Clinical Significance of DNA Variants in Chronic Myeloid Neoplasms: A Report of the Association for Molecular Pathology.

To address the clinical relevance of small DNA variants in chronic myeloid neoplasms (CMNs), an Association for Molecular Pathology Working Group comprehensively reviewed published literature, summarized key findings that support clinical utility, and defined critical gene inclusions for high-throughput sequencing testing panels. This review highlights the biological complexity of CMNs [including myelodysplastic syndromes, myeloproliferative neoplasms, entities with overlapping features (myelodysplastic syndromes/myeloproliferative neoplasms), and systemic mastocytosis], the genetic heterogeneity within diagnostic categories, and similarities between apparently disparate diagnostic entities. The founding variant's hematopoietic differentiation compartment, specific genes and variants present, order of variant appearance, individual subclone dynamics, and therapeutic intervention all contribute to the clinicopathologic features of CMNs. Selection and efficacy of targeted therapies are increasingly based on DNA variant profiles present at various time points; therefore, high-throughput sequencing remains critical for patient management. The following genes are a minimum recommended list to provide relevant clinical information for the management of most CMNs: ASXL1, BCOR, BCORL1, CALR, CBL, CEBPA, CSF3R, DNMT3A, ETV6, EZH2, FLT3, IDH1, IDH2, JAK2, KIT, KRAS, MPL, NF1, NPM1, NRAS, PHF6, PPM1D, PTPN11, RAD21, RUNX1, SETBP1, SF3B1, SMC3, SRSF2, STAG2, TET2, TP53, U2AF1, and ZRSR2. This list is not comprehensive for all myeloid neoplasms and will evolve as insights into effects of combinations of relevant biomarkers on specific clinicopathologic characteristics of CMNs accumulate.

Morphologically occult systemic mastocytosis in bone marrow: clinicopathologic features and an algorithmic approach to diagnosis.

OBJECTIVES: Bone marrow (BM) biopsy specimens involved by systemic mastocytosis (SM) typically show multifocal, compact, dense aggregates of spindled mast cells (MCs). However, some cases lack aggregate formation and fulfill the World Health Organization 2008 criteria for SM, based on minor criteria. METHODS: We identified 26 BM cases of KIT D816V-mutated, morphologically occult SM in the BM. RESULTS: All patients had some combination of allergic/MC activating symptoms. Peripheral blood counts were generally normal. BM aspirates showed 5% or less MCs, which were only occasionally spindled. BM biopsy specimens showed no morphologic classic MC lesions. Tryptase immunohistochemistry (IHC) demonstrated interstitial, individually distributed MCs (up to 5%) with prominent spindling, lacking aggregate formation. MCs coexpressed CD25 by IHC and/or flow cytometry. Spindled MCs constituted more than 25% of total MCs in all cases and more than 50% in 20 of 26 cases. CONCLUSIONS: Morphologically occult involvement of normal-appearing BM by SM will be missed without appropriate clinical suspicion and pathologic evaluation by tryptase and CD25 IHC and KIT D816V mutation analysis. On the basis of these findings, we propose a cost-effective, data-driven, evidence-based algorithmic approach to the workup of these cases.

Mucosal CD30-positive T-cell lymphoproliferations of the head and neck show a clinicopathologic spectrum similar to cutaneous CD30-positive T-cell lymphoproliferative disorders.

CD30-positive T-cell lymphoproliferative disorders are classified as cutaneous (primary cutaneous anaplastic large cell lymphoma and lymphomatoid papulosis) or systemic. As extent of disease dictates prognosis and treatment, patients with skin involvement need clinical staging to determine whether systemic lymphoma also is present. Similar processes may involve mucosal sites of the head and neck, constituting a spectrum that includes both neoplasms and reactive conditions (eg, traumatic ulcerative granuloma with stromal eosinophilia). However, no standard classification exists for mucosal CD30-positive T-cell lymphoproliferations. To improve our understanding of these processes, we identified 15 such patients and examined clinical presentation, treatment and outcome, morphology, phenotype using immunohistochemistry, and genetics using gene rearrangement studies and fluorescence in situ hybridization. The 15 patients (11 M, 4 F; mean age, 57 years) had disease involving the oral cavity/lip/tongue (9), orbit/conjunctiva (3) or nasal cavity/sinuses (3). Of 14 patients with staging data, 7 had mucosal disease only; 2 had mucocutaneous disease; and 5 had systemic anaplastic large cell lymphoma. Patients with mucosal or mucocutaneous disease only had a favorable prognosis and none developed systemic spread (follow-up, 4-93 months). Three of five patients with systemic disease died of lymphoma after 1-48 months. Morphologic and phenotypic features were similar regardless of extent of disease. One anaplastic lymphoma kinase-positive case was associated with systemic disease. Two cases had rearrangements of the DUSP22-IRF4 locus on chromosome 6p25.3, seen most frequently in primary cutaneous anaplastic large cell lymphoma. Our findings suggest mucosal CD30-positive T-cell lymphoproliferations share features with cutaneous CD30-positive T-cell lymphoproliferative disorders, and require clinical staging for stratification into primary and secondary types. Primary cases have clinicopathologic features closer to primary cutaneous disease than to systemic anaplastic large cell lymphoma, including indolent clinical behavior. Understanding the spectrum of mucosal CD30-positive T-cell lymphoproliferations is important to avoid possible overtreatment resulting from a diagnosis of overt T-cell lymphoma.

PAX5-positive T-cell anaplastic large cell lymphomas associated with extra copies of the PAX5 gene locus.

Cell lineage is the major criterion by which lymphomas are classified. Immunohistochemistry has greatly facilitated lymphoma diagnosis by detecting expression of lineage-associated antigens. However, loss or aberrant expression of these antigens may present diagnostic challenges. Anaplastic large cell lymphoma is a T-cell lymphoma that shows morphologic and phenotypic overlap with classical Hodgkin's lymphoma, which is a tumor of B-cell derivation. Staining for the B-cell transcription factor, paired box 5 (PAX5), has been suggested to be helpful in this differential, as it is positive in most classical Hodgkin's lymphomas, but absent in anaplastic large cell lymphomas. In this study we report four systemic T-cell anaplastic large cell lymphomas that were positive for PAX5 by immunohistochemistry, with weak staining intensity similar to that observed in classical Hodgkin's lymphoma. All diagnoses were confirmed by a combination of morphologic, phenotypic, and molecular criteria. Three cases were anaplastic lymphoma kinase (ALK) negative and one was ALK positive. PAX5 immunohistochemistry was negative in 198 additional peripheral T-cell lymphomas, including 66 anaplastic large cell lymphomas. Unexpectedly, although PAX5 translocations were absent, all evaluable PAX5-positive anaplastic large cell lymphomas showed extra copies of the PAX5 gene locus by fluorescence in situ hybridization (FISH). In contrast, only 4% of PAX5-negative peripheral T-cell lymphomas had extra copies of PAX5. We conclude that aberrant expression of PAX5 occurs rarely in T-cell anaplastic large cell lymphomas, and may be associated with extra copies of the PAX5 gene. PAX5-positive lymphomas with morphologic features overlapping different lymphoma types should be evaluated with an extensive immunohistochemical panel and/or molecular studies to avoid diagnostic errors that could lead to inappropriate treatment. As PAX5 overexpression causes T-cell neoplasms in experimental models, PAX5 may have contributed to lymphomagenesis in our cases.

Epstein-Barr virus-associated diffuse large B-cell lymphoma arising on cardiac prostheses.

Primary cardiac lymphoma is extremely rare and lymphoma arising in association with prosthetic valves has been described in only 3 case reports. We describe 3 patients with diffuse large B-cell lymphoma (DLBCL) involving prosthetic heart valves and a synthetic tube graft. All 3 specimens showed shallow layering of acellular fibrinous debris over the prosthetic or synthetic materials, with tumor lymphocytes present at the luminal surface. There were frequent mitoses and abundant karyorrhectic debris. All demonstrated a nongerminal center B-cell phenotype. All 3 cases were positive for Epstein-Barr virus, but there was no staining for human herpes virus 8. There was no other evidence of distant disease at the time of diagnosis and no recurrence or dissemination occurred after surgical removal of the prosthesis, though follow-up was limited. On the basis of 2008 World Health Organization diagnostic criteria, we believe these cases should be classified as DLBCL associated with chronic inflammation. However, unlike the characteristically poor prognosis reported in this entity, we hypothesize that the disease resectability in these cardiac sites, in many cases, may allow for a better prognosis than DLBCL with chronic inflammation at other less resectable sites.

Amiodarone-induced bone marrow granulomas: an unusual cause of reversible pancytopenia.

Bone marrow infiltration by granulomas rarely presents with cytopenias and is usually a result of atypical infections, lymphomas, or sarcoidosis. Drugs are also an important but often overlooked causal agent of bone marrow granulomas. Although rare, amiodarone has been associated with bone marrow granuloma formation. This case report describes a 73-year-old male who presented with pancytopenia during a preoperative evaluation. Amiodarone therapy was suspected to be the causal agent after diagnostic evaluation and exclusion of other causes. After cessation of amiodarone, the patient's pancytopenia gradually resolved over a period of several months. Our report illustrates an often overlooked yet important cause of reversible pancytopenia owing to suspected amiodarone-induced bone marrow granuloma formation, and guides clinicians in an expected timeline for blood count improvement after cessation of this drug.

Prognostically relevant breakdown of 123 patients with systemic mastocytosis associated with other myeloid malignancies.

The prognostic heterogeneity of the World Health Organization category of "systemic mastocytosis with associated clonal hematologic nonmast cell lineage disease" (SM-AHNMD) has not been systematically validated by primary data. Among 138 consecutive cases with SM-AHNMD, 123 (89%) had associated myeloid neoplasm: 55 (45%) myeloproliferative neoplasm (SM-MPN), 36 (29%) chronic myelomonocytic leukemia, 28 (23%) myelodysplastic syndrome (SM-MDS), and 4 (3%) acute leukemia. Of the myeloid subgroups, SM-MPN displayed a 2- to 3-fold better life expectancy (P = .003), whereas leukemic transformation was more frequent in SM-MDS (29%; P = .02). The presence of eosinophilia, although prevalent (34%), was prognostically neutral, and the overall results were not affected by exclusion of FIP1L1-PDGFRA-positive cases. We conclude that it is clinically more useful to consider specific entities, such as SM-MPN, systemic mastocytosis with chronic myelomonocytic leukemia, SM-MDS, and systemic mastocytosis with-acute leukemia, rather than their broad reference as SM-AHNMD.

Systemic mastocytosis in 342 consecutive adults: survival studies and prognostic factors.

Clinical phenotype in systemic mastocytosis (SM) is markedly variable, which complicates prognostication and decision making regarding the choice and timing of therapy. In a retrospective study of 342 consecutive adult patients with SM seen at the Mayo Clinic between 1976 and 2007, disease subdesignation according to the World Health Organization (WHO) proposal was indolent (ISM) in 159 (46%), with associated clonal hematologic non-mast cell lineage disease (SM-AHNMD) in 138 (40%), aggressive (ASM) in 41 (12%), and mast cell leukemia in 4 (1%). KITD816V was detected in bone marrow-derived DNA by allele-specific polymerase chain reaction (PCR) in 68% of 165 patients evaluated (ISM, 78%; ASM, 82%; SM-AHNMD, 60%; P = .03); JAK2V617F was detected in 4%, all in SM-AHNMD. Compared with those with nonindolent SM, life expectancy in ISM was superior and not significantly different from that of the age- and sex-matched US population. In addition, multivariable analysis identified advanced age, weight loss, anemia, thrombocytopenia, hypoalbuminemia, and excess bone marrow blasts as independent adverse prognostic factors for survival. The current study validates the prognostic relevance of the WHO subclassification of SM and provides additional information of value in terms of both risk stratification and interpretation of clinical presentation and laboratory results.

Primary cutaneous CD4-positive small/medium-sized pleomorphic T-cell lymphoma: a clonal T-cell lymphoproliferative disorder with indolent behavior.

Primary cutaneous CD4-positive small/medium-sized pleomorphic T-cell lymphoma, a provisional entity in the 2005 WHO-EORTC classification for cutaneous lymphomas, is not well characterized. Fifteen cases meeting the definition of this entity were identified. Fourteen represented solitary lesions on the head/neck (n=9), upper extremity (n=4), or trunk (n=1). One patient presented with multiple lesions on the trunk and extremities. Histologically, the infiltrate showed a nodular pattern in the dermis and subcutis without epidermotropism, and had a polymorphous composition with a predominance of small to medium-sized CD4-positive T cells. Most cases showed normal T-cell antigen expression; diminished/absent expression of CD7 was seen in three cases and CD2 expression was absent in one case. All cases showed a notable reactive infiltrate including frequent B cells, plasma cells, and histiocytes. Clonal TCR gene rearrangements were detected in each case. No clonal Ig gene rearrangements were detected. Out of the 11 patients with follow-up, none showed systemic disease. The majority resolved without relapse, one without treatment, four with excision, and four with radiation therapy. One patient developed local recurrence. The patient with multiple lesions had disease progression despite chemotherapy and stem cell transplant. These cases highlight the polymorphous histology and prominent reactive B-cell component of this entity. Diagnosis requires molecular genetic analysis, as prominent cytologic atypia and immunophenotypic aberrancy are rare. The differential diagnosis includes reactive lymphoid hyperplasia, mycosis fungoides and cutaneous B-cell lymphomas. In patients with isolated cutaneous lesions, the indolent behavior of this rare T-cell neoplasm should be recognized to avoid unnecessary treatment.

20+ yr without leukemic or fibrotic transformation in essential thrombocythemia or polycythemia vera: predictors at diagnosis.

OBJECTIVES: The current study identified patients with either essential thrombocythemia (ET) or polycythemia vera (PV) who have survived for at least 20 yr without the development of either acute myeloid leukemia/myelodysplastic syndrome (AML/MDS) or myelofibrosis (MF) and compared their presenting features with those in whom these complications occurred in the first 10 yr of disease. METHODS: The study patients were selected from an institutional database of 1061 patients with either ET (n = 603) or PV (n = 458). In both instances, three distinct groups were delineated and their presenting features compared; group A included patients who have remained AML/MDS/MF free after a minimum follow-up of 20 yr; groups B and C included patients who developed either AML/MDS or MF, respectively, in the first decade of their disease. RESULTS: The respective number of patients who fulfilled the above-mentioned criteria for inclusion in groups A, B and C were 40, 12 and 8 for ET and 23, 18 and 12 for PV. In ET, compared with both groups B and C, group A displayed significantly fewer patients with less than normal hemoglobin level (P < 0.0001 and =0.02) or male sex (P = 0.005 and 0.05), respectively. On multivariable analysis, only anemia sustained its significance. A similar analysis in PV revealed an association between group B and leukocytosis using a leukocyte count threshold of either 10 or 15 x 10(9)/L (P = 0.02). CONCLUSION: The current study identifies PV patients with leukocytosis and ET patients with anemia as the most likely to undergo leukemic or fibrotic transformation.

Tumor suppressor p16 methylation in multiple myeloma: biological and clinical implications.

The biological and clinical implications of p16 gene methylation in multiple myeloma (MM) are still unclear despite previous studies. In this comprehensive study, using methylation-specific PCR (MS-PCR), we show that p16 methylation is relatively common and occurs in monoclonal gammopathy of undetermined significance (MGUS; n=17), smoldering multiple myeloma (SMM; n=40), and MM (n=522) at a prevalence of 24%, 28%, and 34%, respectively. However, p16 methylation does not appear to affect gene expression level. In a large cohort of patients with long-term follow-up information (n=439), there was no difference in overall survival between patients with or without p16 methylation. We also found no association between p16 methylation and the main cytogenetic categories, although it was more common among patients with 17p13.1 deletions (p53 locus), a genetic progression event in MM. In addition, p16 methylation has no apparent effect on the cycle because there was also no difference in the plasma cell labeling index (a direct measurement of proliferation) between patients with and without p16 methylation. Our results question a major role for p16 methylation in the oncogenesis of the PC neoplasm, and we now believe p16 methylation may be a marker for overall epigenetic changes associated with disease progression, with no obvious direct biological or clinical consequences.

MPL W515 and JAK2 V617 mutation analysis in patients with refractory anemia with ringed sideroblasts and an elevated platelet count.

Discovery of a constitutively activating point mutation of the Janus kinase 2 (JAK2) receptor-associated tyrosine kinase in patients with polycythemia vera (PV) and other BCR/ABL-negative myeloproliferative disorders prompted many groups around the world to examine diverse subsets of patients with myeloid diseases for the prevalence of the JAK2 V617F mutation and its clinical and pathological associations.

Concurrent MPL515 and JAK2V617F mutations in myelofibrosis: chronology of clonal emergence and changes in mutant allele burden over time.

MPLW515L/K and JAK2V617F can co-exist in myelofibrosis with myeloid metaplasia (MMM). The chronology of clonal emergence was studied in three such cases using serially stored bone marrow. At diagnosis, a major MPL515 mutant clone was accompanied by a minor JAK2V617F clone in all three instances. At 25 time points over a period of 4-8 years, allele burden fluctuated but remained high for MPLW515L/K and low for JAK2V617F. We conclude that MPLW515L/K and JAK2V617F are both early events in MMM and allele burden, rather than the mere presence of these mutations, might be relevant to phenotypic variation in myeloproliferative disorders.

The JAK2 V617F mutation is absent in patients with erythrocytosis due to high oxygen affinity hemoglobin variants.

High oxygen affinity hemoglobin (Hb) variants are an important and well characterized cause of secondary erythrocytosis. We tested 22 patients with high oxygen affinity beta chain variants for the presence of the JAK2 V617F mutation that has been reported in chronic myeloproliferative disorders, particularly polycythemia vera. All specimens showed the absence of this mutation. This observation contributes to the overall clinical specificity of the JAK2 V617F mutation.

MPL515 mutations in myeloproliferative and other myeloid disorders: a study of 1182 patients.

Recently, a gain-of-function MPL mutation, MPLW515L, was described in patients with JAK2V617F-negative myelofibrosis with myeloid metaplasia (MMM). To gain more information on mutational frequency, disease specificity, and clinical correlates, genomic DNA from 1182 patients with myeloproliferative and other myeloid disorders and 64 healthy controls was screened for MPL515 mutations, regardless of JAK2V617F mutational status: 290 with MMM, 242 with polycythemia vera, 318 with essential thrombocythemia (ET), 88 with myelodysplastic syndrome, 118 with chronic myelomonocytic leukemia, and 126 with acute myeloid leukemia (AML). MPL515 mutations, either MPLW515L (n = 17) or a previously undescribed MPLW515K (n = 5), were detected in 20 patients. The diagnosis of patients with mutant MPL alleles at the time of molecular testing was de novo MMM in 12 patients, ET in 4, post-ET MMM in 1, and MMM in blast crisis in 3. Six patients carried the MPLW515L and JAK2V617F alleles concurrently. We conclude that MPLW515L or MPLW515K mutations are present in patients with MMM or ET at a frequency of approximately 5% and 1%, respectively, but are not observed in patients with polycythemia vera (PV) or other myeloid disorders. Furthermore, MPL mutations may occur concurrently with the JAK2V617F mutation, suggesting that these alleles may have functional complementation in myeloproliferative disease.

Lenalidomide therapy in myelofibrosis with myeloid metaplasia.

We present results of 2 similarly designed but separate phase 2 studies involving single-agent lenalidomide (CC-5013, Revlimid) in a total of 68 patients with symptomatic myelofibrosis with myeloid metaplasia (MMM). Protocol treatment consisted of oral lenalidomide at 10 mg/d (5 mg/d if baseline platelet count < 100 x 10(9)/L) for 3 to 4 months with a plan to continue treatment for either 3 or 24 additional months, in case of response. Overall response rates were 22% for anemia, 33% for splenomegaly, and 50% for thrombocytopenia. Response in anemia was deemed impressive in 8 patients whose hemoglobin level normalized from a baseline of either transfusion dependency or hemoglobin level lower than 100 g/L. Additional treatment effects in these patients included resolution of leukoerythroblastosis (4 patients), a decrease in medullary fibrosis and angiogenesis (2 patients), and del(5)(q13q33) cytogenetic remission accompanied by a reduction in JAK2(V617F) mutation burden (1 patient). Grade 3 or 4 adverse events included neutropenia (31%) and thrombocytopenia (19%). We conclude that lenalidomide engenders an intriguing treatment activity in a subset of patients with MMM that includes an unprecedented effect on peripheral blood and bone marrow abnormalities.

Essential thrombocythemia beyond the first decade: life expectancy, long-term complication rates, and prognostic factors.

OBJECTIVE: To describe the long-term natural history of essential thrombocythemia (ET) in terms of life expectancy, risk of disease transformation Into a more aggressive myeloid disorder, and prognostic factors for both survival and disease complications. PATIENTS AND METHODS: The study population consisted of a consecutive cohort of patients seen at the Mayo Clinic In Rochester, Minn, in whom a diagnosis of ET was established before 1992, thus allowing a minimum of 10 years of potential follow-up. The conventional criteria-based diagnosis was confirmed by bone marrow biopsy in all Instances. RESULTS: A total of 322 patients were studied (median age, 54 years; median follow-up, 13.6 years). With a median survival time of 18.9 years, survival in the first decade of disease was similar to that of the control population (risk ratio, 0.72; 95% confidence interval, 0.50-0.99) but became significantly worse thereafter (risk ratio, 2.21; 95% confidence Interval, 1.74-2.76). Multivariable analysis identified age at diagnosis of 60 years or older, leukocytosis, tobacco use, and diabetes mellitus as Independent predictors of poor survival. A 2-variable model based on an age cutoff of 60 years and leukocyte count of 15 x 10(9)/L resulted in 3 risk groups with significant difference in survival. In addition, age at diagnosis of 60 years or older, leukocytosis, and history of thrombosis were independent predictors of major thrombotic events. The risk of leukemic or any myeloid disease transformation was low in the first 10 years (1.4% and 9.1%, respectively) but increased substantially in the second (8.1% and 28.3%, respectively) and third (24.0% and 58.5%, respectively) decades of the disease. CONCLUSION: Life expectancy in patients with ET is significantly worse than that of the control population. Leukocytosis is identified as a novel independent risk factor for both inferior survival and thrombotic events.