Pathology updates and diagnostic approaches to haemophagocytic lymphohistiocytosis.

Haemophagocytic lymphohistiocytosis (HLH) is a complex, often under-recognised hyperinflammatory immune dysregulation syndrome arising in a diverse range of clinical scenarios and conditions. The accurate and timely diagnosis of HLH is crucial for patient survival, and usually requires a high level of clinical suspicion. The histological corollary to clinical HLH-haemophagocytosis-is neither necessary nor sufficient for the diagnosis of HLH, as it may be seen in a variety of reactive conditions and may be absent in true HLH. Nevertheless, the finding of haemophagocytosis in specific clinical situations should prompt consideration of HLH and further testing to exclude the condition. Although haemophagocytosis is traditionally described in bone marrow, identification of it in other tissues, including lymphoid, splenic, liver or neural tissue, can contribute importantly to the overall recognition of HLH. In this review we discuss the underlying pathophysiology and aetiologies of HLH, and the morphological aspects of haemophagocytosis and its associated histological findings in different tissues, and give a brief overview of diagnostic criteria and clinical evaluation.

LIM domain only 2 (LMO2) expression distinguishes T-lymphoblastic leukemia/lymphoma from indolent T-lymphoblastic proliferations.

AIMS: An indolent T-lymphoblastic proliferation (iT-LBP) is a benign, reactive expansion of immature terminal deoxynucleotidyl transferase (TdT)-positive T cells found in extrathymic tissues. iT-LBP can be challenging to distinguish from malignant processes, specifically T-lymphoblastic lymphoma (T-LBL), given the overlapping clinical and histological features. Recently, it has been shown that LIM domain only 2 (LMO2) is overexpressed in T-LBL but not in reactive immature TdT+ T cells in the thymus. On the basis of these findings, the aim of this study was to investigate the expression of LMO2 by using immunohistochemistry and its role in differentiating iT-LBPs from T-LBLs. METHODS AND RESULTS: We retrospectively identified cases of iT-LBP and T-LBL from the pathology archives of four institutions. Seven iT-LBP cases (including five new cases that have not been reported in the literature) and 13 T-LBL cases were analysed. Clinical, morphological, immunophenotypic and molecular data were analysed. Immunohistochemical staining with LMO2 was performed on all iT-LBP and T-LBL cases. A review of five new iT-LBP cases showed similar morphological, immunophenotypic and molecular features to those of previously reported cases. All iT-LBP cases were negative for LMO2 (0/7), whereas 92% of T-LBL cases (12/13) expressed LMO2; the sensitivity was 92% (confidence interval 64-100%) and the specificity was 100% (confidence interval 59-100%). CONCLUSION: We confirm previously published findings that iT-LBP cases show highly overlapping morphological and immunophenotypic features with T-LBL. Importantly, LMO2 expression is a sensitive and specific marker with which to rule out iT-LBP.

Artificial Intelligence in Pathology: A Simple and Practical Guide.

Artificial intelligence (AI) is having an increasing impact on the field of pathology, as computation techniques allow computers to perform tasks previously performed by people. Here, we offer a simple and practical guide to AI methods used in pathology, such as digital image analysis, next-generation sequencing, and natural language processing. We not only provide a comprehensive review, but also discuss relevant history and future directions of AI in pathology. We additionally provide a short tabular dictionary of AI terminology which will help practicing pathologists and researchers to understand this field.

A Long-Term Study of Persistent Sézary Syndrome: Evidence for Antigen Shift by Multiparameter Flow Cytometry and Its Significance in Overall Survival.

Sézary syndrome (SS) is a peripheral T-cell lymphoma characterized by erythroderma, diffuse lymphadenopathy, and circulating neoplastic T cells, which classically show a helper T-cell immunophenotype with loss of CD7 and CD26. Flow cytometry is often used to identify and enumerate populations of Sézary cells in the peripheral blood; however, the significance and frequency of antigen shift over time is unclear. In this article, we follow the immunophenotype of the neoplastic T-cell population from 28 patients with SS across 415 flow cytometry studies. Antigen shift for each patient was assigned as none, minimal = 1-2 markers by 1°, moderate = up to 3 markers, or marked ≥ 4 markers. Sixty-four percent (18/28) of patients showed antigen shift, and among those with antigen shift, the majority showed minimal (8/18) or moderate antigen shift (7/18) with fewer demonstrating marked shift (3/18). Patients without antigen shift showed a trend toward improved overall survival in comparison with patients demonstrating any degree of antigen shift. Antigen shift is seen in a significant proportion of cases of SS with long-term follow-up and may be a marker of more aggressive disease.

Complexities in the diagnosis of large B-cell lymphomas, classic Hodgkin lymphomas and overlapping peripheral T-cell lymphomas simplified: An evidence-based guide.

The diagnosis of a large B-cell lymphoma and classic Hodgkin lymphoma (CHL) is often straightforward. However, in select circumstances, these simple diagnoses can be quite complex. In part, diagnostic difficulty may be due to uncertainty in the evaluation of morphologic and immunophenotypic features along a biologic continuum, or alternatively arise from uncertainty in predicting the behavior and outcomes of patients. Here, we systematically discuss and review areas of diagnostic difficulty in the diagnosis of large B-cell lymphomas (LBCL), classic Hodgkin lymphomas (CHL) and peripheral T-cell lymphomas (PTCL). We provide careful data-driven analyses and evidence-based approaches to help guide pathologists and clinicians. We discuss: 1) marginal zone lymphomas with increased large cells versus diffuse large B-cell lymphoma (DLBCL), 2) chronic lymphocytic leukemia with expanded proliferation centers versus diffuse large B-cell lymphoma (DLBCL), 3) chronic lymphocytic leukemia with Hodgkin/Reed-Sternberg-like cells versus CHL arising from chronic lymphocytic leukemia, 4) complex cases of follicular lymphoma versus DLBCL, 5) PTCL with large B-cell proliferations versus PTCL with LBCL, 6) PTCL with Hodgkin/Reed-Sternberg-like cells versus CHL, and finally 7) blastoid/pleomorphic mantle cell lymphoma versus DLBCL. Our evidence and data driven approach may serve as a useful diagnostic guide.

Mutations in the signal transducer and activator of transcription family of genes in cancer.

In recent years, it has become clear that members of the signal transducer and activator of transcription (STAT) family of genes play an important role in cancer. The STAT family consists of seven genes, STAT1-4, STAT5A, STAT5B and STAT6, that are involved in regulating cellular proliferation, apoptosis, angiogenesis and the immune system response. Constitutive activation of STAT3, via mutational changes, is important in oncogenesis in both solid and hematopoietic cancers. In the case of hematopoietic neoplasms, STAT3 driver mutations have been described in T-cell large granular lymphocytic (T-LGL) leukemia and chronic natural killer lymphoproliferative disorders (CLPD-NK) and are seen in 30%-40% of T-LGL leukemia patients. STAT5B is also mutated in T-LGL leukemia and CLPD-NK, but in a much smaller proportion. Here we review past and current research on STAT genes in hematopoietic and solid cancers with emphasis on STAT3 and STAT5B and their roles in the pathogenesis of hematopoietic malignancies, particularly T-LGL leukemia and CLPD-NK.

An analysis of blastic plasmacytoid dendritic cell neoplasm with translocations involving the MYC locus identifies t(6;8)(p21;q24) as a recurrent cytogenetic abnormality.

AIMS: Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an aggressive neoplasm with leukaemic features and frequent skin involvement. Translocations involving the MYC locus have been recently identified as recurrent cytogenetic abnormalities in this entity. The aim of this study was to assess the clinicopathological, immunophenotypic and genetic features in MYC-rearranged BPDCN cases. METHODS AND RESULTS: Pathology archives from six major institutes were queried for cases of BPDCN with 8q24 MYC translocations, and two cases were identified. A literature review identified 14 cases. Clinicopathological features, immunophenotype and cytogenetic and molecular data were reviewed. In these 16 MYC-rearranged cases, the median age at diagnosis was 70.5 years, and there was a male predominance. Whereas all cases showed marrow involvement, skin lesions (62.5%) and lymphadenopathy (50%) were variably seen. The median survival was 11 months. The median percentage of blasts in peripheral blood was 9%. All cases showed expression of CD4, with 10 of 16 being positive for CD56. HLA-DR, CD123, TCL1 and CD303 were positive in all cases tested. Cytogenetic analysis revealed a single recurrent translocation partner of MYC at 6p21 in 11 cases (69%), whereas four cases showed different MYC translocation partners (2p12, Xq24, 3p25, and 14q32). Interestingly, the group of patients with t(6;8)(p21;q24) showed an older median age at diagnosis (74 years) and a remarkably shorter median survival (3 months). CONCLUSIONS: Translocations involving the 8q24 MYC locus more frequently manifest as t(6;8)(p21;q24), and, given its association with specific clinicopathological features suggesting even more aggressive behaviour, t(6;8)(p21;q24) indicate a genetically defined subgroup within BPDCN.

Case Report: Clinical Variation in Children With Thrombopoietin Receptor (C-MPL) Mutations: Report of 2 Cases.

Congenital amegakaryocytic thrombocytopenia (CAMT, MIM# 604498) is a rare congenital bone marrow failure syndrome which presents early in life with abnormal bleeding because of thrombocytopenia. Classically, megakaryocytes are decreased to absent in the bone marrow. The development of aplastic anemia early in childhood has led to the recommendation for early stem cell transplantation. Quantitative or loss-of-function mutations in the myeloproliferative leukemia gene (c-mpl), whose gene product functions as the thrombopoietin receptor, have been identified as causative for CAMT. Approximately 100 cases of CAMT are published in the medical literature. We describe 2 cases of CAMT who demonstrate disparate clinical courses, thereby highlighting phenotypic differences and increasing awareness of this clinical entity.

Immunohistochemistry for p53 is a useful tool to identify cases of acute myeloid leukemia with myelodysplasia-related changes that are TP53 mutated, have complex karyotype, and have poor prognosis.

In this study, we evaluate the expression of p53 in core biopsies with acute myeloid leukemia and correlate the level of expression with acute myeloid leukemia subtype, TP53 mutation status, karyotype, and survival. Of the 143 cases evaluated, 71 fulfilled the WHO 2016 criteria for acute myeloid leukemia with myelodysplasia-related changes, 40 were acute myeloid leukemia-not otherwise specified, 25 were acute myeloid leukemia with recurrent genetic abnormalities, and 7 were therapy-related acute myeloid leukemia. By immunohistochemistry, 17% showed p53 expression in >5% of the cells. Of the 24 cases with >5% p53-positive cells, 17 were acute myeloid leukemia with myelodysplasia-related changes, 5 were acute myeloid leukemia-not otherwise specified, 1 was acute myeloid leukemia with recurrent genetic abormalities, and 1 was therapy-related acute myeloid leukemia. In cases for which data was available, expression of >5% p53-positive cells was significantly associated with genotype (n=67) and/or karyotype (n=130). Among the 115 cases for which clinical follow up was available, the overall survival of cases with p53 expression >15% (Median=102 days) was significantly shorter compared with cases with p53 expression ≤15% (Median=435 days). Within the acute myeloid leukemia with myelodysplasia-related changes group, this association remained significant, with cases with ≤15% p53-positive cells having a median overall survival of 405 days versus 102 days for cases with >15% p53-positive cells. Among acute myeloid leukemia with myelodysplasia-related changes cases with a complex karyotype, the finding of >15% p53-positive cells was significantly associated with worse overall survival. The poor prognosis associated with more than 15% p53-positive cells was independent of age and karyotype. In acute myeloid leukemia with myelodysplasia-related changes, p53 expression may be useful to infer TP53 mutation status, complex karyotype, and/or poor prognosis in situations where other modalities are not readily available.

Noninvasive monitoring of diffuse large B-cell lymphoma by immunoglobulin high-throughput sequencing.

Recent studies have shown limited utility of routine surveillance imaging for diffuse large B-cell lymphoma (DLBCL) patients achieving remission. Detection of molecular disease by immunoglobulin high-throughput sequencing (Ig-HTS) from peripheral blood provides an alternate strategy for surveillance. We prospectively evaluated the utility of Ig-HTS within 311 blood and 105 tumor samples from 75 patients with DLBCL, comparing Ig-HTS from the cellular (circulating leukocytes) and acellular (plasma cell-free DNA) compartments of peripheral blood to clinical outcomes and (18)fluoro-deoxyglucose positron emission tomography combined with computed tomography (PET/CT; n = 173). Clonotypic immunoglobulin rearrangements were detected in 83% of patients with adequate tumor samples to enable subsequent monitoring in peripheral blood. Molecular disease measured from plasma, compared with circulating leukocytes, was more abundant and better correlated with radiographic disease burden. Before treatment, molecular disease was detected in the plasma of 82% of patients compared with 71% in circulating cells (P = .68). However, molecular disease was detected significantly more frequently in the plasma at time of relapse (100% vs 30%; P = .001). Detection of molecular disease in the plasma often preceded PET/CT detection of relapse in patients initially achieving remission. During surveillance time points before relapse, plasma Ig-HTS demonstrated improved specificity (100% vs 56%, P < .0001) and similar sensitivity (31% vs 55%, P = .4) compared with PET/CT. Given its high specificity, Ig-HTS from plasma has potential clinical utility for surveillance after complete remission.

Pediatric-type Follicular Lymphoma and Pediatric Nodal Marginal Zone Lymphoma: Recent Clinical, Morphologic, Immunophenotypic, and Genetic Insights.

Pediatric-type follicular lymphoma and pediatric nodal marginal zone lymphoma are 2 of the rarest B-cell lymphomas. Although they are both predominantly seen in children, they can manifest in the adult population as well. Our understanding of these lymphomas has advanced rapidly in recent years such that we not only have a firm grasp of the morphologic and immunophenotypic findings, but also have a deeper insight into critical genetic and molecular pathways of these diseases. This review will cover the clinical and pathologic characteristics, treatment, prognosis, and important differential diagnoses of these entities.

Hematolymphoid lesions of the breast.

Hematolymphoid malignancies of the breast are most commonly neoplasms of mature B-lymphocytes, and may arise as a primary disease or by secondary involvement of a systemic disease. Primary breast lymphomas (PBL) account for 0.04-0.5% of breast malignancies, less than 1% of all non-Hodgkin's lymphomas (NHL), and less than 5% of extranodal lymphomas (Lakhani et al., 2012; Swerdlow et al., 2008; Joks et al., 2011; Barista et al., 2000; Giardini et al., 1992; Brogi and Harris, 1999; Topalovski et al., 1999).1-7 Secondary breast lymphomas (SBL) are also rare, with an estimated annual incidence of 0.07% (Domchek et al., 2002; Talwalkar et al., 2008).8,9 Recognition of breast lesions as hematolymphoid is critical to distinguish them from other entities that can occur in the breast.

Significance of myelodysplastic syndrome-associated somatic variants in the evaluation of patients with pancytopenia and idiopathic cytopenias of undetermined significance.

In this study, we set out to evaluate the frequency of mutations in 20 myelodysplastic syndrome-associated genes in 53 individuals with pancytopenia in which bone marrow evaluation failed to meet standard criteria for a diagnosis of myelodysplastic syndrome. These idiopathic pancytopenia cases were associated with no specific cause for their pancytopenia (n=28), aplastic anemia (n=13), pancytopenia attributable to liver disease (n=4), pancytopenia associated with autoimmune disease (n=4), and pancytopenia attributed to drug effect (n=4). We also selected 38 bone marrow aspirates from patients presenting with pancytopenia and meeting criteria for a diagnosis of myelodysplastic syndrome (n=21) or acute myeloid leukemia (n=17) as malignant comparison cases. Targeted sequencing of the 20 genes was performed on all cases. The idiopathic pancytopenia group had a lower average age (46 vs 66 years, P<0.0001) and a lower number of mutations per case that were statistically significant (0.81 vs 1.18, P=0.045). The frequency of cases with at least one mutation was higher for cases with a diagnosable myeloid neoplasm (68 vs 38%, P=0.012). Except for mutations in U2AF1, which was mutated in 5 of the 38 malignant cases (13.2%) and in none of the idiopathic pancytopenia cases (P=0.011), the frequency of mutations in the genes evaluated was not significantly different between idiopathic pancytopenia and malignant cases. Median and mean clinical follow-up for the idiopathic pancytopenia group was available for 444 and 739 days, respectively. Over this time frame, none of the idiopathic pancytopenia patients was diagnosed with a myelodysplastic syndrome or an acute myeloid leukemia. These findings provide further evidence that identification of mutations in several genes associated with myelodysplastic syndromes should not be used alone to support a diagnosis of a myelodysplastic syndrome.

A study of the mutational landscape of pediatric-type follicular lymphoma and pediatric nodal marginal zone lymphoma.

Pediatric-type follicular lymphoma and pediatric marginal zone lymphoma are two of the rarest B-cell lymphomas. These lymphomas occur predominantly in the pediatric population and show features distinct from their more common counterparts in adults: adult-type follicular lymphoma and adult-type nodal marginal zone lymphoma. Here we report a detailed whole-exome deep sequencing analysis of a cohort of pediatric-type follicular lymphomas and pediatric marginal zone lymphomas. This analysis revealed a recurrent somatic variant encoding p.Lys66Arg in the transcription factor interferon regulatory factor 8 (IRF8) in 3 of 6 cases (50%) of pediatric-type follicular lymphoma. This specific point mutation was not detected in pediatric marginal zone lymphoma or in adult-type follicular lymphoma. Additional somatic point mutations in pediatric-type follicular lymphoma were observed in genes involved in transcription, intracellular signaling, and cell proliferation. In pediatric marginal zone lymphoma, no recurrent mutation was identified; however, somatic point mutations were observed in genes involved in cellular adhesion, cytokine regulatory elements, and cellular proliferation. A somatic variant in AMOTL1, a recurrently mutated gene in splenic marginal zone lymphoma, was also identified in a case of pediatric marginal zone lymphoma. The overall non-synonymous mutational burden was low in both pediatric-type follicular lymphoma and pediatric marginal zone lymphoma (4.6 mutations per exome). Altogether, these findings support a distinctive genetic basis for pediatric-type follicular lymphoma and pediatric marginal zone lymphoma when compared with adult subtypes and to one another. Moreover, identification of a recurrent point mutation in IRF8 provides insight into a potential driver mutation in the pathogenesis of pediatric-type follicular lymphoma with implications for novel diagnostic or therapeutic strategies.

Next-generation sequencing of acute myeloid leukemia identifies the significance of TP53, U2AF1, ASXL1, and TET2 mutations.

We assessed the frequency and clinicopathologic significance of 19 genes currently identified as significantly mutated in myeloid neoplasms, RUNX1, ASXL1, TET2, CEBPA, IDH1, IDH2, DNMT3A, FLT3, NPM1, TP53, NRAS, EZH2, CBL, U2AF1, SF3B1, SRSF2, JAK2, CSF3R, and SETBP1, across 93 cases of acute myeloid leukemia (AML) using capture target enrichment and next-generation sequencing. Of these cases, 79% showed at least one nonsynonymous mutation, and cases of AML with recurrent genetic abnormalities showed a lower frequency of mutations versus AML with myelodysplasia-related changes (P<0.001). Mutational analysis further demonstrated that TP53 mutations are associated with complex karyotype AML, whereas ASXL1 and U2AF1 mutations are associated with AML with myelodysplasia-related changes. Furthermore, U2AF1 mutations were specifically associated with trilineage morphologic dysplasia. Univariate analysis demonstrated that U2AF1 and TP53 mutations are associated with absence of clinical remission, poor overall survival (OS), and poor disease-free survival (DFS; P<0.0001), whereas TET2 and ASXL1 mutations are associated with poor OS (P<0.03). In multivariate analysis, U2AF1 and TP53 mutations retained independent prognostic significance in OS and DFS, respectively. Our results demonstrate unique relationships between mutations in AML, clinicopathologic prognosis, subtype categorization, and morphologic dysplasia.

Identification of a ferrireductase required for efficient transferrin-dependent iron uptake in erythroid cells.

The reduction of iron is an essential step in the transferrin (Tf) cycle, which is the dominant pathway for iron uptake by red blood cell precursors. A deficiency in iron acquisition by red blood cells leads to hypochromic, microcytic anemia. Using a positional cloning strategy, we identified a gene, six-transmembrane epithelial antigen of the prostate 3 (Steap3), responsible for the iron deficiency anemia in the mouse mutant nm1054. Steap3 is expressed highly in hematopoietic tissues, colocalizes with the Tf cycle endosome and facilitates Tf-bound iron uptake. Steap3 shares homology with F(420)H(2):NADP(+) oxidoreductases found in archaea and bacteria, as well as with the yeast FRE family of metalloreductases. Overexpression of Steap3 stimulates the reduction of iron, and mice lacking Steap3 are deficient in erythroid ferrireductase activity. Taken together, these findings indicate that Steap3 is an endosomal ferrireductase required for efficient Tf-dependent iron uptake in erythroid cells.

Utility of PD-1, PD-L1, and IDO-1 Stains in Ocular Extranodal Marginal Zone Lymphoma (MZL) and Diffuse Large B-cell Lymphoma (DLBCL).

Extranodal marginal zone lymphoma (EMZL) is the most common subtype of ocular lymphomas. Diffuse large B-cell lymphoma (DLBCL) and EMZL with large-cell transformation present diagnostic challenges. Radiotherapy is the standard treatment for ocular lymphomas, but complications and relapse are common. Diagnostic utility in challenging cases, as well as treatment options using immune checkpoint inhibitors, are unclear in ocular lymphomas. We herein investigated the PD-1, PD-L1, and IDO1 staining patterns in 20 cases of ocular lymphomas, including EMZL (n=14), EMZL with increased large cells (n=2), and DLBCL (n=4). PD-1, PD-L1, and IDO1 staining was not detected in lymphoma cells in any cases but was observed within the tumor microenvironment in all cases. Positivity for PD-1, PD-L1, and IDO1 in inflammatory cells was seen either intratumorally or peritumorally. In all 6 cases with significantly more large B cells, the density of PD-1, PD-L1, and IDO1 expression in the tumor microenvironment was higher than that of the remaining 14 cases without large B cells (P-value<0.0001), whereas other clinicopathologic features showed no statistical correlation. Increased expression of PD-1, PD-L1, and IDO1 in the inflammatory milieu in cases with large cells may provide diagnostic utility in small biopsies as well as therapeutic potential.

Primary renal leukaemia in a young adult male as an extramedullary presentation of T cell acute lymphoblastic leukaemia.

Primary renal involvement by T lymphoblasts is rare among adults with T acute lymphoblastic leukaemia. We report a 28-year-old man presenting with acute renal failure due to infiltration by T lymphoblasts and his response to paediatric-inspired modified BFM-90 protocol. The patient achieved an initial complete remission (CR) but developed central nervous system relapse. He achieved CR2 with cranial irradiation and intrathecal chemotherapy. He underwent a haploidentical transplant in CR2 and remains in remission post-transplant day 330. An early kidney biopsy helped confirm the diagnosis. Such presentations remain responsive to modified BFM-90. An early allotransplant in CR2 remains the standard of care.