EAHP 2020 workshop proceedings, pediatric myeloid neoplasms.

The first section of the bone marrow workshop of the European Association of Haematopathology (EAHP) 2020 Virtual Meeting was dedicated to pediatric myeloid neoplasms. The section covered the whole spectrum of myeloid neoplasms, including myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), myelodysplastic/myeloproliferative neoplasms (MDS/MPN), and acute myeloid leukemia (AML). The workshop cases are hereby presented, preceded by an introduction on these overall rare diseases in this age group. Very rare entities such as primary myelofibrosis, pediatric MDS with fibrosis, and MDS/MPN with JMML-like features and t(4;17)(q12;q21); FIP1L1::RARA fusion, are described in more detail.

Myelodysplastic/myeloproliferative neoplasms: are morphology and immunophenotyping still relevant?

The term myelodysplastic/myeloproliferative neoplasm (MDS/MPN) refers to a group of clonal hematopoietic neoplasms with overlapping clinical, morphologic and genetic myelodysplastic and myeloproliferative features observed at the time of first presentation. Impaired hematopoiesis morphologically associated with evidence of myelodysplasia manifests clinically with cytopenia/s. Simultaneously, myeloproliferation is seen within the bone marrow and leads to cytosis in the peripheral blood. The diagnostic category of MDS/MPN encompasses a heterogeneous group of diseases which share similarities among them, but at the same time have distinct clinical and pathologic features and eventually diverse prognosis; such differences justify their separation in a classification scheme. In the era of genetic and genomic tests, their distinction from conventional myelodysplastic syndromes or myeloproliferative neoplasms still relies on close clinocopathological correlation, with evaluation of both peripheral blood and bone marrow samples being essential in this sense. A multiparametric integration of clinicopathologic data and cytogenetics and molecular genetics results is the preferred diagnostic approach.

A study of disseminated intravascular coagulation in acute leukemia reveals markedly elevated D-dimer levels are a sensitive indicator of acute promyelocytic leukemia.

INTRODUCTION: While the presence of disseminated intravascular coagulation (DIC) has been implicated in worse clinical outcome in acute leukemia, the relationship between different subtypes of acute leukemia and the clinicopathologic features of DIC has not been systematically well studied. METHODS: In this study, we retrospectively reviewed 149 cases of newly diagnosed acute leukemia and assessed the utility of evaluating red blood cell morphologic features, and coagulation parameters in determining the presence of DIC as well as differentiating subtypes of acute leukemia. RESULTS: Review of our cohort demonstrates a novel finding, that elevated D-dimer concentrations ≥19 000 ng/mL fibrinogen equivalent units (FEU) are a sensitive diagnostic indicator of acute promyelocytic leukemia (APL) with moderate specificity, sensitivity 96%, specificity 92% in acute leukemia subtyping. Similar to other studies, APL showed an increased incidence of DIC (P < 0.01) compared to other subtypes of acute leukemia. Surprisingly, the presence of schistocytes on the peripheral blood smear was not a statistically significant indicator of DIC, sensitivity of 36% and specificity of 89%. Finally, the presence of DIC was not a significant indicator of poorer prognosis amongst all patients with AML. CONCLUSION: Overall we identify elevated D-dimer concentrations ≥19 000 ng/mL FEU are a sensitive indicator of acute promyelocytic leukemia (APL), with a sensitivity of 96% and specificity of 92% in the subtyping of acute leukemias, and that the presence of schistocytes in peripheral blood smears is not a diagnostically sensitive screening test for DIC with a sensitivity of 36%.

The clinicopathologic significance of lymphocyte subsets in acute myeloid leukemia.

INTRODUCTION: While the role of the immune system in altering and modulating the progression of solid tumors is well studied, the impact of the immune system on the outcome and progression of hematolymphoid neoplasms is still poorly understood. METHODS: Here, we report a retrospective study detailing our analysis of 130 patients with acute myeloid leukemia (AML), with flow cytometry immunophenotypic evaluation of major lymphocyte subsets including B cells, T cells, and NK cells. RESULTS: Our study identifies differential signatures of lymphocyte subsets pertaining to distinct subcategories of AML, and prognostic correlations in patients. In multivariate analysis, NK cells (specifically CD56+/CD16+ NK cells at a cutoff of ≥5%) were found to be an independent indicator of improved overall and disease-free survival; cytogenetic risk was also shown to be critical in stratifying patients with AML. CONCLUSIONS: In total, we demonstrate that in AML, the subset distribution of immune system lymphocytes is nonrandom, and suggest an important role for distinct lymphocyte subsets, particularly NK cells, in this disease.

The diagnostic and clinical impact of genetics and epigenetics in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a complex disease, for which our understanding of the role of genetic and epigenetic changes has undergone significant advancements. Newer diagnostic and prognostic classifications have increasingly incorporated such information, and novel therapies have been developed to target specific genes, processes, and pathways based on this growing understanding. Given the rapid evolution of this field, it is critical for physicians and translational researchers to have a more in-depth understanding of this evolving landscape. Here, we review both genetics and epigenetics in acute myeloid leukemia from a practical standpoint.

Refining the diagnosis of T-cell large granular lymphocytic leukemia by combining distinct patterns of antigen expression with T-cell clonality studies.

T-cell large granular lymphocytic (LGL) leukemia is a complex diagnosis, requiring persistent clonal expansions of LGLs, and cytopenias. Often the diagnosis is unclear as non-clonal expansions of LGLs commonly occur in reactive conditions. To better understand T-LGL leukemia, we performed a comprehensive clinicopathologic analysis of 85 patients with LGL expansions. Interestingly, distinct CD8+(dim)/CD57+ populations, seen by flow cytometry, were significantly associated with clonal T-LGL leukemia (P < 0.001) as well as neutropenia (median absolute neutrophil count (ANC) 1.45 vs 3.19 × 10(9)/l; P = 0.0017). Furthermore, cases with distinct CD8+(dim)/CD57+ populations and monoclonal T cells had even lower ANCs (median ANC 1.41 × 10(9)/l; P = 0.001) compared with cases without these dual criteria. Additionally, complete or partial loss of CD5 expression was independently associated with clonal T-LGL leukemia (P<0.001) and neutropenia (median ANC 1.41 vs 2.70 × 10(9)/l; P = 0.002). This study describes specific immunophenotypic parameters to better define clonal cases of T-LGL leukemia associated with significant neutropenia.

Mixed-phenotype acute leukemia: historical overview and a new definition.

Acute leukemia with a mixed phenotype is a rare disease and comprises 2-5% of all acute leukemias. These disorders have been known historically by a variety of names, such as mixed lineage leukemia, bilineal leukemia and biphenotypic leukemia, and the criteria for diagnosis have often been arbitrary. The scoring criteria proposed by the European Group for the Immunological Characterization of Leukemias represented a major attempt to define this disorder. However, the relative weight given to some markers and the lack of lineage specificity of most markers have raised questions regarding the significance of this approach. In 2008, the World Health Organization classification of hematopoietic and lymphoid tumors proposed a simpler diagnostic algorithm, which relies on fewer and more lineage-specific markers to define mixed-phenotype acute leukemia (MPAL). MPAL with t(9;22) and MLL rearrangement have been separated. Several studies have suggested that patients with acute leukemia of mixed phenotype have a worse clinical outcome when compared with matched controls with acute myeloid leukemia or acute lymphoblastic leukemia. Further studies are needed to confirm the significance of MPAL as currently defined, to determine a standardized treatment approach and to better understand the biological and clinical aspects of this disease.

Features of hemolysis due to Clostridium perfringens infection.

Infection by Clostridium perfringens can be an unsuspected cause of hemolysis in emergency room patients. Historically, this condition has been associated with wound contamination and other tissue infections. We report the case of an autistic patient who presented to our emergency department with a distended abdomen and hemolysis of unknown etiology. The patient had no history of recent surgery. Exploration of the abdomen revealed a hepatic abscess. Blood cultures tested culture positive for C. perfringens. We present images demonstrating the salient features of the peripheral blood smear in cases of this uncommon but deadly cause of hemolysis.

High frequency of pro-B acute lymphoblastic leukemia in adults with secondary leukemia with 11q23 abnormalities.

To evaluate the frequency and cytogenetic and immunophenotypic features of therapy-related, precursor B-cell acute lymphoblastic leukemia (ALL), 152 cases of immature B-cell ALL were reviewed. These were compared to the frequency of therapy-related acute myeloid leukemia (t-AML) during the same time period. Eight ALL cases with a prior diagnosis of malignancy were identified, including six (4.0%) with prior therapy considered to be therapy-related ALL (t-ALL). The t-ALL cases followed treatment for breast carcinoma (two cases), lung carcinoma (two cases), lymphocyte predominance Hodgkin's disease and follicular lymphoma with a latency period of 13 months to 8 years. All t-ALL cases had a pro-B (CD10-negative) immunophenotype with significantly higher expression of CD15 and CD65, compared to the de novo CD10-positive ALL cases. All six t-ALL cases had MLL abnormalities by fluorescence in situ hybridization, and four showed t(4;11)(q21;q23). These represented half of all 11q23-positive adult ALL cases. During the same time period, 4.9% of all AML cases were considered t-AML. There was a 16.7% frequency of 11q23 abnormalities in the t-AML group. Despite the similar frequency in therapy-related disease among ALL and AML cases, there were differences in the frequency of the diseases and t-ALL represented 12% of all therapy-related leukemias. However, t-ALL represented 46% of all 11q23-positive therapy-related leukemias. The immunogenetic features of t-ALL appear distinct and may aid in identifying more cases of this disease type in the future.

Primary myelodysplasia occurring in adults under 50 years old: a clinicopathologic study of 52 patients.

Although myelodysplastic syndromes (MDSs) are generally thought to be diseases of elderly patients, younger patients also have rarely been diagnosed with MDS. This is a report of the clinical, morphologic and cytogenetic features of 52 cases of primary MDS occurring in adults under the age of 50 years. Cases secondary to chemotherapy or radiotherapy were excluded. There were 31 males and 21 females. The median age at presentation was 39 years (range, 18 to 49 years). The interval between onset of symptoms and diagnosis was brief (median, 4 weeks; range, 1-32 weeks). Of the 49 patients for whom information about duration of symptoms was available, 13 (27%) were asymptomatic. Forty-two (81%) of the patients were classified using FAB criteria for blood and bone marrow morphology: refractory anemia (RA), 11; refractory anemia with ringed sideroblasts (RARS), four; refractory anemia with excess blasts (RAEB), 12; chronic myelomonocytic leukemia (CMML), three; refractory anemia with excess blasts in transformation (RAEB-T), 12 patients. Ten patients could not be categorized. Abnormalities involving chromosome 7 was the most frequent cytogenetic abnormality (31%). Partial chromosomal deletion and chromosome gain were also common abnormalities (22% and 9%, respectively). Translocations accounted for only 9% of the main cytogenetic abnormalities encountered in this patient population. For the 49 patients for whom information regarding AML transformation was available, 23 (47%) progressed to acute myeloid leukemia, with an overall median time to progression of 2 months (range 3 weeks to 3 years). In each category except for RARS, approximately half of the patients progressed, with a slightly less median time to progression in RAEB-T than for the other subtypes of MDS. Thirteen patients underwent bone marrow transplantation at the time of presentation of their disease.

Histological and immunohistochemical characterization of extranodal diffuse large-cell lymphomas with prominent spindle cell features.

AIMS: To describe five cases of diffuse large-cell lymphoma with prominent spindle cell components involving skin, nasal-ocular mucosa, and soft tissue. Because of the spindle cell morphology, such cases must be differentiated from true sarcomas arising in or metastasizing to soft tissue, skin, bone, lymph node, or other organs and sites. METHODS AND RESULTS: Formalin-fixed paraffin-embedded archival tissue from five consultation cases of diffuse large-cell lymphoma with prominent spindle cell features involving the skin, nasal-ocular mucosa, and soft tissue in three male and two female patients was studied by histology and immunohistochemistry. Clinicopathological findings were also reviewed for all the patients. By morphology, initial evaluation of the cases suggested spindle cell sarcoma in two cases, inflammatory pseudotumour in one case, large-cell lymphoma in another case, and one case was considered suspicious for malignant lymphoma. Immunohistochemistry demonstrated a B-cell lineage in four of the spindle cell lesions, with a diagnosis of primary cutaneous CD30+ anaplastic large cell lymphoma made for the fifth case. Four of five cases also showed actin reactivity. CONCLUSIONS: Although extremely rare, lymphomas with prominent spindle cell morphology can be encountered in daily surgical pathology practice, and should be included in the differential diagnosis of spindle cell lesions in skin and soft tissue. The observed actin reactivity in four of the five spindle cell lymphomas may lead to a misdiagnosis of leiomyosarcoma if lymphoid markers are not included in the immunohistochemical panel.

Angiotropic lymphoma: an immunophenotypically and clinically heterogeneous lymphoma.

Angiotropic lymphoma (AL) is an uncommon lymphoma often presenting with nonspecific clinical features and having a high mortality rate. Although not specifically recognized by the Revised European-American Classification of Lymphoid Neoplasms, it likely will appear as a subtype of diffuse large B-cell lymphoma in the upcoming WHO classification. Some authors may also consider it to be a subtype of cutaneous lymphomas. Recent studies have reported an immunophenotypic heterogeneity of AL, and in rare instances, an association with other NHL. To further characterize AL, we studied the immunophenotype by immunohistochemistry for CD5, CD10, CD20, bcl-2, and bcl-6 in 18 cases of B-cell AL identified at three medical centers in North America. Bcl-2 gene rearrangement status by polymerase chain reaction and Epstein Barr virus status by in situ hybridization also were evaluated. Eight men and 10 women were identified with AL (median age 71 years). Eleven patients were diagnosed in life and seven were diagnosed at autopsy. Neurologic symptoms were the most common presentation, seen in six patients. Skin was the most commonly biopsied site. All showed classic intravascular localization; in two cases, there was also a minor diffuse large cell lymphoma component observed in some organs. Most (89%) of the cases expressed bcl-2 protein; CD10, bcl-6 and CD5 were each expressed in 22% of cases. Based on CD5 and CD10 expression, three major groups were evident: CD5-, CD10- (11 cases); CD5+, CD10- (3 cases), and CD5-, CD10+ (3 cases). Even though a follicle center lymphoma preceded the AL in one patient, we did not detect bcl-2 gene rearrangement in any of these cases. All cases were negative for Epstein Barr virus. Of the five treated with chemotherapy, two achieved a complete remission. Based on these findings, we conclude that ALs are clinically and immunophenotypically heterogeneous and may represent more than one pathogenetic entity. In some instances AL may be preceded by another lymphoproliferative disorder, raising the possibility that some cases of AL may represent a transformation from another type of lymphoma. Cutaneous manifestations of AL are common; however, it appears to be a systemic lymphoma. Although often fatal, patients with AL who are diagnosed early and treated with chemotherapy may achieve remission.

Evaluation of T cell receptor testing in lymphoid neoplasms: results of a multicenter study of 29 extracted DNA and paraffin-embedded samples.

To evaluate current diagnostic methods used for the evaluation of T cell receptor (TCR) gene rearrangements, 24 different laboratories analyzed 29 lymphoid neoplasm samples of extracted DNA and paraffin-embedded tissue and were asked to complete a technical questionnaire related to the testing. Participating laboratories performed Southern blot and polymerase chain reaction (PCR) testing for rearrangements of the TCRbeta chain gene and PCR for the TCRgamma chain gene rearrangements. Of 14 laboratories performing TCRbeta Southern blot analysis, there was complete agreement in 10 of 14 cases, with some false negative results obtained in 4 cases. No false positive results were obtained by Southern blot analysis. TCRbeta PCR analysis was only performed by two laboratories, and only 47.1% of positive samples were detected. Twenty-one laboratory results were obtained for TCRgamma PCR. This method showed an overall detection rate of 77.9% for T cell gene rearrangements with a 4.1% false positive rate, as compared to both TCRgamma Southern blot analysis results and immunophenotyping. The detection rate for TCRgamma PCR, however, significantly differed when extracted DNA samples from frozen tissue were compared to paraffin-embedded tissue (85.4% versus 65.9%; P = 0.0005). Significant differences in true positive results were obtained when laboratories using primers directed against multiple TCRgamma variable regions (V1-8 plus one to three other primer sets) were compared to laboratories that used only a single set of TCR primers directed against the V1-8 (P < 0.0001). Other technical factors significantly affecting results were also identified. These findings provide useful data on the current state of diagnostic TCR testing, highlight the risk of false negative results for TCR testing directed against only portions of the TCRgamma gene, and identify limitations of testing of paraffin-embedded tissues in some laboratories.

Large solitary fibrous tumor of the kidney: report of two cases and review of the literature.

Solitary fibrous tumors are spindle cell neoplasms frequently arising in the serosal surface as well as a variety of other sites. We report two cases of large solitary fibrous tumor arising in the kidney, clinically thought to be renal cell carcinoma, in 41- and 72-year-old men. Although large in size (13.0 and 14.0 cm in greatest dimension, respectively), both lesions were well circumscribed and composed of a mixture of spindle cells and dense collagenous bands with no areas of necrosis or cystic changes noted macroscopically or microscopically. Immunohistochemical studies revealed reactivity for vimentin, CD34, collagen IV, and bcl-2 protein in both cases, with no staining for keratin, S-100 protein, or muscle markers, confirming the diagnosis of solitary fibrous tumor of the kidney. Solitary fibrous tumor of the kidney is rare but may present as a large mass that may be clinically confused with carcinoma or sarcoma.

Myeloperoxidase immunoreactivity in adult acute lymphoblastic leukemia.

To evaluate the frequency and significance of myeloperoxidase positivity in adult acute lymphoblastic leukemia (ALL), bone marrow biopsy material from 82 adults with ALL was evaluated with a polyclonal myeloperoxidase (pMPO) antibody. Nineteen cases (23%) demonstrated evidence of pMPO immunoreactivity. Positive cases were precursor B-cell lineage, and CD13 or CD15 expression was more frequent than in the pMPO-negative cases. A subset of pMPO-positive cases studied with a monoclonal MPO antibody was negative. Western blot analysis using the pMPO antibody showed the expected 55-kd band for myeloperoxidase in pMPO-positive and pMPO-negative ALLs, suggesting a lack of specificity of this antibody in ALL. Forty-two percent (8/19) of the pMPO-positive ALL cases demonstrated evidence of t(9;22) by either karyotype or polymerase chain reaction analysis. The pMPO-positive ALLs had a lower frequency of extramedullary disease than the pMPO-negative group and a trend toward improved overall survival compared with the pMPO-negative group. Immunoreactivity with pMPO in adult ALL may lead to an incorrect interpretation of biphenotypic acute leukemia using a recently described scoring system, and a revision to that scoring system is proposed to accommodate pMPO-positive ALL.

CD79: a review.

CD79 is composed of CD79a and CD79b components expressed almost exclusively on B cells and B-cell neoplasms. CD79a and CD79b expression precedes immunoglobulin (Ig) heavy-chain gene rearrangement and CD20 expression during B-cell ontogeny and disappears later than CD20 in the late (plasma cell) stage of B-cell differentiation. Therefore, antibodies to CD79a and CD79b are useful in the differential diagnosis of B-cell neoplasms from T-cell neoplasms or myeloid neoplasms, or L and H lymphocyte predominance Hodgkin's lymphoma from classic Hodgkin's lymphoma. In addition, CD79a and CD79b antibodies are useful markers in the diagnosis of precursor B-acute lymphoblastic leukemia (pre-B-ALL) because many of these tumors are negative for other B-cell markers, such as CD20 and CD45RA. Furthermore, for B-cell neoplasms, wherein CD20 expression is aberrantly lost, such as in diffuse large B-cell lymphoma, or for B-cell neoplasms after CD20-antibody therapy, CD79a may be used as a first-line B-cell marker for the diagnosis. In this review, the authors discuss the molecular biology of CD79 and the frequency and usefulness of CD79 expression in these neoplasms.