Artificial Intelligence in Bone Marrow Histological Diagnostics: Potential Applications and Challenges.

The expanding digitalization of routine diagnostic histological slides holds a potential to apply artificial intelligence (AI) to pathology, including bone marrow (BM) histology. In this perspective, we describe potential tasks in diagnostics that can be supported, investigations that can be guided, and questions that can be answered by the future application of AI on whole-slide images of BM biopsies. These range from characterization of cell lineages and quantification of cells and stromal structures to disease prediction. First glimpses show an exciting potential to detect subtle phenotypic changes with AI that are due to specific genotypes. The discussion is illustrated by examples of current AI research using BM biopsy slides. In addition, we briefly discuss current challenges for implementation of AI-supported diagnostics.

Erythroid Predominance in Bone Marrow Biopsies of Acute Myeloid Leukemia Patients after Decitabine Treatment Correlates with Mutation Profile and Complete Remission.

INTRODUCTION: Acute myeloid leukemia (AML) patients may receive hypomethylating agents such as decitabine (DAC) as part of their treatment. Not all patients respond to this therapy, and if they do, the clinical response may occur only after 3-6 courses of treatment. Hence, early biomarkers predicting response would be very useful. METHODS: We retrospectively analyzed a cohort of 22 AML patients who were treated with DAC. Histology of the bone marrow biopsy, pathogenic mutations, and methylation status were related to the treatment response. RESULTS: In 8/22 (36%) patients, an erythroid dominant response (EDR) pattern, defined as a ratio of myeloid cells/erythroid cells <1, was observed. In the remaining 14 cases, a myeloid predominance was preserved during treatment. No difference in the hypomethylating effect of DAC treatment was observed in patients with and without EDR, as global 5-methylcytosine levels dropped similarly in both groups. Mutational analysis by NGS using a panel of commonly mutated genes in AML showed that patients with an early EDR harbored on average less mutations, with U2AF1 mutations occurring more frequently, whereas RUNX1 mutations were underrepresented compared to non-EDR cases. Interestingly, the development of an EDR correlated with complete remission (7/8 cases with an EDR vs. only 2/14 cases without an EDR). CONCLUSION: We conclude that early histological bone marrow examination for the development of an EDR may be helpful to predict response in AML patients during treatment with DAC.

Detection of Second Primary Lymphoma in Late Diffuse Large B-cell Lymphoma Recurrences.

Approximately one-third of patients with diffuse large B-cell lymphoma (DLBCL) relapse and often require salvage chemotherapy followed by autologous stem cell transplantation. In most cases, the clonal relationship between the first diagnosis and subsequent relapse is not assessed, thereby potentially missing the identification of second primary lymphoma. In this study, the clonal relationship of 59 paired DLBCL diagnoses and recurrences was established by next-generation sequencing-based detection of immunoglobulin gene rearrangements. Among 50 patients with interpretable results, 43 patients (86%) developed clonally related relapsed disease. This was observed in 100% of early recurrences (<2 years), 80% of the recurrences with an interval between 2 and 5 years, and 73% of late recurrences (≥5 years). On the other hand, 7 (14%) out of 50 patients displayed different dominant clonotypes in primary DLBCL and clinical recurrences, confirming the occurrence of second primary DLBCL; 37% of DLBCL recurrences that occurred ≥4 years after diagnosis were shown to be second primary lymphomas. The clonally unrelated cases were Epstein-Barr virus positive in 43% of the cases, whereas this was only 5% in the relapsed DLBCL cases. In conclusion, next-generation sequencing-based clonality testing in late recurrences should be considered in routine diagnostics to distinguish relapse from second primary lymphoma, as this latter group of patients with DLBCL may benefit from less-intensive treatment strategies.

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.

Clonality assessment and detection of clonal diversity in classic Hodgkin lymphoma by next-generation sequencing of immunoglobulin gene rearrangements.

Clonality analysis in classic Hodgkin lymphoma (cHL) is of added value for correctly diagnosing patients with atypical presentation or histology reminiscent of T cell lymphoma, and for establishing the clonal relationship in patients with recurrent disease. However, such analysis has been hampered by the sparsity of malignant Hodgkin and Reed-Sternberg (HRS) cells in a background of reactive immune cells. Recently, the EuroClonality-NGS Working Group developed a novel next-generation sequencing (NGS)-based assay and bioinformatics platform (ARResT/Interrogate) to detect immunoglobulin (IG) gene rearrangements for clonality testing in B-cell lymphoproliferations. Here, we demonstrate the improved performance of IG-NGS compared to conventional BIOMED-2/EuroClonality analysis to detect clonal gene rearrangements in 16 well-characterized primary cHL cases within the IG heavy chain (IGH) and kappa light chain (IGK) loci. This was most obvious in formalin-fixed paraffin-embedded (FFPE) tissue specimens, where three times more clonal cases were detected with IG-NGS (9 cases) compared to BIOMED-2 (3 cases). In total, almost four times more clonal rearrangements were detected in FFPE with IG-NGS (N = 23) as compared to BIOMED-2/EuroClonality (N = 6) as judged on identical IGH and IGK targets. The same clonal rearrangements were also identified in paired fresh frozen cHL samples. To validate the neoplastic origin of the detected clonotypes, IG-NGS clonality analysis was performed on isolated HRS cells, demonstrating identical clonotypes as detected in cHL whole-tissue specimens. Interestingly, IG-NGS and HRS single-cell analysis after DEPArray™ digital sorting revealed rearrangement patterns and copy number variation profiles indicating clonal diversity and intratumoral heterogeneity in cHL. Our data demonstrate improved performance of NGS-based detection of IG gene rearrangements in cHL whole-tissue specimens, providing a sensitive molecular diagnostic assay for clonality assessment in Hodgkin lymphoma.

Proteogenomic analysis of the autoreactive B cell repertoire in blood and tissues of patients with Sjögren's syndrome.

OBJECTIVE: To comparatively analyse the aberrant affinity maturation of the antinuclear and rheumatoid factor (RF) B cell repertoires in blood and tissues of patients with Sjögren's syndrome (SjS) using an integrated omics workflow. METHODS: Peptide sequencing of anti-Ro60, anti-Ro52, anti-La and RF was combined with B cell repertoire analysis at the DNA, RNA and single cell level in blood B cell subsets, affected salivary gland and extranodal marginal zone lymphomas of mucosa-associated lymphoid tissue (MALT) of patients with SjS. RESULTS: Affected tissues contained anti-Ro60, anti-Ro52, anti-La and RF clones as a small part of a polyclonal infiltrate. Anti-Ro60, anti-La and anti-Ro52 clones outnumbered RF clones. MALT lymphoma tissues contained monoclonal RF expansions. Autoreactive clones were not selected from a restricted repertoire in a circulating B cell subset. The antinuclear antibody (ANA) repertoires displayed similar antigen-dependent and immunoglobulin (Ig) G1-directed affinity maturation. RF clones displayed antigen-dependent, IgM-directed and more B cell receptor integrity-dependent affinity maturation. This coincided with extensive intra-clonal diversification in RF-derived lymphomas. Regeneration of clinical disease manifestations after rituximab coincided with large RF clones, which not necessarily belonged to the lymphoma clone, that displayed continuous affinity maturation and intra-clonal diversification. CONCLUSION: The ANA and RF repertoires in patients with SjS display tissue-restricted, antigen-dependent and divergent affinity maturation. Affinity maturation of RF clones deviates further during RF clone derived lymphomagenesis and during regeneration of the autoreactive repertoire after temporary disruption by rituximab. These data give insight into the molecular mechanisms of autoreactive inflammation in SjS, assist MALT lymphoma diagnosis and allow tracking its response to rituximab.

Improved survival for adolescents and young adults with Hodgkin lymphoma and continued high survival for children in the Netherlands: a population-based study during 1990-2015.

Population-based studies that assess long-term patterns of incidence, major aspects of treatment and survival are virtually lacking for Hodgkin lymphoma (HL) at a younger age. This study assessed the progress made for young patients with HL (<25 years at diagnosis) in the Netherlands during 1990-2015. Patient and tumour characteristics were extracted from the population-based Netherlands Cancer Registry. Time trends in incidence and mortality rates were evaluated with average annual percentage change (AAPC) analyses. Stage at diagnosis, initial treatments and site of treatment were studied in relation to observed overall survival (OS). A total of 2619 patients with HL were diagnosed between 1990 and 2015. Incidence rates increased for 18-24-year-old patients (AAPC + 1%, P = 0·01) only. Treatment regimens changed into less radiotherapy and more 'chemotherapy only', different for age group and stage. Patients aged 15-17 years were increasingly treated at a paediatric oncology centre. The 5-year OS for children was already high in the early 1990s (93%). For patients aged 15-17 and 18-24 years the 5-year OS improved from 84% and 90% in 1990-1994 to 96% and 97% in 2010-2015, respectively. Survival for patients aged 15-17 years was not affected by site of treatment. Our present data demonstrate that significant progress in HL treatment has been made in the Netherlands since 1990.

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.

Histiocytic cell neoplasms involving the bone marrow: summary of the workshop cases submitted to the 18th Meeting of the European Association for Haematopathology (EAHP) organized by the European Bone Marrow Working Group, Basel 2016.

The bone marrow is a preferential site for both reactive and neoplastic histiocytic proliferations. The differential diagnosis ranges from reactive histiocyte hyperplasia in systemic infections, vaccinations, storage diseases, post myeloablative therapy, due to increased cell turnover, and in hemophagocytic lymphohistiocytosis, through extranodal Rosai-Dorfman disease to neoplasms derived from histiocytes, including histiocytic sarcomas (HS), Langerhans cell histiocytoses (LCH), Erdheim-Chester disease (ECD), and disseminated juvenile xanthogranuloma (JXG). One of the most important recent developments in understanding the biology of histiocytic neoplasms and in contributing to diagnosis was the detection of recurrent mutations of genes of the Ras/Raf/MEK/ERK signaling pathway, in particular the BRAFV600E mutation, in LCH and ECD. Here, we summarize clinical and pathological findings of 17 histiocytic neoplasms that were presented during the bone marrow symposium and workshop of the 18th European Association for Haematopathology (EAHP) meeting held in Basel, Switzerland, in 2016. A substantial proportion of these histiocytic neoplasms was combined with clonally related lymphoid (n = 2) or myeloid diseases (n = 5, all ECD). Based on the latter observation, we suggest excluding co-existent myeloid neoplasms at initial staging of elderly ECD patients. The recurrent nature of Ras/Raf/MEK/ERK signaling pathway mutations in histiocytic neoplasms was confirmed in 6 of the 17 workshop cases, illustrating their diagnostic significance and suggesting apotential target for tailored treatments.

A dominant-negative GFI1B mutation in the gray platelet syndrome.

The gray platelet syndrome is a hereditary, usually autosomal recessive bleeding disorder caused by a deficiency of alpha granules in platelets. We detected a nonsense mutation in the gene encoding the transcription factor GFI1B (growth factor independent 1B) that causes autosomal dominant gray platelet syndrome. Both gray platelets and megakaryocytes had abnormal marker expression. In addition, the megakaryocytes had dysplastic features, and they were abnormally distributed in the bone marrow. The GFI1B mutant protein inhibited nonmutant GFI1B transcriptional activity in a dominant-negative manner. Our studies show that GFI1B, in addition to being causally related to the gray platelet syndrome, is key to megakaryocyte and platelet development.

Recurrent mutations in genes involved in nuclear factor-κB signalling in nodal marginal zone lymphoma-diagnostic and therapeutic implications.

AIMS: To investigate the spectrum of mutations in 20 genes involved in B-cell receptor and/or Toll-like receptor signalling resulting in activation of nuclear factor-κB (NF-κB) in 20 nodal marginal zone lymphomas (NMZLs), 20 follicular lymphomas (FLs), and 11 cases of B-cell lymphoma, unclassifiable (BCL-u). METHODS AND RESULTS: Nodal marginal zone lymphomas were diagnosed according to strict criteria, including the expression of at least one putative marginal zone marker (MNDA and/or IRTA1). Cases that showed features of NMZL but did not fulfil all criteria were included as BCL-u. All FLs were required to have a BCL2 rearrangement. Mutations were found in: nine NMZLs, with recurrent mutations in TNFAIP3 and CD79B; 12 FLs, with recurrent mutations in TNFRSF14, TNFAIP3, and CARD11; and five cases of BCL-u, with recurrent mutations in TNFRSF14. TNFRSF14 mutations were present in FL and BCL-u, but not in any of the NMZLs. In the BCL-u group, TNFRSF14 mutations clustered with a FL immunophenotype. CONCLUSIONS: These results suggest that TNFRSF14 mutations point towards a diagnosis of FL, and can be used in the sometimes difficult distinction between NMZL and FL, but to apply this in diagnostics would require confirmation in an independent cohort. In addition, the presence or absence of specific mutations in pathways converging on NF-κB could be important for decisions regarding targeted treatment.

Immunoglobulin rearrangement analysis from multiple lesions in the same patient using next-generation sequencing.

AIMS: For patients who have multiple lymphomas with discordant pathology, it is relevant to determine whether there is one disseminated lymphoma or two unrelated lymphomas. Patients with disseminated, clonally related lymphomas are usually treated with the most powerful drugs available, while patients with unrelated (primary) lymphomas receive mainly standard first-line therapies. METHODS AND RESULTS: We used next-generation sequencing on the Ion Torrent Personal Genome Machine to characterize the immunoglobulin heavy gene V-D-J rearrangements in two diagnostic tissue samples, including formalin-fixed and paraffin-embedded tissue, of two patients with iatrogenic immunodeficiency-associated Epstein-Barr virus lymphoproliferative disorder, with ulcerative colitis as underlying disease. The immunoglobulin rearrangement sequences obtained by next-generation sequencing revealed undoubtedly clonally related lesions in two tissue biopsies that were taken over time in the first patient, which is concordant with disseminated lymphoma. The other patient showed two clonally unrelated lesions, which is incompatible with clonal dissemination. This information was not inferred from evaluation of the heavy and light chain rearrangements by fragment analysis, which is currently the gold standard. CONCLUSION: Our study demonstrates the diagnostic application of next-generation sequencing of immunoglobulin rearrangement assessment in pathology for clinical decision-making in patients with several simultaneous or subsequent lymphoproliferations.

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.

Homing characteristics of donor T cells after experimental allogeneic bone marrow transplantation and posttransplantation therapy for multiple myeloma.

Relapse and graft-versus-host disease remain major problems associated with allogeneic bone marrow (BM) transplantation (allo-BMT) and posttransplantation therapy in patients with multiple myeloma (MM) and other hematologic malignancies. A possible strategy for selectively enhancing the graft-versus-myeloma response and possibly reducing graft-versus-host disease is to increase the migration of alloreactive T cells toward the MM-containing BM. In the present study, we characterized the BM-homing behavior of donor-derived effector T cells in a novel allo-BMT model for the treatment of MM. We observed that posttransplantation immunotherapy consisting of donor lymphocyte infusion (DLI) and vaccination with minor histocompatibility antigen-loaded dendritic cells (DCs) was associated with prolonged survival compared with allo-BMT with no further treatment. Moreover, CD8(+) effector T cells expressing inflammatory homing receptors, including high levels of CD44, LFA-1, and inflammatory chemokine receptors, were recruited to MM-bearing BM. This was paralleled by strongly increased expression of IFN-γ and IFN-γ-inducible chemokines, including CXCL9, CXCL10, and CXCL16, especially in mice treated with DLI plus minor histocompatibility antigen-loaded DC vaccination. Remarkably, expression of the homeostatic chemokine CXCL12 was reduced. Furthermore, IFN-γ and TNF-α induced BM endothelial cells to express high levels of the inflammatory chemokines and reduced or unaltered levels of CXCL12. Finally, presentation of CXCL9 by multiple BM endothelial cell-expressed heparan sulfate proteoglycans triggered transendothelial migration of effector T cells. Taken together, our data demonstrate that both post-transplantation DLI plus miHA-loaded DC vaccination and MM growth result in an increased expression of inflammatory homing receptors on donor T cells, decreased levels of the homeostatic BM-homing chemokine CXCL12, and strong induction of inflammatory chemokines in the BM. Thus, along with increasing the population of alloreactive T cells, post-transplantation immunotherapy also might contribute to a more effective graft-versus-tumor response by switching homeostatic T cell migration to inflammation-driven migration.

Read the clonotype: Next-generation sequencing-based lymphocyte clonality analysis and perspectives for application in pathology.

Clonality assessment using the unique rearrangements of immunoglobulin (IG) and T-cell receptor (TR) genes in lymphocytes is a widely applied supplementary test for the diagnosis of B-cell and T-cell lymphoma. To enable a more sensitive detection and a more precise comparison of clones compared with conventional clonality analysis based on fragment analysis, the EuroClonality NGS Working Group developed and validated a next-generation sequencing (NGS)-based clonality assay for detection of the IG heavy and kappa light chain and TR gene rearrangements for formalin-fixed and paraffin-embedded tissues. We outline the features and advantages of NGS-based clonality detection and discuss potential applications for NGS-based clonality testing in pathology, including site specific lymphoproliferations, immunodeficiency and autoimmune disease and primary and relapsed lymphomas. Also, we briefly discuss the role of T-cell repertoire of reactive lymphocytic infiltrations in solid tumors and B-lymphoma.

Extranodal marginal zone lymphoma clonotypes are detectable prior to eMZL diagnosis in tissue biopsies and peripheral blood of Sjögren's syndrome patients through immunogenetics.

Introduction: Activated B cells play a key role in the pathogenesis of primary Sjögren's syndrome (pSS) through the production of autoantibodies and the development of ectopic germinal centers in the salivary glands and other affected sites. Around 5-10% of pSS patients develop B-cell lymphoma, usually extranodal marginal zone lymphomas (eMZL) of the mucosa-associated lymphoid tissue (MALT). The aim of the current study is to investigate if the eMZL clonotype is detectable in prediagnostic blood and tissue biopsies of pSS patients. Methods/Results: We studied prediagnostic tissue biopsies of three pSS patients diagnosed with eMZL and four pSS controls through immunoglobulin (IG) gene repertoire sequencing. In all three cases, we observed the eMZL clonotype in prediagnostic tissue biopsies. Among controls, we observed transient elevation of clonotypes in two pSS patients. To evaluate if eMZL clonotypes may also be detected in the circulation, we sequenced a peripheral blood mononuclear cell (PBMC) sample drawn at eMZL diagnosis and two years prior to eMZL relapse in two pSS patients. The eMZL clonotype was detected in the peripheral blood prior to diagnosis in both cases. Next, we selected three pSS patients who developed eMZL lymphoma and five additional pSS patients who remained lymphoma-free. We sequenced the IG heavy chain (IGH) gene repertoire in PBMC samples taken a median of three years before eMZL diagnosis. In two out of three eMZL patients, the dominant clonotype in the prediagnostic PBMC samples matched the eMZL clonotype in the diagnostic biopsy. The eMZL clonotypes observed consisted of stereotypic IGHV gene combinations (IGHV1-69/IGHJ4 and IGHV4-59/IGHJ5) associated with rheumatoid factor activity, a previously reported feature of eMZL in pSS. Discussion: In conclusion, our results indicate that eMZL clonotypes in pSS patients are detectable prior to overt eMZL diagnosis in both tissue biopsies and peripheral blood through immunogenetic sequencing, paving the way for the development of improved methods of early detection of eMZL.

Adventitial adaptive immune cells are associated with ascending aortic dilatation in patients with a bicuspid aortic valve.

Background: Bicuspid aortic valve (BAV) is associated with ascending aorta aneurysms and dissections. Presently, genetic factors and pathological flow patterns are considered responsible for aneurysm formation in BAV while the exact role of inflammatory processes remains unknown. Methods: In order to objectify inflammation, we employ a highly sensitive, quantitative immunohistochemistry approach. Whole slides of dissected, dilated and non-dilated ascending aortas from BAV patients were quantitatively analyzed. Results: Dilated aortas show a 4-fold increase of lymphocytes and a 25-fold increase in B lymphocytes in the adventitia compared to non-dilated aortas. Tertiary lymphoid structures with B cell follicles and helper T cell expansion were identified in dilated and dissected aortas. Dilated aortas were associated with an increase in M1-like macrophages in the aorta media, in contrast the number of M2-like macrophages did not change significantly. Conclusion: This study finds unexpected large numbers of immune cells in dilating aortas of BAV patients. These findings raise the question whether immune cells in BAV aortopathy are innocent bystanders or contribute to the deterioration of the aortic wall.