Ketterling, Rhett P.

SummaryAmong 826 patients with primary myelofibrosis (PMF) and analysable metaphases on cytogenetic studies, 352 (42·6%) had abnormal karyotype, of which 240 (68·2%) were sole aberrations and 48 (13·6%) were complex; the most frequent abnormalities were 20q− (23·3%), 13q− (18·2%), +8 (11·1%), +9 (9·9%), chromosome 1q+ (9·7%) and −7/7q− (7·1%). Phenotypic correlates included: abnormal karyotype with anaemia (P = 0·02), leucopenia (P < 0·01) and thrombocytopenia (P < 0·01); complex karyotype with younger age (P = 0·04) and thrombocytopenia (P < 0·01); leucopenia with 20q−, +8 and −7/7q− and thrombocytopenia with 20q− and −7/7q−. Cytopenias were less likely to occur with 13q−. 476 patients were annotated for JAK2/CALR/MPL mutations; abnormal karyotype frequencies were 43% in JAK2, 42% CALR, 33% MPL mutated and 34% triple‐negative cases (P = 0·3). A proportion of patients were also screened for ASXL1, EZH2, IDH1, IDH2, SRSF2, U2AF1 and SF3B1 mutations; in all instances, mutational frequencies were higher in patients with normal karyotype, reaching significance for ASXL1 (P = 0·02) and U2AF1 (P = 0·01). 13q− was associated with mutant CALR (P = 0·03), +9 with mutant JAK2 (P = 0·02) and 20q− with mutant SRSF2 (P = 0·02). The current PMF study provides detailed cytogenetic information and correlations with mutations and clinical phenotype.

Four hundred and nine patients with chronic myelomonocytic leukemia (CMML) were included in the international collaborative study (268 (66%) and 141 (34%) from Mayo clinic and French consortium respectively). Thirty percent displayed an abnormal karyotype, including; 72% sole, 16% two, and 11% complex abnormalities. The most common abnormalities included; +8 (23%), ‐Y (20%), ‐7/7q‐(14%), 20q‐ (8%), +21 (8%), and der(3q) (8%). Patients with an abnormal karyotype were more likely to be elderly (P = 0.03), be anemic (P = 0.0009), have leukocytosis (P = 0.02) with neutrophilia (P = 0.03), demonstrate increased circulating immature myeloid cells (P = 0.0003), peripheral blood blasts (P < 0.0001), and bone marrow blasts (P < 0.0001). ASXL1 (P = 0.04) and SF3B1 (P = 0.03) mutations clustered with an abnormal karyotype, whereas SRSF2 (P = 0.02) mutations occurred more commonly with a normal karyotype. A step‐wise survival analysis resulted in three distinct cytogenetic risk categories: high (complex and monosomal karyotypes), intermediate (all abnormalities not in the high or low risk groups) and low [normal, sole ‐Y and sole der (3q)] with median survivals of 3 [hazard ratio (HR) = 8.1, 95% confidence interval (CI) = 4.6–14.2], 20 (HR = 1.7, 95% CI = 1.2–2.3) and 41 months, respectively. In multivariable analysis, this particular cytogenetic risk stratification remained significant in the context of the Molecular Mayo Model (P < 0.0001), MD Anderson prognostic model (P < 0.0001), the GFM CMML model (P < 0.0001) and was effective in predicting leukemic transformation (P = 0.004). Am. J. Hematol. 89:1111–1115, 2014. © 2014 Wiley Periodicals, Inc.

Abstract Background: Peripheral T-cell lymphomas (PTCLs) represent a heterogeneous group of generally aggressive malignancies, with 5-year overall survival (OS) rates of ∼35%. Only ALK translocations, which define ALK-positive anaplastic large T-cell lymphoma (ALCL), have emerged as a genetic biomarker predicting favorable outcome following conventional chemotherapy (e.g. CHOP). Using next-generation sequencing, we discovered additional recurrent chromosomal translocations in PTCLs involving either the DUSP22 locus on 6p25.3 or TP63 on 3q28. The purpose of this study was to evaluate the prognostic utility of combined testing for both these events in PTCL patients. Methods: We examined the clinicopathologic features of 134 systemic PTCLs using WHO criteria, including 49 ALCLs, 23 angioimmunoblastic PTCLs, 50 PTCLs of “not otherwise specified” type, and 12 PTCLs of other subtypes. Immunohistochemistry was used to determine ALK expression, an established surrogate for ALK translocations in ALCL. Fluorescent in situ hybridization (FISH) was performed to detect translocations of DUSP22 or TP63 in all cases. OS was plotted using the Kaplan-Meier method. Comparisons were made using the log-rank test. Results: There were 83 males and 51 females, with a mean age of 58 years (range, 6 to 90 years). Median OS for all patients was 22 months (5-year OS, 36%) with a median follow-up of 24 months (range, 0.1 to 354 months). Translocations of DUSP22, TP63, and ALK were mutually exclusive. Patients with ALK-positive ALCL (n=17) had a median OS of 176 months (median follow-up, 68 months; 5-year OS, 69%). Despite being ALK-negative, cases with translocations of DUSP22 or TP63 shared pathologic features with ALCL. Median OS was not reached in patients with DUSP22 translocations (n=7; median follow-up, 88 months; 5-year OS, 100%). Patients with tumors harboring TP63 translocations (n=9) had a median OS of 13 months (all dead at 76 months; 5-year OS, 11%). Patients with none of these abnormalities (n=101) had a median OS of 17 months (median follow-up, 13 months; 5-year OS, 29%). Differences in OS among these groups were highly significant (p=0.0002). Conclusions: PTCLs with DUSP22 translocations had a favorable prognosis similar to ALK-positive ALCLs. In contrast, TP63 were associated with very poor prognosis. FISH for these biomarkers is a promising tool for risk stratification, particularly since both translocations occurred in cases with overlapping pathologic features. PTCLs with DUSP22 translocations may represent a genetically defined subset of ALK-negative ALCLs. Like patients with ALK-positive ALCLs, these patients may not derive additional benefit from intensified up-front strategies such as early transplantation. Patients with none of these abnormalities comprised 75% of patients studied and had an intermediate prognosis, indicating the need for additional biomarkers in this group to improve risk stratification. Citation Format: Edgardo R. Parrilla Castellar, George Vasmatzis, Ryan A. Knudson, Rhett P. Ketterling, Karen L. Grogg, William R. Macon, Mark E. Law, Stephen M. Ansell, Ahmet Dogan, Andrew L. Feldman. Genetic biomarkers for risk stratification of patients with peripheral T-cell lymphoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3538. doi:10.1158/1538-7445.AM2013-3538

Abstract Background: Cutaneous T-cell neoplasms display markedly heterogeneous clinical behavior despite often similar pathologic features. For example, transformed mycosis fungoides (tMF) has a poor prognosis and requires intensive therapy whereas lymphomatoid papulosis (LyP) is an indolent lymphoproliferative disorder with spontaneously resolving lesions. These entities have overlapping histologic features and no molecular tools currently exist to aid in subclassification. Methods: We identified 11 T-cell neoplasms with histologic appearances distinct from previously described cutaneous T-cell neoplasms but with features overlapping those of both tMF and LyP. The phenotypic and genetic characteristics of the tumors were studied using immunohistochemistry and fluorescence in situ hybridization, respectively. Clinical data on presentation, treatment, evolution of the lesions, and follow-up were obtained. Results: All patients were older adults (mean age, 75 yr; range, 67-88 yr; 9M:2F). Clinical presentations were similar, with papulonodular skin lesions (0.3-1.0 cm) restricted to a single body area in all but two patients. Histologically there was an extensive atypical lymphoid infiltrate in the epidermis and a dermal tumor containing large transformed cells with numerous mitotic figures and apoptotic bodies. All tumors had a T-cell phenotype and expressed CD30 in both dermal and epidermal components. No case expressed ALK. Most cases had a high proliferative rate (&gt;80%) by Ki67 staining. FISH analysis demonstrated rearrangements of the DUSP22-IRF4 locus on 6p25.3 in all cases. No patient had evidence of systemic disease on imaging studies. In 4 untreated patients, papules began to regress spontaneously a few weeks after presentation, with complete resolution within a month or two. Seven patients were treated with local excision (with or without cryotherapy) or radiotherapy. All non-excised lesions ultimately regressed. All patients were alive at last follow-up (median, 21 mo; range, 7-150 mo). Cutaneous recurrences occurred in 5 patients. At last follow-up only 1 patient had active lesions, which were confined to the presenting site. The remaining 10 patients had been disease-free from 2 to 32 months. Conclusions: Despite pathologic features mimicking an aggressive lymphoma, the benign clinical course observed in all 11 patients suggests these cases represent a newly recognized subtype of LyP. These neoplasms are characterized by 6p25.3 rearrangements, representing the first recurrent genetic abnormality identified in LyP. In contrast, we previously have shown absence of these rearrangements in tMF. Distinction of LyP from tMF is critical in this older patient population unlikely to tolerate intensive therapy. Thus, detection of 6p25.3 rearrangements represents a new tool for molecular classification of cutaneous T-cell neoplasms. Citation Format: Andrew L. Feldman, Laszlo J. Karai, Marshall E. Kadin, Eric D. Hsi, Jason C. Sluzevich, Rhett P. Ketterling, Ryan A. Knudson. Discovery of a previously undescribed cutaneous T-cell neoplasm with chromosomal rearrangements of 6p25.3. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1208. doi:10.1158/1538-7445.AM2013-1208

Abstract Despite the established use of poly-chemotherapy, relapse continues to be the most common cause of death in AML and MDS and cure rates remain below 20%. AML/MDS arise following the accumulation of stepwise genetic and epigenetic changes in hematopoietic stem and progenitor cells (HSPC). Utilizing a novel strategy of parallel transcriptional analysis of sorted HSPC populations in distinct subtypes of AML, we compared the gene expression in AML HSPC with identical compartments from age-matched healthy controls and identified Interleukin 1 receptor accessory protein (IL1RAP) as one of the most significantly upregulated genes in HSPC in all examined subtypes of AML. Fluorescence in situ hybridization of sorted IL1RAP+ and IL1RAP- cells from patients with monosomy 7 AML (-7) indicated that the aberrant clone was restricted to IL1RAP+ cells, demonstrating that IL1RAP overexpression is a distinguishing feature of the -7 clone. Multivariate analysis of a large cohort of patients with normal karyotype AML showed that patients with high IL1RAP levels had inferior overall survival than patients with lower IL1RAP levels, suggesting an independent prognostic value for this molecule in AML. IL1RAP expression levels in MDS were found elevated on stem cells of patients with high risk disease, proposing a role of IL1RAP in higher risk MDS and progression to AML. Downregulation of IL1RAP expression by lentivirally expressed shRNAs decreased clonogenicity in cell lines and AML/MDS primary patient samples, induced apoptosis of AML cells, and reduced proliferation of AML cells and infiltration of hematopoietic organs in vivo. IL1RAP is a transmembrane protein required for signaling through several receptors of the IL1 family. Downregulation of IL1RAP expression in AML cells led to phosphorylation changes in several kinases and their substrates suggesting participation of IL1RAP in multiple signaling pathways and highlighting its potential as therapeutic target. We investigated whether inhibition of IL1RAP with pharmacological compounds is feasible and effective. Antibody-mediated inhibition of IL1RAP led to inhibition of AML cell growth in vitro. In addition, we designed peptides to interfere with IL1RAP-receptor interactions which lead to inhibition of AML cell growth. Both types of agents are being further tested and optimized. In summary, our study reveals IL1RAP as aberrantly expressed on HSPC of AML and high-risk MDS patients. Inhibition of IL1RAP is feasible and functionally effective, and thus has the potential to lead to novel therapies specifically directed at such stem cells. Beyond IL1RAP, our study provides a map of dysregulated transcripts in HSPC from patients with AML, which may offer further opportunities for therapeutic intervention. The strategy of comparative analysis of sorted stem and progenitor cells in cancer versus healthy controls may be applicable to other type of cancers with a suspected stem cell origin, and instrumental for the identification of targets for stem cell-directed therapy. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C225. Citation Format: Laura Barreyro, Kelly Mitchell, Britta Will, Boris Bartholdy, Li Zhou, Tihomira Todorova, Robert Stanley, Susana Ben-Neriah, Cristina Montagna, Samir Parekh, Andrea Pellagatti, Jacqueline Boultwood, Elisabeth Paietta, Rhett Ketterling, Larry Cripe, Hugo Fernandez, Peter Greenberg, Jacob Rowe, Martin Tallman, Christian Steidl, Constantine Mitsiades, Amit Verma, Ulrich Steidl. IL1RAP as functionally relevant target for stem-cell directed therapy in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C225.

Key Points Survival in ET is superior to that of PV, regardless of mutational status, but remains inferior to the sex- and age-matched US population. JAK2/CALR/MPL mutational status is prognostically informative in PMF, regarding overall and leukemia-free survival.

Key Points ALK-negative ALCLs have chromosomal rearrangements of DUSP22 or TP63 in 30% and 8% of cases, respectively. DUSP22-rearranged cases have favorable outcomes similar to ALK-positive ALCLs, whereas other genetic subtypes have inferior outcomes.

AbstractBackgroundVascular events in essential thrombocythemia (ET) are associated with advanced age and thrombosis history. Recent information suggests additional effect from the presence of specific mutations.ObjectivesTo examine the influence of age and thrombosis history on the reported association between mutational status and thrombosis‐free survival in ET.Patients and MethodsAnalysis was performed using a Mayo Clinic cohort of 300 ET patients, and key findings were reanalyzed by including additional 102 Italian patients.ResultsAmong 300 Mayo patients with ET (median age 55 yr, 60% females), mutational frequencies were 53% JAK2, 32% CALR, 3% MPL, and 12% JAK2, CALR and MPL wild type. One hundred and six (35%) patients experienced arterial (n = 75) or venous (n = 43) events, before (n = 55) or after (n = 71) diagnosis. In univariate analysis, compared to JAK2‐mutated cases, JAK2, CALR and MPL wild type (HR 0.31, 95% CI 0.11–0.86), and CALR‐mutated (0.53, 95% CI 0.30–0.92) patients displayed better thrombosis‐free survival. JAK2, CALR, and MPL wild type remained significant (P = 0.03; HR 0.32, 95% CI 0.11–0.9) during multivariable analysis that included age (P = 0.01) and thrombosis history (P = 0.0006); a favorable impact from CALR mutations was of borderline significance (P = 0.1; HR 0.62, 95% CI 0.35–1.1), but became significant (P = 0.02) when multivariable analysis including thrombosis history (P = 0.02) was performed on patients younger than 60 yr of age.ConclusionsThe favorable impact of mutational status on thrombosis‐free survival in ET might be most evident for JAK2, CALR, and MPL wild type patients, whereas the favorable effect from CALR mutations might be confined to young patients.

CALR (calreticulin) trails JAK 2 as the second most mutated gene in essential thrombocythemia (ET). Mutant CALR in ET is a result of frameshift mutations, caused by exon 9 deletions or insertions; type‐1, 52‐bp deletion (p.L367fs*46), and type‐2, 5‐bp TTGTC insertion (p.K385fs*47) variants constitute more than 80% of these mutations. The current study includes a total of 1027 patients divided into test ( n = 402) and validation ( n = 625) cohorts. Among the 402 ET patients in the test cohort, 227 (57%) harbored JAK 2, 11 (3%) Myeloproliferative leukemia virus oncogene ( MPL ), and 114 (28%) CALR mutations; 12% were wild‐type for all three mutations (i.e., triple‐negative). Among the 114 patients with CALR mutations, 51 (45%) displayed type‐1 and 44 (39%) type‐2 variants; compared to mutant JAK 2, both variants were associated with higher platelet and lower hemoglobin and leukocyte counts. However, male sex was associated with only type‐1 ( P = 0.005) and younger age with type‐2 ( P = 0.001) variants. Notably, platelet count was significantly higher in type‐2 vs. type‐1 CALR ‐mutated patients ( P = 0.03) and the particular observation was validated in the validation cohort that included 111 CALR ‐mutated ET patients ( P = 0.002). These findings, coupled with the recent demonstration of preferential expression of mutant and wild‐type CALR in megakaryocytes, suggest differential effects of CALR variants on thrombopoiesis. Am. J. Hematol. 89:E121–E124, 2014. © 2014 Wiley Periodicals, Inc.