Standards of Pathology in the Diagnosis of Systemic Mastocytosis: Recommendations of the EU-US Cooperative Group.

Pathology plays a central role in the diagnosis of systemic mastocytosis (SM), its delineation from other neoplasms and reactive conditions, and in monitoring of SM under therapy. The morphologic hallmark of SM is the accumulation of spindle-shaped, hypogranulated mast cells (MCs) in bone marrow (BM) and other extracutaneous tissues. Four of the 5 World Health Organization-defined diagnostic criteria (ie, compact MC aggregates [=major criterion]; atypical MC morphology; activating KIT point mutations; aberrant expression of CD25 and/or CD2 and/or CD30 in MCs [=minor criteria]) can be addressed by the pathologist. The final classification of SM variants as either BM mastocytosis, indolent SM, smoldering SM, aggressive SM (ASM), SM with an associated hematologic neoplasm (SM-AHN), or MC leukemia (MCL) has important prognostic significance and requires the integration of certain morphological, clinical, radiological, and biochemical data, referred to as B- and C-findings. Substantial diagnostic challenges may be posed to the pathologist and clinician especially in the so-called advanced SM variants, that is, ASM, MCL, and SM-AHN. In this article, updated recommendations of the EU-US Cooperative Group regarding standards of pathology in the diagnosis of SM, presented during the year 2020 Working Conference held in September in Vienna, are reported.

Case report of a clinically indolent but morphologically high-grade cutaneous mast cell tumor in an adult: Atypical cutaneous mastocytoma or mast cell sarcoma?

We present a case of an adult male with a solitary mast cell tumor of the skin with unusual nuclear pleomorphism and mitotic activity. The tumor was excised, recurred within 2 years, was reexcised after 4 years and did not recur >6 years after diagnosis. The tumor showed progressive cytonuclear atypia and a high mitotic and proliferation rate by Ki67-staining from the onset. No KIT mutations were identified in the tumor and bone marrow. Serum tryptase levels and a bone marrow aspirate and trephine biopsy were normal. Although the histomorphology of the skin tumor was consistent with mast cell sarcoma, the clinical behavior without systemic progression argued against this diagnosis. The tumor was finally considered as atypical mastocytoma, borderline to mast cell sarcoma. Currently, the patient is in close follow-up and still in complete remission.

Secondary cytogenetic abnormalities in core-binding factor AML harboring inv(16) vs t(8;21).

Patients with core-binding factor (CBF) acute myeloid leukemia (AML), caused by either t(8;21)(q22;q22) or inv(16)(p13q22)/t(16;16)(p13;q22), have higher complete remission rates and longer survival than patients with other subtypes of AML. However, ∼40% of patients relapse, and the literature suggests that patients with inv(16) fare differently from those with t(8;21). We retrospectively analyzed 537 patients with CBF-AML, focusing on additional cytogenetic aberrations to examine their impact on clinical outcomes. Trisomies of chromosomes 8, 21, or 22 were significantly more common in patients with inv(16)/t(16;16): 16% vs 7%, 6% vs 0%, and 17% vs 0%, respectively. In contrast, del(9q) and loss of a sex chromosome were more frequent in patients with t(8;21): 15% vs 0.4% for del(9q), 37% vs 0% for loss of X in females, and 44% vs 5% for loss of Y in males. Hyperdiploidy was more frequent in patients with inv(16) (25% vs 9%, whereas hypodiploidy was more frequent in patients with t(8;21) (37% vs 3%. In multivariable analyses (adjusted for age, white blood counts at diagnosis, and KIT mutation status), trisomy 8 was associated with improved overall survival (OS) in inv(16), whereas the presence of other chromosomal abnormalities (not trisomy 8) was associated with decreased OS. In patients with t(8;21), hypodiploidy was associated with improved disease-free survival; hyperdiploidy and del(9q) were associated with improved OS. KIT mutation (either positive or not tested, compared with negative) conferred poor prognoses in univariate analysis only in patients with t(8;21).

Mantle cell lymphomas with concomitant MYC and CCND1 breakpoints are recurrently TdT positive and frequently show high-grade pathological and genetic features.

Chromosomal breakpoints involving the MYC gene locus, frequently referred to as MYC rearrangements (MYC - R+), are a diagnostic hallmark of Burkitt lymphoma and recurrent in many other subtypes of B-cell lymphomas including follicular lymphoma, diffuse large B-cell lymphoma and other high-grade B-cell lymphomas and are associated with an aggressive clinical course. In remarkable contrast, in MCL, only few MYC - R+ cases have yet been described. In the current study, we have retrospectively analysed 16 samples (MYC - R+, n = 15, MYC - R-, n = 1) from 13 patients and describe their morphological, immunophenotypic and (molecular) genetic features and clonal evolution patterns. Thirteen out of fifteen MYC - R+ samples showed a non-classical cytology including pleomorphic (centroblastic, immunoblastic), anaplastic or blastoid. MYC translocation partners were IG-loci in 4/11 and non-IG loci in 7/11 analysed cases. The involved IG-loci included IGH in 3 cases and IGL in one case. PAX5 was the non-IG partner in 2/7 patients. The MYC - R+ MCL reported herein frequently displayed characteristics associated with an aggressive clinical course including high genomic-complexity (6/7 samples), frequent deletions involving the CDKN2A locus (7/10 samples), high Ki-67 proliferation index (12/13 samples) and frequent P53 expression (13/13 samples). Of note, in 4/14 samples, SOX11 was not or only focally expressed and 3/13 samples showed focal or diffuse TdT-positivity presenting a diagnostic challenge as these features could point to a differential diagnosis of diffuse large B-cell lymphoma and/or lymphoblastic lymphoma/leukaemia.

High pATM is Associated With Poor Local Control in Supraglottic Cancer Treated With Radiotherapy.

OBJECTIVES: Most early stage laryngeal squamous cell carcinomas (LSCC) are treated with radiotherapy. Discovery of new biomarkers are needed to improve prediction of outcome after radiotherapy and to identify potential targets for systemic targeted therapy. The ataxia telangiectasia mutated (ATM) gene plays a critical role in DNA damage response induced by ionizing radiation. METHODS: The prognostic value of immunohistochemical expression of pATM, pChk2, and p53 were investigated in 141 patients with T1-T2 LSCC curatively treated with external beam radiotherapy. Uni- and multivariable Cox regression analyses were performed to examine the relation between expression levels of markers and local control. RESULTS: Local control was significantly worse in cases with high levels of pATM (HR 2.14; 95% CI, 1.08-4.24; P = .03). No significant associations with local control were found for pChk2 and p53 expression. The association of high pATM expression with poor local control was only found for supraglottic LSCC (HR 10.9; 95% CI, 1.40-84.4; P = .02). CONCLUSION: Our findings suggest a potential role for ATM in response to radiotherapy in early stage supraglottic LSCC and imply ATM inhibition as a possibility to improve response to radiotherapy. LEVEL OF EVIDENCE: NA Laryngoscope, 130: 1954-1960, 2020.

Mutational Evolution in Relapsed Diffuse Large B-Cell Lymphoma.

Current genomic models in diffuse large B-cell lymphoma (DLBCL) are based on single tumor biopsies, which might underestimate heterogeneity. Data on mutational evolution largely remains unknown. An exploratory study using whole exome sequencing on paired (primary and relapse) formalin fixed paraffin embedded DLBCL biopsies (n = 14) of 6 patients was performed to globally assess the mutational evolution and to identify gene mutations specific for relapse samples from patients treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone. A minority of the mutations detected in the primary sample (median 7.6%, range 4.8⁻66.2%) could not be detected in the matching relapse sample. Relapsed DLBCL samples showed a mild increase of mutations (median 12.5%, range 9.4⁻87.6%) as compared to primary tumor biopsies. We identified 264 genes possibly related to therapy resistance, including tyrosine kinases (n = 18), (transmembrane) glycoproteins (n = 73), and genes involved in the JAK-STAT pathway (n = 7). Among the potentially resistance related genes were PIM1, SOCS1, and MYC, which have been reported to convey a risk for treatment failure. In conclusion, we show modest temporal heterogeneity between paired tumor samples with the acquisition of new mutations and identification of genes possibly related to therapy resistance. The mutational evolution could have implications for treatment decisions and development of novel targeted drugs.

Core-binding factor acute myeloid leukemia with t(8;21): Risk factors and a novel scoring system (I-CBFit).

BACKGROUND: Although the prognosis of core-binding factor (CBF) acute myeloid leukemia (AML) is better than other subtypes of AML, 30% of patients still relapse and may require allogeneic hematopoietic cell transplantation (alloHCT). However, there is no validated widely accepted scoring system to predict patient subsets with higher risk of relapse. METHODS: Eleven centers in the US and Europe evaluated 247 patients with t(8;21)(q22;q22). RESULTS: Complete remission (CR) rate was high (92.7%), yet relapse occurred in 27.1% of patients. A total of 24.7% of patients received alloHCT. The median disease-free (DFS) and overall (OS) survival were 20.8 and 31.2 months, respectively. Age, KIT D816V mutated (11.3%) or nontested (36.4%) compared with KIT D816V wild type (52.5%), high white blood cell counts (WBC), and pseudodiploidy compared with hyper- or hypodiploidy were included in a scoring system (named I-CBFit). DFS rate at 2 years was 76% for patients with a low-risk I-CBFit score compared with 36% for those with a high-risk I-CBFit score (P < 0.0001). Low- vs high-risk OS at 2 years was 89% vs 51% (P < 0.0001). CONCLUSIONS: I-CBFit composed of readily available risk factors can be useful to tailor the therapy of patients, especially for whom alloHCT is not need in CR1 (ie, patients with a low-risk I-CBFit score).

Validation of a whole-slide image-based teleconsultation network.

AIMS: Most validation studies on digital pathology diagnostics have been performed in single institutes. Because rapid consultation on cases with extramural experts is one of the most important uses for digital pathology laboratory networks, the aim of this study was to validate a whole-slide image-based teleconsultation network between three independent laboratories. METHODS AND RESULTS: Each laboratory contributed 30 biopsies and/or excisions, totalling 90 specimens (776 slides) of varying difficulty and covering a wide variety of organs and subspecialties. All slides were scanned centrally at ×40 scanning magnification and uploaded, and subsequently assessed digitally by 16 pathologists using the same image management system and viewer. Each laboratory was excluded from digital assessment of their own cases. Concordance rates between the two diagnostic modalities (light microscopic versus digital) were compared. Loading speed of the images, zooming latency and focus quality were scored. Leaving out eight minor discrepancies without any clinical significance, the concordance rate between remote digital and original microscopic diagnoses was 97.8%. The two cases with a major discordance (for which the light microscopic diagnoses were deemed to be the better ones) resulted from a different interpretation of diagnostic criteria in one case and an image quality issue in the other case. Average scores for loading speed of the images, zooming latency and focus quality were 2.37 (on a scale up to 3), 2.39 (scale up to 3) and 3.06 (scale up to 4), respectively. CONCLUSIONS: This validation study demonstrates the suitability of a teleconsultation network for remote digital consultation using whole-slide images. Such networks may contribute to faster revision and consultation in pathology while maintaining diagnostic standards.

Evaluating the benefits of digital pathology implementation: time savings in laboratory logistics.

BACKGROUND: The benefits of digital pathology for workflow improvement and thereby cost savings in pathology, at least partly outweighing investment costs, are being increasingly recognised. Successful implementations in a variety of scenarios have started to demonstrate the cost benefits of digital pathology for both research and routine diagnosis, contributing to a sound business case encouraging further adoption. To further support new adopters, there is still a need for detailed assessment of the impact that this technology has on the relevant pathology workflows, with an emphasis on time-saving. AIMS: To assess the impact of digital pathology adoption on logistic laboratory tasks (i.e. not including pathologists' time for diagnosis-making) in the Laboratorium Pathologie Oost Nederland, a large regional pathology laboratory in The Netherlands. METHODS AND RESULTS: To quantify the benefits of digitisation, we analysed the differences between the traditional analogue and new digital workflows, carried out detailed measurements of all relevant steps in key analogue and digital processes, and compared the time spent. We modelled and assessed the logistic savings in five workflows: (i) routine diagnosis; (ii) multidisciplinary meeting; (iii) external revision requests; (iv) extra stainings; and (v) external consultation. On average, >19 working hours were saved on a typical day by working digitally, with the highest savings in routine diagnosis and multidisciplinary meeting workflows. CONCLUSIONS: By working digitally, a significant amount of time could be saved in a large regional pathology laboratory with a typical case mix. We also present the data in each workflow per task and concrete logistic steps to allow extrapolation to the context and case mix of other laboratories.

MYC expression and translocation analyses in low-grade and transformed follicular lymphoma.

AIMS: Low-grade follicular lymphoma (FL) (grade 1/2, FL1/2) has an annual risk of transformation of ≈3%, which is associated with aberrations in CDKN2A/B, TP53, and MYC. As in diffuse large B-cell lymphoma, high MYC expression in transformed FL (tFL) might predict a MYC breakpoint. METHODS AND RESULTS: We quantified MYC expression by immunohistochemistry and digital analysis in 41 paired biopsies from 20 patients with FL1/2 with subsequent transformation and in four isolated biopsies of tFL. As controls, 28 biopsies of FL1/2 without transformation (median follow-up of 105 months) and nine biopsies of FL3A/B were analysed. In the 20 FL1/2-tFL pairs, MYC expression was significantly higher in tFL than in the initial FL1/2 biopsies (median 54% versus 6%; 7% in FL3A, and 35% in FL3B). MYC breaks (MYC-R) were detected in eight of 21 (38%) tFLs analysed by fluorescence in-situ hybridization (FISH), with a median MYC score of 86%. In two of the analysed tFL cases, the translocation was already detected in antecedent FL1/2. MYC partners were immunoglobulin (IG) loci in three of eight cases (one IGL, one IGH, and one IGK) and non-IG in five of eight cases (two PAX5, one BCL6, and two unknown). Of the eight MYC-R+ cases, six were BCL2+/MYC+ double-hit, one was BCL2+/BCL6+/MYC+ triple-hit, and one was MYC+ single-hit. All three IG-MYC+ cases showed a MYC expression level of >85%, whereas the five cases with a non-IG MYC partner had a wider range of expression (median 68%, range 13-86%). Among the 13 MYC-R- tFLs, two groups with almost dichotomous MYC expression could be observed (three cases showed ≥90% MYC expression), suggesting alternative mechanisms of MYC activation. CONCLUSIONS: we show an increase in MYC expression from FL1/2 to tFL. MYC breakpoints were present in ≈40% of the cases, which is markedly higher than in de novo DLBCL. MYC expression was uniformly high in cases with an IG-MYC translocation but much more heterogeneous and in part independent of the presence of a MYC break in non-IG-MYC and MYC-negative cases.

HLA dependent immune escape mechanisms in B-cell lymphomas: Implications for immune checkpoint inhibitor therapy?

Antigen presentation by tumor cells in the context of Human Leukocyte Antigen (HLA) is generally considered to be a prerequisite for effective immune checkpoint inhibitor therapy. We evaluated cell surface HLA class I, HLA class II and cytoplasmic HLA-DM staining by immunohistochemistry (IHC) in 389 classical Hodgkin lymphomas (cHL), 22 nodular lymphocyte predominant Hodgkin lymphomas (NLPHL), 137 diffuse large B-cell lymphomas (DLBCL), 39 primary central nervous system lymphomas (PCNSL) and 19 testicular lymphomas. We describe a novel mechanism of immune escape in which loss of HLA-DM expression results in aberrant membranous invariant chain peptide (CLIP) expression in HLA class II cell surface positive lymphoma cells, preventing presentation of antigenic peptides. In HLA class II positive cases, HLA-DM expression was lost in 49% of cHL, 0% of NLPHL, 14% of DLBCL, 3% of PCNSL and 0% of testicular lymphomas. Considering HLA class I, HLA class II and HLA-DM together, 88% of cHL, 10% of NLPHL, 62% of DLBCL, 77% of PCNSL and 87% of testicular lymphoma cases had abnormal HLA expression patterns. In conclusion, an HLA expression pattern incompatible with normal antigen presentation is common in cHL, DLBCL, PCNSL and testicular lymphoma. Retention of CLIP in HLA class II caused by loss of HLA-DM is a novel immune escape mechanism, especially prevalent in cHL. Aberrant HLA expression should be taken into account when evaluating efficacy of checkpoint inhibitors in B-cell lymphomas.

FcRL4+ B-cells in salivary glands of primary Sjögren's syndrome patients.

Fc receptor-like protein 4 (FcRL4) is normally expressed on a small subset of mucosa-associated B-cells, as well as on mucosa-associated lymphoid tissue (MALT) lymphoma B-cells. Primary Sjögren's syndrome (pSS) patients have an increased risk of developing MALT lymphomas, preferentially in the parotid glands. For this reason we studied here by immunohistochemistry and mRNA analysis whether FcRL4 expressing B-cells are present in salivary gland tissue (labial and parotid) of pSS patients (n = 54) and non-pSS sicca patients (n = 16) and whether parotid gland MALT lymphomas in pSS patients (n = 49) also express this receptor. We also studied the effect of treatment (rituximab and abatacept) on the presence of FcRL4+ B-cells, and whether numbers in labial gland biopsies at time of diagnosis of pSS can predict whether patients are at risk for MALT lymphoma development. We demonstrate that FcRL4+ cells are present in salivary gland tissue of pSS patients where they are closely associated with ductal epithelial cells forming lymphoepithelial lesions. The glandular FcRL4+ cells are highly proliferative, genuine PAX5+ B-cells. These FcRL4+ B-cells are far more frequent in parotid gland than in labial gland tissue (p = 0.003). We further show that expression of FcRL4 is present in pSS-related parotid MALT lymphomas. The FcRL4 mRNA expression level in parotid MALT lymphoma is increased compared to parotid gland tissue of pSS patients without lymphoma (p = 0.017). However, numbers of FcRL4+ B-cells in labial gland biopsies taken at the time of pSS diagnosis, are not predictive for later development of MALT lymphoma. Reduction of parotid gland FcRL4+ B-cells by rituximab, but not abatacept is accompanied by restoration of the glandular epithelium, illustrating the crosstalk between these B-cells with the ductal cells. In conclusion, intraepithelial FcRL4+ B-cells are present in the salivary glands of pSS patients. These cells are likely involved in the epithelial changes seen in pSS. Their enrichment in parotid glands may explain why MALT lymphomas in pSS patients preferentially develop at this specific location.

B-Lymphoblastic Lymphomas Evolving from Follicular Lymphomas Co-Express Surrogate Light Chains and Mutated Gamma Heavy Chains.

Follicular lymphoma (FL) is an indolent B-cell non-Hodgkin lymphoma able to transform into germinal center-type diffuse large B-cell lymphoma. We describe four extraordinary cases of FL, which progressed to TdT+CD20- precursor B-lymphoblastic lymphoma (B-LBL). Fluorescence in situ hybridization analysis showed that all four B-LBLs had acquired a MYC translocation on transformation. Comparative genomic hybridization analysis of one case demonstrated that in addition to 26 numerical aberrations that were shared between the FL and B-LBL, deletion of CDKN2A/B and 17q11, 14q32 amplification, and copy-neutral loss of heterozygosity of 9p were gained in the B-LBL cells. Whole-exome sequencing revealed mutations in FMN2, NEB, and SYNE1 and a nonsense mutation in KMT2D, all shared by the FL and B-LBL, and TNFRSF14, SMARCA2, CCND3 mutations uniquely present in the B-LBL. Remarkably, all four FL-B-LBL pairs expressed IgG. In two B-LBLs, evidence was obtained for ongoing rearrangement of IG light chain variable genes and expression of the surrogate light chain. IGHV mutation analysis showed that all FL-B-LBL pairs harbored identical or near-identical somatic mutations. From the somatic gene alterations found in the IG and non-IG genes, we conclude that the FLs and B-LBLs did not develop in parallel from early t(14;18)-positive IG-unmutated precursors, but that the B-LBLs developed from preexistent FL subclones that accumulated additional genetic damage.

Proteomics Based Identification of Proteins with Deregulated Expression in B Cell Lymphomas.

Follicular lymphoma and diffuse large B cell lymphomas comprise the main entities of adult B cell malignancies. Although multiple disease driving gene aberrations have been identified by gene expression and genomic studies, only a few studies focused at the protein level. We applied 2 dimensional gel electrophoresis to compare seven GC B cell non Hodgkin lymphoma (NHL) cell lines with a lymphoblastoid cell line (LCL). An average of 130 spots were at least two folds different in intensity between NHL cell lines and the LCL. We selected approximately 38 protein spots per NHL cell line and linked them to 145 unique spots based on the location in the gel. 34 spots that were found altered in at least three NHL cell lines when compared to LCL, were submitted for LC-MS/MS. This resulted in 28 unique proteins, a substantial proportion of these proteins were involved in cell motility and cell metabolism. Loss of expression of B2M, and gain of expression of PRDX1 and PPIA was confirmed in the cell lines and primary lymphoma tissue. Moreover, inhibition of PPIA with cyclosporine A blocked cell growth of the cell lines, the effect size was associated with the PPIA expression levels. In conclusion, we identified multiple differentially expressed proteins by 2-D proteomics, and showed that some of these proteins might play a role in the pathogenesis of NHL.

Sequential karyotyping in Burkitt lymphoma reveals a linear clonal evolution with increase in karyotype complexity and a high frequency of recurrent secondary aberrations.

Typical Burkitt lymphoma is characterized by an IG-MYC translocation and overall low genomic complexity. Clinically, Burkitt lymphoma has a favourable prognosis with very few relapses. However, the few patients experiencing disease progression and/or relapse have a dismal outcome. Here we report cytogenetic findings of seven cases of Burkitt lymphoma in which sequential karyotyping was performed at time of diagnosis and/or disease progression/relapse(s). After case selection, karyotype re-review and additional molecular analyses were performed in six paediatric cases, treated in Berlin-Frankfurt-Münster-Non-Hodgkin lymphoma study group trials, and one additional adult patient. Moreover, we analysed 18 cases of Burkitt lymphoma from the Mitelman database in which sequential karyotyping was performed. Our findings show secondary karyotypes to have a significant increase in load of cytogenetic aberrations with a mean number of 2, 5 and 8 aberrations for primary, secondary and third investigations. Importantly, this increase in karyotype complexity seemed to result from recurrent secondary chromosomal changes involving mainly trisomy 21, gains of 1q and 7q, losses of 6q, 11q, 13q, and 17p. In addition, our findings indicate a linear clonal evolution to be the predominant manner of cytogenetic evolution. Our data may provide a biological framework for the dismal outcome of progressive and relapsing Burkitt lymphoma.

Paediatric nodal marginal zone B-cell lymphadenopathy of the neck: a Haemophilus influenzae-driven immune disorder?

Many hyperplasias and lymphomas of marginal zone B-cells are associated with infection. We identified six children and one adolescent with cervical lymphadenopathy showing prominent polyclonal nodal marginal zone hyperplasia (pNMZH) and four adolescents with monoclonal paediatric nodal marginal zone lymphoma (pNMZL). The clonality status was assessed using BIOMED-2-IG PCR analysis. Haemophilus influenzae was identified in all six cases of pNMZH that could be tested by direct culture (N = 3) or a very sensitive PCR for the H. influenzae gyrase gene in frozen materials (N = 5). H. influenzae was not detected in three pNMZLs and 28 non-specific reactive cervical lymph nodes of age-matched controls, except for a single control node that was obtained during oropharyngeal surgery for a cleft palate showing very low copy numbers of H. influenzae. pNMZH patients were younger than pNMZL patients (median age 12 versus 21 years). pNMZH showed a prominent nodular appearance with variable fibrosis without acute inflammation. Within the nodules, the expanded germinal centres and variably sized marginal zones were colonized by activated B-cells with weak expression of IgD and lack of CD10 and/or BCL6 expression. Some areas showed skewed light chain expression in plasma cells (4/5 cases lambda). In four cases tested, this was confirmed by flow cytometry for surface Ig (3/4 cases lambda). In contrast, pNMZL showed more extensive expansion of marginal zones by centrocytoid cells and often expression of BCL2 protein. Several H. influenzae strains are known to interact with the constant part of IgD on human B-cells, leading to their polyclonal proliferation and activation. We speculate that in vivo stimulation of IgD+ marginal zone B-cells by this bacterium may be implicated in this particular lymphadenopathy that should be distinguished from monoclonal pNMZL.