Significance of MYD88 L265P Mutation Status in the Subclassification of Low-Grade B-Cell Lymphoma/Leukemia.

CONTEXT: Lymphoplasmacytic lymphoma (LPL), marginal zone lymphoma (MZL), and chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL) are well-defined clinicopathologic entities. However, distinguishing LPL from MZL and from atypical cases of CLL can sometimes be difficult because of overlapping features. Recent studies have identified a recurrent L265P mutation in the MYD88 gene in most cases of LPL. Although this represents a promising diagnostic marker for LPL, the mutation is also reported in rare cases of MZL and CLL (as well as other types of B-cell lymphoma). Detection rates for this mutation have varied depending on the analytic methodology. OBJECTIVE: To assess the diagnostic utility of MYD88 L265P mutation in diagnosing low-grade B-cell lymphomas. DESIGN: We developed a novel pyrosequencing assay for the MYD88 L265P mutation and assessed its diagnostic utility in 317 cases of low-grade B-cell lymphoma (45 LPL [14%], 53 MZL [17%], and 219 CLL [69%]). We incorporated formal clinical and pathologic review of selected cases to ensure the most accurate diagnosis and subclassification. RESULTS: The MYD88 L265P mutation was identified in 43 cases of LPL (96%), including 3 nonimmunoglobulin-M LPL cases. In contrast, the mutation was present in only 2 cases of MZL (4%), and 5 cases of CLL (2%). Thus, pyrosequencing for the MYD88 L265P mutation demonstrates a high clinical sensitivity and specificity to distinguish LPL from MZL and CLL. CONCLUSIONS: This study confirms the strong association of the MYD88 L265P mutation with LPL, as well as the existence of rare cases of small B-cell lymphoma that complicate this association.

Extramedullary Manifestations of Myeloid Neoplasms.

OBJECTIVES: This session of the 2013 Society of Hematopathology/European Association for Haematopathology workshop focused on extramedullary manifestations of myeloid neoplasms. METHODS: We divided the submitted cases into four subgroups: (1) isolated myeloid sarcoma (MS); (2) MS with concurrent acute myeloid leukemia (AML), with a focus on karyotypic and molecular findings; (3) extramedullary relapse of AML, including relapse in the posttransplant setting; and (4) blast phase/transformation of a myeloproliferative neoplasm or chronic myelomonocytic leukemia. RESULTS: Establishing a diagnosis of isolated MS requires a high index of suspicion and use of immunophenotypic methods. Recurrent cytogenetic abnormalities or gene mutations that occur in MS mirror those known to occur in AML. CONCLUSIONS: In the era of targeted therapy and sophisticated risk stratification, every attempt must be made to perform a complete workup on MS cases (or concurrent AML) since the diagnosis of MS, in itself, is no longer adequate for patient management. Cases of blastic plasmacytoid dendritic cell neoplasm were also included and discussed in this session.

Scoring of MYC protein expression in diffuse large B-cell lymphomas: concordance rate among hematopathologists.

Recent studies have shown that immunohistochemical evaluation of MYC protein expression in diffuse large B-cell lymphoma is a useful prognostic tool with high concordance rate among pathologists. Concordance in these studies was assessed among few pathologists from one institution by scoring tissue microarrays. In daily practice, MYC evaluation is performed on entire tumor sections by a diverse group of pathologists. In our study, nine hematopathologists from two institutions scored whole-tissue sections of two sets of cases. The training set included 13 cases of diffuse large B-cell lymphoma and 4 cases of Burkitt lymphoma. The validation set included 18 cases of diffuse large B-cell lymphoma and 1 case of Burkitt lymphoma. MYC positivity was defined as ≥40% of tumor cells demonstrating nuclear staining similar to prior studies. The mean score for each case was used to determine MYC status with discrepant cases defined as having any score causing a different MYC status designation. Discrepant cases from the training set were characterized by staining heterogeneity, extensive necrosis or crush artifact and had mean scores within 15 percentage points of 40%. Cases from the validation set that demonstrated any of these features were scored twice on two different days. Overall concordance was moderate (Kappa score: 0.68, P-value<0.001) with no significant change between the two sets (Kappa scores: 0.69 vs 0.67). Thirty-nine percent of cases were discrepant. The findings indicate that a significant number of diffuse large B-cell lymphomas are inherently difficult to score due to staining heterogeneity. The effect of heterogeneity can be under-represented when concordance is measured among few pathologists scoring tissue microarrays. Careful scoring strategy in our study failed to improve concordance. In the absence of specific instructions on how to deal with heterogeneity, caution is advised when evaluating MYC expression in diffuse large B-cell lymphoma.

Gene expression profiles predictive of outcome and age in infant acute lymphoblastic leukemia: a Children's Oncology Group study.

Gene expression profiling was performed on 97 cases of infant ALL from Children's Oncology Group Trial P9407. Statistical modeling of an outcome predictor revealed 3 genes highly predictive of event-free survival (EFS), beyond age and MLL status: FLT3, IRX2, and TACC2. Low FLT3 expression was found in a group of infants with excellent outcome (n = 11; 5-year EFS of 100%), whereas differential expression of IRX2 and TACC2 partitioned the remaining infants into 2 groups with significantly different survivals (5-year EFS of 16% vs 64%; P < .001). When infants with MLL-AFF1 were analyzed separately, a 7-gene classifier was developed that split them into 2 distinct groups with significantly different outcomes (5-year EFS of 20% vs 65%; P < .001). In this classifier, elevated expression of NEGR1 was associated with better EFS, whereas IRX2, EPS8, and TPD52 expression were correlated with worse outcome. This classifier also predicted EFS in an independent infant ALL cohort from the Interfant-99 trial. When evaluating expression profiles as a continuous variable relative to patient age, we further identified striking differences in profiles in infants less than or equal to 90 days of age and those more than 90 days of age. These age-related patterns suggest different mechanisms of leukemogenesis and may underlie the differential outcomes historically seen in these age groups.

Identification of novel cluster groups in pediatric high-risk B-precursor acute lymphoblastic leukemia with gene expression profiling: correlation with genome-wide DNA copy number alterations, clinical characteristics, and outcome.

To resolve the genetic heterogeneity within pediatric high-risk B-precursor acute lymphoblastic leukemia (ALL), a clinically defined poor-risk group with few known recurring cytogenetic abnormalities, we performed gene expression profiling in a cohort of 207 uniformly treated children with high-risk ALL. Expression profiles were correlated with genome-wide DNA copy number abnormalities and clinical and outcome features. Unsupervised clustering of gene expression profiling data revealed 8 unique cluster groups within these high-risk ALL patients, 2 of which were associated with known chromosomal translocations (t(1;19)(TCF3-PBX1) or MLL), and 6 of which lacked any previously known cytogenetic lesion. One unique cluster was characterized by high expression of distinct outlier genes AGAP1, CCNJ, CHST2/7, CLEC12A/B, and PTPRM; ERG DNA deletions; and 4-year relapse-free survival of 94.7% ± 5.1%, compared with 63.5% ± 3.7% for the cohort (P = .01). A second cluster, characterized by high expression of BMPR1B, CRLF2, GPR110, and MUC4; frequent deletion of EBF1, IKZF1, RAG1-2, and IL3RA-CSF2RA; JAK mutations and CRLF2 rearrangements (P < .0001); and Hispanic ethnicity (P < .001) had a very poor 4-year relapse-free survival (21.0% ± 9.5%; P < .001). These studies reveal striking clinical and genetic heterogeneity in high-risk ALL and point to novel genes that may serve as new targets for diagnosis, risk classification, and therapy.

Gene expression classifiers for relapse-free survival and minimal residual disease improve risk classification and outcome prediction in pediatric B-precursor acute lymphoblastic leukemia.

To determine whether gene expression profiling could improve outcome prediction in children with acute lymphoblastic leukemia (ALL) at high risk for relapse, we profiled pretreatment leukemic cells in 207 uniformly treated children with high-risk B-precursor ALL. A 38-gene expression classifier predictive of relapse-free survival (RFS) could distinguish 2 groups with differing relapse risks: low (4-year RFS, 81%, n = 109) versus high (4-year RFS, 50%, n = 98; P < .001). In multivariate analysis, the gene expression classifier (P = .001) and flow cytometric measures of minimal residual disease (MRD; P = .001) each provided independent prognostic information. Together, they could be used to classify children with high-risk ALL into low- (87% RFS), intermediate- (62% RFS), or high- (29% RFS) risk groups (P < .001). A 21-gene expression classifier predictive of end-induction MRD effectively substituted for flow MRD, yielding a combined classifier that could distinguish these 3 risk groups at diagnosis (P < .001). These classifiers were further validated on an independent high-risk ALL cohort (P = .006) and retainedindependent prognostic significance (P < .001) in the presence of other recently described poor prognostic factors (IKAROS/IKZF1 deletions, JAK mutations, and kinase expression signatures). Thus, gene expression classifiers improve ALL risk classification and allow prospective identification of children who respond or fail current treatment regimens. These trials were registered at http://clinicaltrials.gov under NCT00005603.

Systemic mastocytosis associated with t(8;21)(q22;q22) acute myeloid leukemia.

Although KIT mutations are present in 20-25% of cases of t(8;21)(q22;q22) acute myeloid leukemia (AML), concurrent development of systemic mastocytosis (SM) is exceedingly rare. We examined the clinicopathologic features of SM associated with t(8;21)(q22;q22) AML in ten patients (six from our institutions and four from published literature) with t(8;21) AML and SM. In the majority of these cases, a definitive diagnosis of SM was made after chemotherapy, when the mast cell infiltrates were prominent. Deletion 9q was an additional cytogenetic abnormality in four cases. Four of the ten patients failed to achieve remission after standard chemotherapy and seven of the ten patients have died of AML. In the two patients who achieved durable remission after allogeneic hematopoietic stem cell transplant, recipient-derived neoplastic bone marrow mast cells persisted despite leukemic remission. SM associated with t(8;21) AML carries a dismal prognosis; therefore, detection of concurrent SM at diagnosis of t(8;21) AML has important prognostic implications.

Clinical manifestations of hemoglobin Chico at high altitude.

Hemoglobin Chico is a rare hemoglobinopathy characterized by low oxygen affinity and a right-shifted oxygen dissociation curve. Detailed clinical evaluations of affected individuals have not been previously reported. We therefore report on the clinical features of Hemoglobin Chico in a Latino male living at high altitude, who desired to participate in school sports. As a young boy with asthma, he had the unusual finding of growth delay and digital clubbing which improved with asthma control. At 16 years of age, he had mild anemia and a decreased pulse oximetry (83%) but sufficient pulmonary reserve to participate in physically demanding activities.

Peripheral T-cell lymphoma with extensive dendritic cell network mimicking follicular dendritic cell tumor: a case report with pathologic, immunophenotypic, and molecular findings.

Peripheral T-cell lymphoma (PTCL) with a nodular pattern of growth is uncommon and may be misdiagnosed initially as a B-cell lymphoma or reactive process. We report a case of a rapidly growing PTCL with a distinctly nodular pattern in an axillary lymph node from an 89-year-old man. Immunohistochemical stains for CD21, CD23, and CD35 highlighted an extensive dendritic cell network that imparted the nodular appearance and, in addition, was associated intimately with the neoplastic cells. The neoplastic cells otherwise had an immunophenotype similar to previously reported cases of PTCL with a nodular pattern and germinal center origin (CD3+, CD4+, CD5+, bcl-6+, CD31+, subset CD10+, subset CXCL13+, and subset CD79a+). Molecular studies confirm a clonal T-cell receptor g gene rearrangement. This case emphasizes unusual morphologic features in a PTCL that may be mistaken for follicular lymphoma or a tumor of follicular dendritic cell origin.

Risk for leukemia in infants without Down syndrome who have transient myeloproliferative disorder.

Transient myeloproliferative disorder (TMD) occurs in 10% of infants with Down syndrome (DS). Down syndrome infants with resolved TMD may later develop acute megakaryocytic leukemia (AMKL). In these patients, AMKL is associated with somatic mutations in the X-linked transcription factor gene, GATA1. AMKL also has been described after TMD in children without DS. We report on a non-DS child identified with trisomy 21 mosaicism and a GATA1 mutation in the original blast cells who has been followed for 2 years without exhibiting AMKL. Currently, the risk for such infants developing acute leukemia is uncertain. We recommend that nondysmorphic infants with TMD undergo chromosome analysis for trisomy 21 and testing for GATA1 mutations to aid surveillance for leukemic transformation.

Gene expression profiling of adult acute myeloid leukemia identifies novel biologic clusters for risk classification and outcome prediction.

To determine whether gene expression profiling could improve risk classification and outcome prediction in older acute myeloid leukemia (AML) patients, expression profiles were obtained in pretreatment leukemic samples from 170 patients whose median age was 65 years. Unsupervised clustering methods were used to classify patients into 6 cluster groups (designated A to F) that varied significantly in rates of resistant disease (RD; P < .001), complete response (CR; P = .023), and disease-free survival (DFS; P = .023). Cluster A (n = 24), dominated by NPM1 mutations (78%), normal karyotypes (75%), and genes associated with signaling and apoptosis, had the best DFS (27%) and overall survival (OS; 25% at 5 years). Patients in clusters B (n = 22) and C (n = 31) had the worst OS (5% and 6%, respectively); cluster B was distinguished by the highest rate of RD (77%) and multidrug resistant gene expression (ABCG2, MDR1). Cluster D was characterized by a "proliferative" gene signature with the highest proportion of detectable cytogenetic abnormalities (76%; including 83% of all favorable and 34% of unfavorable karyotypes). Cluster F (n = 33) was dominated by monocytic leukemias (97% of cases), also showing increased NPM1 mutations (61%). These gene expression signatures provide insights into novel groups of AML not predicted by traditional studies that impact prognosis and potential therapy.

Nonsecretory variant of immunoproliferative small intestinal disease: a case report with pathologic, immunophenotypic, and molecular findings.

We report a case of the nonsecretory variant of immunoproliferative small intestinal disease involving the distal small bowel and the mesenteric and retroperitoneal lymph nodes in a 19-year-old woman from Mexico. This variant extranodal marginal zone B-cell lymphoma appeared similar in the different sites of involvement, with more interspersed large cells and greater plasmacytic differentiation present in intestinal specimens. Characteristic lymphoepithelial lesions and follicular colonization were seen in intestinal and lymph node sections, respectively. The neoplastic B cells were cytoplasmic immunoglobulin (Ig) A heavy-chain restricted and lacked surface and cytoplasmic light-chain expression by flow cytometric analysis. Serum and urine protein electrophoresis/immunofixation revealed hypogammaglobulinemia with no paraprotein. Molecular studies showed absence of immunoglobulin heavy-chain (IgH) gene rearrangement, with a nonfunctional clonotypic rearrangement of the kappa light-chain gene. This case highlights the role for kappa light-chain gene evaluation in immunoproliferative small intestinal disease, because IgH gene rearrangement analysis is often negative.

CD4(+) CD56(+) lineage-negative malignancies are rare tumors of plasmacytoid dendritic cells.

CD4(+) CD56(+) lineage-negative malignancies are difficult to diagnose and classify. Recent studies have suggested that these malignancies may derive from plasmacytoid dendritic cells (pDC). In this report, we examine 10 cases of CD4+, CD56+ lineage-negative malignancies that presented in various tissue sites. The goal was to identify the morphologic, immunophenotypic, and genotypic findings to devise a diagnostic approach to tissue biopsies of these lesions and to confirm the proposed cell of origin. The mean age was 66 years (range, 45-80 years) with a male predominance (8 males/2 females). Frequent sites of disease included skin (60%) and peripheral blood/bone marrow (70%). Tumor cells were positive for CD45, CD43, CD4, and CD56 (9 of 10). The pDC markers, CD123 (9 of 10) and CD45RA (10 of 10), were detected by immunoperoxidase staining. Also noted was CD2 positivity (1 case), weak CD7 positivity (4 of 8 cases), weak CD33 (4 of 9 cases), TdT (2 cases), and CD68 (2 cases). All cases were otherwise negative for EBV (EBER), B-cell, T-cell, myeloid, and NK cell markers. T-cell receptor-gamma gene rearrangement was negative in all cases. Complex structural chromosomal abnormalities were seen in 3 of 5 cases, a subset of which may be recurrent in pDC malignancy. Overall prognosis was poor despite multiagent chemotherapy and/or radiation. Our study confirms that CD4+/CD56+ lineage-negative tumors are derived from pDC and have characteristic clinical, histopathologic, and immunophenotypic features. Furthermore, these rare neoplasms can be readily diagnosed using recently developed immunoperoxidase techniques.

Flow cytometric immunophenotypic analysis of 306 cases of multiple myeloma.

Bone marrow aspirates from 306 patients with multiple myeloma were analyzed by flow cytometric immunophenotyping. The plasma cells (PCs) were identified by their characteristic light scatter distribution and reactivity patterns to CD138, CD38, and CD45. Monoclonality was confirmed by immunoglobulin light chain analysis. The immunophenotypic profile of the PCs was determined with a panel of antibodies. Moderate to bright expression of CD56, CD117, CD20, CD45, and CD52 was detected in 71.7%, 17.8%, 9.3%, 8.8%, and 5.2% of cases, respectively. These antigens were expressed by a distinct subpopulation of the PCs in 6.3%, 2.2%, 3.7%, 2.9%, and 2.6% of additional cases. CD19 was negative in more than 99% of cases. The combination of CD38 and CD138 was superior to CD38 alone for identifying CD45+ myeloma and separating CD20+ myeloma from B-cell lymphoma. PC immunophenotyping might be useful for detecting minimal residual disease in cases with aberrant antigen expression and for selection of therapeutic agents that have specific membrane targets.

Waldenstrom macroglobulinemia involving extramedullary sites: morphologic and immunophenotypic findings in 44 patients.

Waldenstrom macroglobulinemia (WM) is a clinicopathologic syndrome in which a B-cell neoplasm involving the bone marrow, usually lymphoplasmacytic lymphoma (LPL), is associated with immunoglobulin M paraprotein in the serum. Extramedullary involvement occurs in a subset of patients and is infrequently examined histologically. The files of M.D. Anderson Cancer Center were searched for patients with WM who underwent biopsy of one or more extramedullary sites during the course of disease. Each biopsy specimen was classified using the criteria of the World Health Organization classification. The study group consisted of 44 patients (26 men and 18 women), with a total of 51 specimens obtained from lymph nodes (n = 36), soft tissue (n = 4), spleen (n = 3), skin (n = 2), lung (n = 2), tonsils (n = 1), colon (n = 1), liver (n = 1), and gallbladder (n = 1). Lymphoplasmacytic lymphoma was the most common histologic type, in 40 (78%) samples. This category was morphologically heterogeneous and was further subclassified as lymphoplasmacytic (n = 21), lymphoplasmacytoid (n = 18), and polymorphous (n = 1). Four of these LPL cases morphologically resembled marginal zone B-cell lymphoma. Four additional samples were involved by diffuse large B-cell lymphoma, probably transformed from LPL. Three more samples were involved by LPL with unusual features: two were CD5-positive and one was a composite tumor with classical Hodgkin's disease. Other categories of lymphoma in this group of patients with WM included small lymphocytic lymphoma/chronic lymphocytic leukemia (n = 2), mantle cell lymphoma (n = 1), and follicular lymphoma (n = 1). Waldenstrom macroglobulinemia is most commonly associated with LPL but can rarely occur with other types of B-cell lymphoma. Lymphoplasmacytic lymphoma in patients with WM is morphologically heterogeneous and can be indistinguishable from marginal zone B-cell lymphoma. CD5+ B-cell lymphomas with features otherwise typical of LPL are rare, and we think these tumors are part of the spectrum of LPL.

Systemic mastocytosis with associated clonal hematological non-mast-cell lineage disease: analysis of clinicopathologic features and activating c-kit mutations.

The majority of patients with systemic mastocytosis with associated clonal, hematological non-mast cell lineage disease (SM-AHNMD) have a myeloid stem cell malignancy including myelodysplastic syndromes (MDS), myelodysplastic/myeloproliferative disorders, acute myeloid leukemia (AML), or chronic myeloproliferative disease. The clinicopathologic features of SM-AHNMD have not been fully characterized. We describe seven cases of this entity: 3 with MDS, 3 with AML, and 1 with chronic myelomonocytic leukemia. In the majority of cases, SM was diagnosed concurrently with the myeloid malignancy and aberrant mast cell morphology was observed. The commonly described c-kit enzymatic site mutation Asp816Val was detected only in 2 cases, while 3 patients carried the Asp816His mutation. Among the 3 cases with AML, 2 patients carried the translocation t(8;21). On the basis of our results and other reported cases, there appears to be a specific association between SM and AML with t(8;21). Concurrent occurrence of SM may define a subset of patients with de novo AML and other myeloid malignancies who have an adverse prognosis. As clinically effective tyrosine kinase inhibitors that inhibit enzymatic-type c-kit mutations are being developed, detection of mast cell proliferation associated with myeloid malignancy may have important therapeutic implications.

Bone marrow transplant engraftment analysis with loss of an informative allele.

Short tandem repeats (STRs) are highly polymorphic DNA sequences in the human genome. STR genotype analysis is used for human identity testing and to monitor bone marrow engraftment after allogeneic transplantation. Engraftment analysis requires one or more informative STR loci that distinguish recipient from donor. The following case illustrates that chromosome loss in tumor cells during the course of disease may cause corresponding loss of an STR locus. This circumstance is a potential source of error in the interpretation of engraftment analysis, especially if only one informative allele is used to monitor engraftment.