Natasha Szuber

Mutations and prognosis in myelodysplastic syndromes: Karyotype-adjusted analysis of targeted sequencing in 300 consecutive cases and development of a genetic risk model

To develop a genetic risk model for primary myelodysplastic syndromes (MDS), we queried the prognostic significance of next‐generation sequencing (NGS)‐derived mutations, in the context of the Mayo cytogenetic risk stratification, which includes high‐risk (monosomal karyotype; MK), intermediate‐risk (non‐MK, classified as intermediate/poor/very poor, per the revised international prognostic scoring system; IPSS‐R), and low‐risk (classified as good/very good, per IPSS‐R). Univariate analysis in 300 consecutive patients with primary MDS identified TP53, RUNX1, U2AF1, ASXL1, EZH2, and SRSF2 mutations as “unfavorable” and SF3B1 as “favorable” risk factors for survival; for the purposes of the current study, the absence of SF3B1 mutation was accordingly dubbed as an “adverse” mutation. Analysis adjusted for age and MK, based on our previous observation of significant clustering between MK and TP53 mutations, confirmed independent prognostic contribution from RUNX1, ASXL1, and SF3B1 mutations. Multivariable analysis that included age, the Mayo cytogenetics risk model and the number of adverse mutations resulted in HRs (95% CI) of 5.3 (2.5‐10.3) for presence of three adverse mutations, 2.4 (1.6‐3.7) for presence of two adverse mutations, 1.5 (1.02‐2.2) for presence of one adverse mutation, 5.6 (3.4‐9.1) for high‐risk karyotype, 1.5 (1.1‐2.2) for intermediate‐risk karyotype and 2.4 (1.8‐3.3) for age >70 years; HR‐weighted risk point assignment generated a three‐tiered genetic risk model: high (Nu2009=u200965; 5‐year survival 2%), intermediate (Nu2009=u2009100; 5‐year survival 18%), and low (Nu2009=u2009135; 5‐year survival 56%). The current study provides a practically simple risk model in MDS that is based on age, karyotype, and mutations only.

Driver mutations and prognosis in primary myelofibrosis: Mayo-Careggi MPN alliance study of 1,095 patients

The 2013 discovery of calreticulin (CALR) mutations in myeloproliferative neoplasms was attended by their association with longer survival in primary myelofibrosis (PMF). Subsequent studies have suggested prognostic distinction between type 1/like and type 2/like CALR mutations and detrimental effect from triple‐negative mutational status. Among 709 Mayo Clinic patients with PMF, 467 (66%) harbored JAK2, 112 (16%) CALR type 1/like, 24 (3.4%) CALR type 2/like, 38 (5.4%) MPL mutations and 68 (10%) were triple‐negative. Survival was longer with type 1/like CALR, compared to JAK2 (HR 2.6, 95% CI 1.9‐3.5), type 2/like CALR (HR 2.5, 95% CI 1.4‐4.5), MPL (HR 1.8, 95% CI 1.1‐2.9) and triple‐negative mutational status (HR 2.4, 95% CI 1.6‐3.6), but otherwise similar between the non‐type 1/like CALR mutational states (Pu2009=u2009.41). In multivariable analysis, the absence of type 1/like CALR (Pu2009<u2009.001; HR 2, 95% CI 1.4‐2.7), presence of ASXL1/SRSF2 mutations (Pu2009<u2009.001; HR 1.9, 95% CI 1.5‐2.4) and DIPSS‐plus (Pu2009<u2009.001) were each predictive of inferior survival. Furthermore, among 210 patients with ASXL1/SRSF2 mutations, survival was significantly longer in the presence vs. absence of type 1/like CALR mutations (median 5.8 vs. 2.9 years; Pu2009<u2009.001). Triple‐negative status did not disclose additional prognostic information for overall or leukemia‐free survival. The observations regarding the prognostic distinction between CALR mutation variants were validated in an external cohort of 386 patients from the University of Florence Careggi hospital. We conclude that type 1/like CALR mutations in PMF not only predict superior survival, but also partially amend the detrimental effect of high molecular risk mutations.

Splanchnic vein thrombosis in patients with myeloproliferative neoplasms: The Mayo clinic experience with 84 consecutive cases

scans to report the visualized needle track as well as any complications. Study protocol was approved by institutional review board. The average age of the 25 study patients was 64 years, 56% were male, and the average BMI was 25.7 kg/m. All patients had successful BMP’s under local anesthesia. In 3 (12%) patients, the needle track traversed the ilium and penetrated the sacro-iliac joint and the sacrum (Figure 1). The needle track in these cases was at approximately 90 degrees to the coronal plane of the body. None of the 3 patients with sacral penetration reported significant pain or neurological symptoms. In 6 cases, the needle tracks were visualized at various angles in the ilium. In 16 scans, no needle track was seen. Of the 25 CT scans, none revealed a significant hematoma or other vascular complication. The lengths of the biopsy cores have been reported elsewhere. There is little data available on the safest techniques for the BMP. In a study on cadavers, Konda et al demonstrated that advancement of the biopsy needle perpendicular to the body’s coronal plane was associated with increased risk of injury to the iliac and iliolumbar arteries, femoral and obturator nerves, and the sacro-iliac joint, if penetration of the inner table of the ilium occurred. Retroperitoneal hemorrhage following a BMP, although uncommon, likely occurs due to traversal of the ilium by the needle. In contrast, penetration of the inner iliac cortex while advancing the biopsy needle laterally towards the ASIS is significantly less likely to result in neurovascular damage or trauma to the sacroiliac joint. It seems reasonable to conclude that fewer neurovascular injuries will occur by targeting the ASIS. In our study, 3 patients (12%) had penetration of the inner table of the ilium through the sacro-iliac joint and into the sacrum. Analysis of CT scans shows that a 9 degree change in needle angulation can lead to penetration of the sacrum (Figure 1). No adverse consequences of inadvertent penetration of the sacrum were noted in this small series. Study limitations include the relatively small sample size and lack of control for objectively comparing the technique and tools used. Despite these limitations, this study presents novel information about real-world visualization of needle tracks after a blind BMP. Further studies are needed to determine the safest and most efficacious technique for the BMP.

The impact of sex on disease phenotype and prognostic thresholds of anemia in myelodysplastic syndromes

To the Editor: Current information on sex-specific genetic (including mutations) and clinical details in myelodysplastic syndromes (MDS) is limited. Similarly, the impact of sex on the use of the conventional 10 g/dl hemoglobin threshold, for prognostication in MDS, has yet to be clarified. The original International Prognostic Scoring System (IPSS) for primary MDS included a hemoglobin level of <10 g/dl as one of its risk variables, alongside of karyotype, bone marrow blast percentage, platelet count of <100 3 10/l and absolute neutrophil count of <1.8 3 10/l). The revised IPSS (IPSS-R), published in 2012, retained anemia as an independent prognostic marker and accounted for its severity by distinguishing hemoglobin level of <8 g/dl from that of 8 to <10 g/dl. However, neither IPSS nor IPSS-R accounted for the significant differences in hemoglobin levels between men and women. We recently addressed the latter issue in the setting of primary myelofibrosis and were able to show that using a hemoglobin threshold of 10 g/dl undermined the prognostic contribution of lesser degrees of anemia in men, but not in women. In the current study, we examined the impact of sex on the distribution of mutations, cytogenetic risk and other clinical features in MDS. In addition, we queried the individual prognostic contribution of red cell transfusion need and hemoglobin level of <10 g/dl, in transfusion-independent cases, in women vs. men with MDS. The current study was approved by the institutional review board of the Mayo Clinic, Rochester, MN, USA. The diagnosis of primaryMDS was according to the 2008 World Health Organization criteria. All patients were evaluated at time of their first referral to theMayoClinic, which coincided, in most instances, with the time of initial diagnosis. Statistical analyses considered clinical and laboratory parameters obtained at time of initial diagnosis or referral. During estimation of overall survival, patients receiving allogeneic stem cell transplantation were censored at the time of their transplant. Overall survival curves were prepared by the Kaplan– Meiermethod and compared by the log-rank test. Cox proportional hazard regression model was used for multivariable analysis. P values of less than .05 were considered significant. The JMP Pro 13.0.0 software from SAS Institute, Cary, NC, USA,was used for all calculations. A total of 357 consecutive patients (median age 74 years; 70% males) with primary MDS were considered; the study population included patients from recently published documents. IPSS-R risk distribution was 11% very high, 15% high, 17% intermediate, 40% low, and 16% very low. Cytogenetic risk distribution, according to the recently described Mayo model, was 11% high, 30% intermediate, and 59% low. Next-generation sequencing-derived mutation information was available in all patients and the most frequent were SF3B1 (32%), ASXL1 (27%), TET2 (24%), U2AF1 (15%), DNMT3A (14%), SRSF2 (13%), TP53 (12%), and RUNX1 (10%). Additional data on clinical and laboratory parameters at presentation are outlined in Table 1. As comprehensively illustrated in Table 1, men with MDS were more likely to display ASXL1 and U2AF1 mutations and higher bone marrow blast percentage while their women counterparts were more likely to display a more favorable karyotype and lower risk disease by IPSS-R. Borderline significance was also noted for an association between male sex and RUNX1 mutations. Of note, we found no difference between the sexes in terms of age distribution, red cell transfusion need or hemoglobin or platelet levels (Table 1). However, although not apparent from the crude comparisons of hemoglobin levels, the incidence of anemia, defined as a hemoglobin level below the sexadjusted lower limit of the institutional reference range, was somewhat higher in males (Table 1). After a median follow-up of 14 months, 217 (61%) deaths, 35 (10%) leukemic transformations, and 19 (5%) stem cell transplants were documented. In order to clarify the individual prognostic contribution of red cell transfusion need and the conventional hemoglobin threshold of 10 g/dl, patients were stratified into 3 groups: (i) red cell transfusiondependent, (ii) transfusion-independent but with hemoglobin level of <10 g/dl, and (iii) hemoglobin level 10 g/dl. Figure 1A (female patients) and Figure 1B (male patients) illustrate the impact of sex on the prognostic value of using a hemoglobin threshold of 10 g/dl, in otherwise transfusion-independent cases. In women, the only thing that mattered for survival, in regards to hemoglobin level, was whether or not the patient was transfusion-dependent. In other words, the hemoglobin threshold of 10 g/dl did not predict survival in transfusion-independent womenwithMDS and the overall scenario was not affected by lowering the hemoglobin threshold to 9 g/dl (data not shown). In univariate analysis of survival in men with MDS, the presence of transfusion need, as well as hemoglobin level of <10 g/dl, in transfusion-independent cases, were associated with inferior survival (Figure 1B). However, in multivariable analysis that included the recently described age-adjusted genetic risk model for MDS, the significance of using the hemoglobin threshold of <10 g/dl, in transfusion-independent cases, was no longer apparent, while transfusion need retained its significance, in both men and women with MDS (data not shown). The current study highlights a number of novel observations. First, women with MDS, compared to their male counterparts, were less likely to harbor adverse genetic features, including ASXL1, U2AF1, and

Myeloproliferative neoplasms in the young: Mayo Clinic experience with 361 patients age 40 years or younger

Between 1967 and 2017, 361 patients with myeloproliferative neoplasms (MPN), ageu2009≤u200940u2009years, were seen at our institution, constituting 12% of all MPN patients (nu2009=u20093023) seen during the same time period; disease‐specific incidences were 12% in polycythemia vera (PV; nu2009=u200979), 20% in essential thrombocythemia (ET; nu2009=u2009219) and 5% in primary myelofibrosis (PMF; nu2009=u200963). Compared to their older counterparts, younger patients were more likely to present with low risk disease (Pu2009<u2009.001) and display female preponderance in ET (Pu2009=u2009.04), lower incidence of arterial events overall (Pu2009<u2009.001), and higher incidence of venous thrombosis in PV (Pu2009=u2009.01). Younger patients were also more likely to express CALR mutations, in ET and PMF, normal karyotype, in PV and PMF, and lower incidence of high molecular risk mutations in PMF (P significant in all instances). Over median follow‐up of 11.3, 13, and 7.1 years for PV, ET, and PMF, leukemic transformations were respectively documented in 4%, 2%, and 10% (P values 0.1‐0.9) while incidences of fibrotic progression in PV (22%) and ET (16%) were expectedly higher in young patients, because of their longer survival (Pu2009<u2009.001). Median survival in young patients was 37u2009years for PV, 35 for ET and 20 for PMF; the corresponding values were 22, 22, and 8u2009years for ages 41‐60u2009years and 10, 11, and 3u2009years for ages >60u2009years (Pu2009<u2009.001). Young MPN patients comprise a unique disease subset defined by an attenuated‐risk cytogenetic and mutational backdrop and conspicuously longer survival compared to their older counterparts, which requires assertion during patient counseling.

Determinants of long-term outcome in type 1 calreticulin-mutated myelofibrosis

1016/S1470-2045(12)70200-0. 9. Ruan J, Martin P, Shah B, Schuster SJ, Smith SM, Furman RR, et al. Lenalidomide plus rituximab as initial treatment for mantlecell lymphoma. N Engl J Med. 2015;373:1835–44. 10. Zinzani PL, Pellegrini C, Gandolfi L, Stefoni V, Quirini F, Derenzini E, et al. Combination of lenalidomide and rituximab in elderly patients with relapsed or refractory diffuse large B-cell lymphoma: a phase 2 trial. Clin Lymphoma Myeloma Leuk. 2011. https://doi.org/10.1016/j.clml.2011.02.001. 11. Ivanov V, Coso D, Chetaille B, Esterni B, Olive D, AurranSchleinitz T, et al. Efficacy and safety of lenalinomide combined with rituximab in patients with relapsed/refractory diffuse large Bcell lymphoma. Leuk Lymphoma 2014. https://doi.org/10.3109/ 10428194.2014.889822. 12. Wang M, Fowler N, Wagner-Bartak N, Feng L, Romaguera J, Neelapu SS, et al. Oral lenalidomide with rituximab in relapsed or refractory diffuse large cell, follicular and transformed lymphoma: a phase II clinical trial. Leukemia. 2013. https://doi.org/10.1038/ leu.2013.95. 13. Zinzani PL, Pellegrini C, Argnani L, Broccoli A. Prolonged disease-free survival in elderly relapsed diffuse large B-cell lymphoma patients treated with lenalidomide plus rituximab. Haematologica. 2016;101:e385–6. 14. Morschhauser F, Salles G, Le Gouill S, Tilly H, Thieblemont C, Bouabdallah K, et al. An open-label, phase Ib study of obinutuzumab plus lenalidomide in relapsed/refractory follicular B-cell lymphoma. Blood. 2018. https://doi.org/10.1182/blood-2018-05853499. 15. Cheson BD, Horning SJ, Coiffier B, Shipp MA, Fisher RI, Connors JM, Lister TA, Vose J, Grillo-López A, Hagenbeek A, Cabanillas F, Klippensten D, Hiddemann W, Castellino R, Harris NL, Armitage JO, Carter W, Hoppe R, Canellos GP. J Clin Oncol. PMID: 10561185. https://doi.org/10.1200/JCO. 1999.17.4.1244. 16. Crump M, Neelapu SS, Farooq U, Van Den Neste E, Kuruvilla J, Westin J, et al. Outcomes in refractory diffuse large B-cell lymphoma: results from the international SCHOLAR-1 study. Blood. 2017;130:1800–8. 17. Vitolo U, Trněný M, Belada D, Burke JM, Carella AM, Chua N, et al. Obinutuzumab or rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone in previously untreated diffuse large B-cell lymphoma. J Clin Oncol. 2017;35:3529–37. JCO.2017.73.340.

CSF3R-mutated chronic neutrophilic leukemia: Long-term outcome in 19 consecutive patients and risk model for survival

Chronic neutrophilic leukemia (CNL) is a rare BCRABL negative myeloproliferative neoplasm (MPN), whose molecular pathogenesis transitioned from obscurity to the limelight with the seminal identification of oncogenic colony stimulating factor 3 receptor (CSF3R) mutations in the vast majority of CNL patients in 2013. The unifying features of CNL consist of sustained mature neutrophil proliferation, bone marrow granulocytic hyperplasia, and hepatosplenomegaly, and while clinical manifestations and disease course remain heterogeneous, prognosis is often unfavorable. Due to both the historically challenging diagnostic confirmation and the rarity of CNL, comprehensive analyses of World Health Organization (WHO)-defined, molecularly annotated populations of CNL patients have been scarce and often limited by small cohorts. Although the more common CSF3RT618I is perceived as the “hallmark” genetic lesion, there is little information on how individuals harboring other CSF3R mutations may be distinguished from their CSF3RT618Imutated counterparts. Furthermore, while there is some evidence that variables such as high leukocyte count, thrombocytopenia, and the presence ASXL1 mutations may define a higher-risk subset of CNL patients, there are limited data on prognostic factors and long-term survival in CNL and consequently, no existing operational risk model or prognostic scoring system. The objective of this study was to determine the characteristics, treatment patterns, and long-term overall survival (OS) in 19 consecutive WHO-defined CSF3R-mutated CNL patients —the largest cohort to date. Furthermore, we sought to identify and integrate adverse prognostic factors into a risk model predictive of inferior overall survival in CNL. Nineteen consecutive patients evaluated at the Mayo Clinic harboring CSF3R mutations and meeting WHO criteria for CNL were retrospectively identified. Details of clinical characteristics, laboratory parameters including molecular and cytogenetics data, treatment regimens and responses, as well as disease evolution were carefully abstracted from medical records. Diagnosis was confirmed in all cases by review of peripheral blood counts and smears, and bone marrow (BM) aspirates and biopsies. Mutation analysis of CSF3R, ASXL1, and SETBP1 was conducted as previously described . Analyses were based on clinical and laboratory parameters obtained at diagnosis. OS was calculated as an interval from the time of diagnosis to last follow-up or death. Survival analysis was performed by the Kaplan–Meier method and differences assessed using the log-rank test. Conventional statistical methods were used for all analyses. Statistical analyses were performed using Stat View software (SAS Institute, Cary, NC, USA). From October 1995 to October 2017, 19 consecutive WHO-defined CSF3R-mutated patients were evaluated at the Mayo Clinic. Detailed clinical and laboratory characteristics of this cohort are presented in Table 1. Mutations in CSF3R included the classical T618I mutation in 14 patients and other mutations including M696T in 2 patients and T640N, c.2215C>T truncation mutation, and I598I SYN in one patient each. Median age was 68 (range 26–87 years) and there was a slight preponderance

Chronic neutrophilic leukemia: new science and new diagnostic criteria

Chronic neutrophilic leukemia (CNL) is a distinct myeloproliferative neoplasm defined by persistent, predominantly mature neutrophil proliferation, marrow granulocyte hyperplasia, and frequent splenomegaly. The seminal discovery of oncogenic driver mutations in CSF3R in the majority of patients with CNL in 2013 generated a new scientific framework for this disease as it deepened our understanding of its molecular pathogenesis, provided a biomarker for diagnosis, and rationalized management using novel targeted therapies. Consequently, in 2016, the World Health Organization (WHO) revised the diagnostic criteria for CNL to reflect such changes in its genomic landscape, now including the presence of disease-defining activating CSF3R mutations as a key diagnostic component of CNL. In this communication, we provide a background on the history of CNL, its clinical and hemopathologic features, and its molecular anatomy, including relevant additional genetic lesions and their significance. We also outline the recently updated WHO diagnostic criteria for CNL. Further, the natural history of the disease is reviewed as well as potential prognostic variables. Finally, we summarize and discuss current treatment options as well as prospective novel therapeutic targets in hopes that they will yield meaningful improvements in patient management and outcomes.

Development of a prognostically relevant cachexia index in primary myelofibrosis using serum albumin and cholesterol levels

Key Points Serum albumin and cholesterol levels predict survival in primary myelofibrosis, independent of each other and contemporary risk models. The cachexia index, determined by serum albumin and cholesterol levels, might further refine current prognostic models in myelofibrosis.

Decreased survival and increased rate of fibrotic progression in essential thrombocythemia chronicled after the FDA approval date of anagrelide

First‐line cytoreductive drug of choice in high risk essential thrombocythemia (ET) is currently hydroxyurea, a practice based on the results of a randomized study; second‐line drugs of choice include pegylated interferon‐α, busulfan and anagrelide. Anagrelide clinical trials were pioneered by the late Murray N. Silverstein (1928‐1998) of the Mayo Clinic whose studies led to FDA approval in March 1997. The current study represents a retrospective examination of the potential impact of anagrelide therapy on survival and disease complications in ET. 1076 patients with ET were considered (median age 58u2009years; females 63%); risk distribution, according to the international prognostic score for ET (IPSET), was 28% high, 42% intermediate, and 30% low. Overall (OS), myelofibrosis‐free (MFFS) and thrombosis‐free survival data were compared for ET patients diagnosed before and after the 1997 FDA approval date for anagrelide; a significant difference was apparent in OS (Pu2009=u2009.006; HR 1.4, 95% CI 1.1‐1.7) and MFFS (Pu2009<u2009.001; HR 4.2, 95% CI 2.7‐6.5), in favor of patients diagnosed prior to 1997; the difference was sustained during multivariable analysis that included IPSET. Similarly stratified survival data in polycythemia vera (nu2009=u2009665) and primary myelofibrosis (nu2009=u20091282) showed no similar impact on survival (Pu2009=u2009.3 and .17, respectively). The current study represents a retrospective analysis and suggests significantly decreased OS and MFFS in ET patients diagnosed after the FDA approval date of anagrelide. Whether or not anagrelide therapy was to blame for the worsening of OS and MFFS over time cannot be assumed and requires validation in a prospective study.