Jennifer L. Oliveira

Molecular Testing for Lipomatous Tumors: Critical Analysis and Test Recommendations Based on the Analysis of 405 Extremity-based Tumors

Ancillary molecular testing has been advocated for diagnostic accuracy in the differentiation of lipomas from atypical lipomatous tumors/well-differentiated liposarcomas (ALT/WDL); however, the implications and specific indications for use are not well-established in the current literature. Herein, we extend previous findings by quantitatively evaluating the impact of molecular testing of lipomatous neoplasms in our routine clinical practice, how it modifies the historical perspective of their clinical course, and the effect of distinct surgical procedures in modulating the risk of local recurrence for these tumors after molecular classification. On the basis of these analyses, we suggest a specific set of basic recommendations for complementary molecular assessment in the diagnosis of lipomatous tumors. Four hundred and five lipomatous neoplasms located in the trunk and extremities were analyzed histologically and for the presence of 12q13-15 amplification on paraffin-embedded tissues by assessing MDM2/CPM amplification. Survival analyses were calculated with Kaplan-Meier and compared with the log-rank. Multivariate analysis was evaluated by the Cox regression method. The 405 tumors were histologically classified as ordinary lipoma (n=324), intramuscular lipoma (n=29), and ALT/WDL (n=52). The level of agreement between the histologic diagnosis and the molecular diagnosis was high (96%) but pathologists showed a tendency to overestimate cytologic atypia and the diagnosis of ALT/WDL (precision, 79%; accuracy, 88%). Molecular assessment led to a major diagnostic reclassification in 18 tumors (4%). Eleven of the tumors histologically classified as ALT/WDL were reclassified as ordinary lipoma (n=5) and intramuscular lipoma (n=6); none of which recurred. Seven ordinary lipomas were reclassified as ALT/WDL, 6 of which were larger than 15 cm and deeply located; 2 recurred locally. After molecular data, the 5-year local recurrence rates for ordinary lipoma, intramuscular lipoma, and ALT/WDL were 1%, 12%, and 44%, respectively. Multivariate analyses after molecular assessment showed tumor type and type of resection to be associated with the risk of local recurrence. Complementary molecular testing refines the histologic classification of lipomatous tumors and better estimates the impact of surgical procedures on the risk of local recurrence. Pathologists tend to overestimate the degree of cytologic atypia and the indiscriminate use of molecular testing should be avoided, especially for extremity-based tumors. Molecular testing should be considered for “relapsing lipomas,” tumors with questionable cytologic atypia (even if widely excised), or for large lipomatous tumors (>15 cm) without diagnostic cytologic atypia.

High-resolution genomic mapping reveals consistent amplification of the fibroblast growth factor receptor substrate 2 gene in well-differentiated and dedifferentiated liposarcoma.

Well‐differentiated liposarcoma (WDLS) is one of the most common malignant mesenchymal tumors and dedifferentiated liposarcoma (DDLS) is a malignant tumor consisting of both WDLS and a transformed nonlipogenic sarcomatous component. Cytogenetically, WDLS is characterized by the presence of ring or giant rod chromosomes containing several amplified genes, including MDM2, TSPAN31, CDK4, and others mainly derived from chromosome bands 12q13‐15. However, the 12q13‐15 amplicon is large and discontinuous. The focus of this study was to identify novel critical genes that are consistently amplified in primary (nonrecurrent) WDLS and with potential relevance for future targeted therapy. Using a high‐resolution (5.0 kb) “single nucleotide polymorphism”/copy number variation microarray to screen the whole genome in a series of primary WDLS, two consistently amplified areas were found on chromosome 12: one region containing the MDM2 and CPM genes, and another region containing the FRS2 gene. Based on these findings, we further validated FRS2 amplification in both WDLS and DDLS. Fluorescence in situ hybridization confirmed FRS2 amplification in all WDLS and DDLS tested (n = 57). Real time PCR showed FRS2 mRNA transcriptional upregulation in WDLS (n = 19) and DDLS (n = 13) but not in lipoma (n = 5) and normal fat (n = 9). Immunoblotting revealed high expression levels of phospho‐FRS2 at Y436 and slightly overexpression of total FRS2 protein in liposarcoma but not in normal fat or preadipocytes. Considering the critical role of FRS2 in mediating fibroblast growth factor receptor signaling, our findings indicate that FRS2 signaling should be further investigated as a potential therapeutic target for liposarcoma.

A comprehensive analysis of hemoglobin variants by high-performance liquid chromatography (HPLC).

High‐performance liquid chromatography (HPLC) is a method commonly used for the detection of hemoglobin (Hb) variants. In addition to providing precise quantitation of Hb A2 and Hb F, the reported retention time and peak shape of a high number of hemoglobin (Hb) variants are very helpful for presumptive identification. However, there is a scarcity of summarized data in the literature of the mobility of Hb variants on this method.

Clinicopathologic features of B-Cell lineage neoplasms with aberrant expression of CD3: a study of 21 cases.

CD3 expression by immunohistochemistry was historically considered restricted to T-lineage or NK-lineage neoplasms but recently has been reported in rare cases of mature B-cell neoplasms, frequently in association with Epstein-Barr virus. Here, we describe the pathologic features of 21 B-cell lineage neoplasms that express CD3 protein by immunohistochemistry: 12 diffuse large B-cell lymphomas (DLBCLs); 2 plasmablastic lymphomas (PBLs); 4 plasma cell neoplasms; 2 Burkitt lymphomas; and 1 nodal follicular lymphoma, grade 3A. CD20 expression was negative or only partially positive in 13/21 cases. Epstein-Barr virus was positive in 3/20 tested cases (2 PBLs and 1 DLBCL). All tested neoplasms (14/14) had clonal immunoglobulin gene rearrangements, and no clonal T-cell gene rearrangements were detected (0/14). The 12 DLBCLs segregated into 2 main groups: 7 demonstrated features of plasmacytic differentiation but did not meet criteria for PBL, and 5 had anaplastic features. In addition to morphology, other features shared among the DLBCLs with plasmacytic differentiation, the plasma cell neoplasms, and the PBLs included extranodal presentation, cytoplasmic localization of CD3, and lack of expression of other T-cell antigens in most cases. In contrast, DLBCLs with anaplastic features and the single follicular lymphoma coexpressed multiple T-cell antigens in a predominantly membranous pattern and presented with nodal disease in a relatively younger patient population. Our data expand the spectrum of morphologic, phenotypic, and clinical features of B-cell neoplasms aberrantly expressing CD3. As these neoplasms often lack typical expression of B-cell antigens, knowledge of these features will help avoid misclassification.

Clinical and laboratory characteristics in congenital ANKRD26 mutation-associated thrombocytopenia: A detailed phenotypic study of a family.

Abstract The clinical and laboratory characteristics of patients with non-syndromic, autosomal dominant thrombocytopenia secondary to germ line ANKRD26 mutations appear to be heterogeneous. Except for a targeted molecular genotyping approach, there is no distinct clinical or laboratory phenotype that has been specifically associated with this particular gene mutation. Such heterogeneity could be due to variations in mutation and genetic background in different families. To understand the phenotypic heterogeneity, we thoroughly studied one affected family using the International Society for Thrombosis and Haemostasis bleeding assessment tool and both clinically validated standard and esoteric platelet testing (electron microscopy (EM) and flow cytometry). We found that decreased platelet aggregation with arachidonic acid and epinephrine agonists was common in affected family members. EM studies demonstrated persistent borderline low mean dense granules per platelet, decreased alpha granules and an increased canalicular network pattern in all affected members. Since these characteristics are subtle or non-pathognomonic, molecular testing for ANKRD26 mutation remains the most reliable test to render a diagnosis and should be considered when evaluating a patient or family with congenital thrombocytopenia, particularly if there is a history of myeloid neoplasms.

ASXL1 mutated chronic myelomonocytic leukemia in a patient with familial thrombocytopenia secondary to germline mutation in ANKRD26

Thrombocytopenia 2 (THC2), a non-syndromic, autosomal dominant thrombocytopenia, is caused by mutations in the ANKRD26 gene, located on chromosome 10p12.1 Thus far, 12 heterozygous single nucleotide substitutions in the 5′-untranslated region of the ANKRD26 gene have been described in 21 families.2, 3 Affected patients usually present with moderate thrombocytopenia with normal platelet size and normal mean platelet volumes. Many patients were reported to have an α-granule deficiency as detected by immunofluorescent labelling without consistent defects in platelet aggregation studies. Evidence for dysmegakaryopoiesis has been observed in a small number of patients who have undergone bone marrow biopsies. Surprisingly, a small subset of individuals can have elevated hemoglobin levels and leukocyte counts, without a clear explanation.3

Bone marrow findings of the newly described TEMPI syndrome: when erythrocytosis and plasma cell dyscrasia coexist

TEMPI syndrome (telangiectasias, elevated erythropoietin level and erythrocytosis, monoclonal gammopathy, perinephric fluid collections, and intrapulmonary shunting) is a recently described syndrome that, owing to erythrocytosis, may be confused with polycythemia vera. It is best classified as a type of plasma cell dyscrasia with paraneoplastic manifestations, similar to POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, M-protein, and skin abnormalities). To date, 11 patients have been identified. This is the first morphologic review of TEMPI syndrome bone marrow samples, in order to define pathologic features that may aid in the recognition of the syndrome and to identify post-therapy changes. Seven bone marrow aspirates and biopsies from three patients, including two post-treatment marrows, were examined. Patients were 36, 49, and 49 years old at time of diagnosis. In all cases, erythropoietin levels were extremely elevated at >5000 IU/l, the paraprotein was IgG kappa, JAK2 V617F was negative and vascular endothelial growth factor levels were normal. In one case, the increase in clonal plasma cells reached levels of smoldering myeloma (18%), but remaining marrows showed few monoclonal plasma cells (<5%). All pre-treatment biopsies showed erythroid hyperplasia, with mild nonspecific megakaryocytic, and erythroid cytologic atypia in one marrow. Prominent plasma cell vacuolization and reactive-appearing lymphoid aggregates were noted in one case. Findings of myeloproliferative neoplasms, including megakaryocyte clusters and fibrosis, were not identified. In conclusion, TEMPI syndrome should be considered when erythrocytosis and plasma cell dyscrasia coexist. The bone marrow findings, although nonspecific, differ significantly from polycythemia vera. Peculiar clinical and laboratorial findings of TEMPI, including elevated erythropoietin and normal vascular endothelial growth factor level, allow the diagnosis and distinction from POEMS syndrome. Significant decrease in erythropoietin level following treatment suggests a role of erythropoietin in monitoring therapeutic response.

Successful treatment of a child with T/myeloid acute bilineal leukemia associated with TLX3/BCL11B fusion and 9q deletion.

Acute bilineal leukemias are rare and are commonly associated with t(9;22) and MLL abnormalities. Herein, we report a pediatric case of bilineal T/myeloid acute leukemia associated with del (9q)(q13q22) and TLX3/BCL11B fusion due to the cryptic t(5;14)(q35;32). FISH studies confirmed the TLX3/BCL11B fusion in both the myeloid and lymphoid blasts, while the 9q deletion was restricted to the lymphoid component. Optimal therapy for such patients remains controversial and it is not clear if they should be treated with ALL or AML‐based chemotherapeutic regimens. Our patient has been in extended remission following ALL‐based chemotherapy and a matched unrelated cord blood transplant. Pediatr Blood Cancer 2011;56:467–469.

Hb Nebraska [β86(F2)Ala→Ile (HBB:c.259G>A;260C>T)]: a unique high oxygen affinity hemoglobin variant with a double nucleotide substitution within the same codon.

A new high oxygen affinity hemoglobin (Hb) variant, Hb Nebraska [β86(F2)Ala→Ile, GCC>ATC; HGVS: HBB: c.259G>A;260C>T] is reported. This variant was not identified by routine methods and was only suspected due to erythrocytosis and an abnormal p50 value. The variant was analyzed by DNA sequencing and mass spectrometry (MS). The β chain variant is unusual in that it has two nucleotide substitutions occurring at the same codon.

HB CAMBRIDGE-MA (β144(HC1)-β146(HC3)Lys-Tyr-His→ 0( HBB c.433 A>T)): A NEW HIGH OXYGEN AFFINITY VARIANT

A new β hemoglobin (Hb) variant, Hb Cambridge-MA [β144(HC1)β146(HC3)Lys-Tyr-His→0 (AAG>TAG) (HGVS: HBB c.433 A>T] is described. The variant was characterized by high performance liquid chromatography (HPLC), alkaline, acid, globin chain and capillary electrophoresis, isoelectric focusing (IEF), heat and isopropanol stability, p50 analyses, intact globin mass spectrometry (MS) and DNA sequencing. The new variant shows high oxygen (O2) affinity and is associated with mild polycythemia.