Impact of clone size with a single cytogenetic abnormality on the revised International Prognostic Scoring System in myelodysplastic syndromes
Authors: Omar Alkharabsheh, Salwa S. Saadeh, Mrinal S. Patnaik, Hassan Alkhateeb, Naseema Gangat, Kebede H. Begna, William J. Hogan, Patricia T. Greipp, Rong He, Phuong L. Nguyen, Mark R. Litzow, Aref Al‐Kali
Journal: American Journal of Hematology (2018)

Myelodysplastic syndromes (MDSs) are a group of heterogeneous hematopoietic clonal stem cell disorders defined by cytopenia and dysplasia. There are multiple classification systems that aid in risk stratification and subsequently treatment selection in MDS. The most common one is the International Prognostic Scoring System (IPSS), this had been revised (IPSS-R) in 2012 to include more cytogenetic data.1 In IPSS-R, cytogenetics has the highest score compared to the other variables, such as bone marrow blasts percentage, degree of cytopenia, anemia, thrombocytopenia, and neutropenia. In addition, cytogenetic testing in this scoring systems is conducted by conventional karyotyping. Karyotyping is considered adequate when the analyzed number of metaphases is 20.2 A clone in karyotyping is defined by a group of cells from a single progenitor that share a similar genetic aberration.3 The International System for Human Cytogenetic Nomenclature (ISCN) in 2016 specified a clone by the presence of a structural rearrangement in at least two metaphases, or in the case of monosomies, three metaphases.3 With the wide range of heterogeneity of chromosomal aberrations that can develop in MDS, we aimed to investigate them by the concept of clone size (ie, number of abnormal metaphases in a conventional karyotype) and its effect on survival as compared to normal karyotype in patients with MDS. We conducted a retrospective study analyzing records of patients seen at Mayo Clinic from October 1993 until March 2016. All patients had a bone marrow biopsy performed at diagnosis that was reviewed by our hematopathologists. Data extraction included complete blood count at diagnosis, bone marrow blast percentage, conventional cytogenetics testing, date of diagnosis, and date of death or date of last follow-up. The Institutional Review Board approved this study. Risk stratification assessment using IPSS-R was calculated, including the total score and cytogenetic category. Overall survival (OS) was calculated from the date of diagnosis until death or last follow-up. Patients with karyotypes that had 20 metaphases only were included. Because of the complexity of analysis, we only looked at patients with a single clone. We classified patients into five groups based on their IPSS-R cytogenetic category: very good, good, intermediate, poor, and very poor. From the good group, we selected patients with a normal karyotype as the comparator arm. Composite chromosomes (variation from cell to cell in the clone abnormality) were allowed if the IPSS-R cytogenetic category was very poor. Regarding IPSS-R cytogenetic categories, the good group other than normal karyotypes, were analyzed for survival difference within each clone at different size cutoffs. The very poor group included single-clone complex karyotypes with more than three abnormalities. The poor group included single del(3q), single monosomy 7 or double −7/7q−, and single-clone complex karyotypes with three abnormalities only. The intermediate group included +8, del(7q), +19, i(17q), any other single clones, or a double clone not including del(5q) or monosomy 7/del(7q). Clone size was calculated by dividing the number of abnormal metaphases by 20 and multiplying by 100%, resulting in an abnormal single clones ranging from 5%, 10%, 15%, 20%, and so forth, up to 100%. Kaplan-Meier estimates were used to calculate OS using log-rank testing. In addition, we investigated this concept at the level of single clones with a single chromosomal abnormality but with a wider range (25, 50, 75, and 100%) because of the small number of patients. We analyzed 1300 patients with MDS confirmed by bone marrow biopsy and aspirate with available cytogenetic data for inclusion and exclusion criteria. Supporting Information Table S1 summarizes the baseline characteristics of the entire cohort. Of this cohort, 843 patients met our criteria. Of the 843 patients, 475 (56%) were normal karyotype, and 368 (44%) had a single-clone abnormality. Supporting Information Table S2 highlights the differences between these two groups and Supporting Information Figure S1 shows their median OS difference. Median OS was 36.4, 54.0, 30.3, 21.1, and 8.7 months for very low, low, intermediate, high, and very high IPSS-R risk scores, respectively (P < .001; Supporting Information Figure S2). In analyzing every cutoff level, starting from 5% up to 100%, 20 levels showed that normal karyotype survival was statistically higher when compared to higher risk scores by IPSS-R cytogenetic category except for the 5% level. Looking at cutoff levels within each group did not show statistical significance. However, the poor group results were not consistent, secondary to low number of patients (Table 1). On multivariate analysis, clone size did not predict survival independent of IPSS-R risk score. We assessed survival for patients with a single chromosomal abnormality according to clone size at cutoff values of 25, 50, 75, and 100% (Supporting Information Table S3). Clone size did not affect survival in any of the groups. One observation found, in patients with trisomy 8, that the higher the clone cutoff value percentage, the better OS. This observation was not seen across other intermediate IPSS-R cytogenetics categories. The goal of this study was to investigate the concept of clone size as defined by the ISCN 2016 and its impact on the IPSS-R survival model. Our hypothesis depended on available data from karyotyping (ie, percentage of abnormal metaphases), and it did not require more sophisticated cytogenetic or molecular laboratory testing to risk stratify patients with MDS. Clone size or percent of abnormal metaphases are complex concepts for the cytogenetics community because of a number of variables that affect the process of reporting results. For example, the number of cells that were cultured, single or multiple clones, stemline or sideline clones, and composite chromosomes are variables that, after being integrated with morphologic and molecular results, factor into the final impression and diagnosis.3 In our analysis, we compared patients based on their IPSS-R cytogenetic groups to increase the cohort number and to make statistical analysis feasible. All groups were compared to the normal karyotype to examine whether patients with small clones behaved like the normal karyotype in terms of survival, making this abnormality clinically not significant. Our results are consistent with the ISCN 2016 definition of a clone. Clone size of 5% represents one abnormal metaphase only. Statistical significance was set at 10% (P < .02). Also, we performed an analysis within each group of single clone that was characterized by a single karyotype abnormality. Those represented every single MDS-defining cytogenetic abnormality. In this intergroup analysis, we used a wider range of cutoff (25, 50, 75, and 100%) because of the small number of patients in many groups. The results, as shown in Supporting Information Table S3, did not indicate a difference in median OS between these clone percentages. The concept of clone size in MDS and its impact on survival outcomes was investigated previously by Mello and his colleges who reported no impact of clone size in −7/7q survival but significant OS difference in 5q and trisomy 8 at 100% clone size versus <100%.4 Also, measuring clone size was performed using fluorescent in situ hybridization (FISH) by counting the number of abnormal interphase nuclei identified with each probe,5 they investigated 81 MDS cases at diagnosis and 28 at follow-up, and they found worse outcomes and higher risk of progression to acute myeloid leukemia with larger clone size (more than 20%) by FISH. However, these results need to be validated in a larger cohort. In addition, with the discovery of drivers mutation for several myeloid malignancies, such as JAK2, TET2, SF3B1, TP53, and others in myeloid neoplasms, many investigators have studied the clone mutant allele burden and its effects on disease phenotype and outcomes.6 There were a number of limitations to our study. The first limitation was the low number of patients with small clone size, especially at the 5%-10% level, and lack of FISH data at the 5% level to confirm or exclude clonality or culture aberrance. The second limitation was including patients from 1993 when next generation sequencing was not available to better define the cohort and correlate our data with modern diagnostic techniques. We concluded that the presence of a clone as defined by the ISCN 2016 is clinically significant regardless of the number of abnormal metaphases in the karyotype. More research is needed to evaluate this concept further. The authors declare that they have no conflicts of interest with the contents of this article. Table S1 Baseline Characteristics for All MDS Patients (N = 1300) Table 2: Characteristics Differences Between Patients With Normal Karyotype and Single-Clonal Abnormality (N = 843) Table 3: Single Clone With 1 Chromosomal Abnormality Survival Difference at 25%, 50%, 75%,and 100% Cutoff Clone Percentage Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.