-year-old man who presented in 2012 with an epidural mass, diagnosed as a diffuse large B-cell lymphoma (DLBCL) with concurrent low-grade follicular lymphoma. Three years later, the patient presented with a cervical mass, diagnosed as a lymphoblastic lymphoma (LBL). Both the DLBCL and LBL contained a "triple hit" with BCL2, BCL6, and cMYC translocations demonstrated by fluorescence in situ hybridization analysis and a complex karyotype by single-nucleotide polymorphism array analysis. Furthermore, the 2 lymphomas were shown to be clonally related by clonality analysis and single-nucleotide polymorphism array analysis. This case report presents a highly unusual case of an LBL with a triple hit, originating from a DLBCL, which has rarely been described in the literature and deserves further exploration.

Chromosome 16q loss--a genetic key to the understanding of breast carcinogenesis.

In the last decade the concepts of breast cancer dedifferentiation and progression have undergone a significant and substantial change. In the past it was widely believed that the detailed associations between genetic and morphological changes defined in the Vogelstein model of colorectal cancer pathogenesis could be transferred to breast carcinogenesis. A multitude of studies seemed to verify this a priori hypothesis. However, with the introduction of global screening techniques, predominantly at the DNA level, it became obvious that this linear model might be oversimplified for breast cancer. It is now widely accepted that losses of chromosomal 16q characterize in-situ and invasive breast cancer tumours with predominantly low tumour grade and estrogen receptor (ER) positivity (luminal breast cancers). In contrast, high grade breast cancers of the HER2, the basal or the non expressor phenotype with 16q-losses are rarely seen and in consequence a concept of multiple, parallel pathways with defined precursor lesions emerged. As a consequence, it became obvious that the hunt for oncogenes/tumour suppressor genes in invasive breast cancer is pathway specific. Whereas high grade breast cancers have been relatively well characterized by several recurrent changes in oncogenes/tumour suppressor genes located on various chromosomal regions (e.g. egfr, p53, HER2), the characterization of a 16q-specific tumour suppressor gene in ER-positive breast cancer is still a tremendous challenge. This review will focus on the role of 16q in breast cancer and aims to give insights into actual research efforts, e.g. alternative explanations in order to unravel the central role of 16q in breast cancer.