Partial tandem duplication of KMT2A (MLL) may predict a subset of myelodysplastic syndrome with unique characteristics and poor outcome

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1 Published Ahead of Print on January 19, 2018, as doi: /haematol Copyright 2018 Ferrata Storti Foundation. Partial tandem duplication of KMT2A (MLL) may predict a subset of myelodysplastic syndrome with unique characteristics and poor outcome by Sarah M. Choi, Rajan Dewar, Patrick W. Burke, and Lina Shao Haematologica 2018 [Epub ahead of print] Citation: Sarah M. Choi, Rajan Dewar, Patrick W. Burke, and Lina Shao. Partial tandem duplication of KMT2A (MLL) may predict a subset of myelodysplastic syndrome with unique characteristics and poor outcome. Haematologica. 2018; 103:xxx doi: /haematol Publisher's Disclaimer. E-publishing ahead of print is increasingly important for the rapid dissemination of science. Haematologica is, therefore, E-publishing PDF files of an early version of manuscripts that have completed a regular peer review and have been accepted for publication. E-publishing of this PDF file has been approved by the authors. After having E-published Ahead of Print, manuscripts will then undergo technical and English editing, typesetting, proof correction and be presented for the authors' final approval; the final version of the manuscript will then appear in print on a regular issue of the journal. All legal disclaimers that apply to the journal also pertain to this production process.

2 Partial tandem duplication of KMT2A (MLL) may predict a subset of myelodysplastic syndrome with unique characteristics and poor outcome Running Title: KMT2A (MLL)-PTD defines a high risk MDS subgroup Sarah M. Choi, 1 Rajan Dewar, 1 Patrick W. Burke, 2 Lina Shao 1 1 Department of Pathology, University of Michigan, Ann Arbor, MI; 2 Department of Internal Medicine, University of Michigan, Ann Arbor, MI Corresponding Author: Sarah M. Choi 5242A Med Sci I 1301 Catherine Street Ann Arbor, Michigan choism@med.umich.edu Phone: Fax: Word counts Text: 1218 Abstract: N/A Figure count: 1 Table count: 1 Reference count: 15

3 Partial tandem duplication (PTD) of the KMT2A (MLL) gene is detected in approximately 5-10% of cases of acute myeloid leukemia (AML) 1-5 and within cases showing normal, confer a worse prognosis. 2,6,7 In these patients, the duplications are variable in size and most commonly involve exons 2 or 3, spanning through exon 6 or exons Unlike many other translocations involving the KMT2A gene, MLL-PTD cannot be detected using conventional cytogenetics. 8 Other methods, such as the cytogenomic array, can be used to identify patients with this genetic alteration, who may be potential candidates for therapy (eg demethylating agents, histone deacetylase inhibitors). 3,9 As in AML, MLL-PTD can also be seen in a subset of patients with myelodysplastic syndrome (MDS) and its acquisition has observed during leukemic transformation, 4,10 however, the clinical significance of MLL-PTD at diagnosis, particularly with regard to survival, has not been well characterized in these patients. In this study, we describe unique pathologic and clinical characteristics in a series of MDS patients with MLL-PTD and analyze the impact of MLL-PTD on therapy response and clinical outcomes. We reviewed the records of consecutive patients with a diagnosis of MDS who had cytogenomic microarray studies performed between March 2014-June 2017 (n=83) Cases with MLL-PTD were identified and compared to a control cohort of patients (n=38) without MLL-PTD also derived from the same original cohort (Supplemental Tables S1-S2). Cases of AML with MLL- PTD were also reviewed. All patients had been evaluated and treated at the same institution during the same time period. Overall Survival (OS) was calculated from the diagnosis date to date of death, censoring for patients alive at the time of study completion. Additional methods are described in Supplemental Methods. A total of 14 cases with MLL-PTD were identified by the cytogenomic microarray (Table 1). Excluding patients with therapy-related myeloid neoplasms, MLL-PTD was detected in 6-7% of patients with MDS that were screened by the array during the same time period. These patients were predominantly male, ranging in age from years of age at diagnosis (mean 59.3 years, median 66.9 years).

4 The majority of MLL-PTD MDS cases were classified as MDS with excess blasts (MDS-EB; by WHO 2016 criteria), 11 showed normal by conventional cytogenetics, and had variable IPSS-R scores ranging from low to very high (Table 1). Cases of MLL-PTD presented with lower absolute neutrophil count (p=0.012) and platelet count (p=0.046) compared to non-mll- PTD MDS cases with high-very high IPSS-RA, but comparable hemoglobin and bone marrow blast percentage (Figure 1A). Flow cytometric immunophenotyping did not reveal specific immunophenotypic aberrations distinguishing MLL-PTD cases from non-mll-ptd cases (data not shown). MLL-PTD MDS patients showed worse OS compared to MDS patients without MLL-PTD, even when compared to those with high-very high IPSS-RA (Figure 1B, p<0.0001; Figure 1C, p=0.002). The median OS for MLL-PTD MDS was 9.85 months from the time of diagnosis compared to 31.5 months for the high-very high IPSS-RA group. OS was also worse when comparing cases of MDS with poor-very poor cytogenetic features, which had a median OS of 18.2 months (Figure 1D, p=0.027). MLL-PTD MDS patients, with and without acute leukemic transformation, also showed worse OS compared to patients with MLL-PTD who presented with de novo AML (Figure 1E, p=0.043). All four patients with de novo AML were alive at the conclusion of this study. Patients with MLL-PTD MDS who transformed to AML as well as de novo MLL-PTD AML received either or FLAG 13 induction therapy (Table 1). Three of the five MLL-PTD patients who received 7+3 induction therapy showed residual disease in day 14 marrows, whereas two de novo AML cases, both of which also had a FLT3- ITD mutation, achieved CR and were subsequently transplanted. All cases that failed initial 7+3 induction therapy showed complete response to FLAG re-induction therapy. One patient with t- MDS failed initial FLAG induction therapy. Two additional MDS patients received initial FLAG induction therapy but passed away due to infection before disease response evaluation could be completed.

5 All MLL-PTD patients who proceeded to transplant were still alive at the conclusion of this study, except for one patient with t-mds who relapsed with AML (Table 1). All patients who did not receive a transplant passed away with the longest survival time of 16.7 months. The single MLL-PTD MDS patient who received a transplant remains alive at the time of this writing. This study identifies a subset of MDS defined by the presence of MLL-PTD that is associated with more advanced disease with excess blasts and worse outcome, compared to non-mll PTD MDS, even those with high risk IPSS scores and complex. Though the sample size is small, the effect size is pronounced and statistically significant in all comparisons. The overall prevalence of MLL-PTD in MDS (6-7% of cases) is similar to that observed in AML and slightly higher than reported in a previous study of mutational analysis in MDS. 10 It is of note that the control cohort of high and very high risk IPSS-RA in our study shows a better median overall survival of 31.5 months than has been reported in the larger multinational IPSS-R study, which showed a median survival of 19 months for high risk and 10.8 months for very high risk groups. 14 This finding could potentially be reflective of differences in therapy and clinical practice or of the smaller numbers of patients in this single institution review. It is also significant because despite improved survival of high and very high risk MDS patients being treated at the same institution during the same time interval, patients with MLL-PTD still fared very poorly. Furthermore, their median survival of 9.85 months is even lower than very high risk MDS patients as predicted by the IPSS-R study. 14 This finding suggests that the finding of MLL- PTD in an MDS patient is indeed a very poor prognostic factor and taken a step further, these patients may potentially benefit from expeditious transplantation if feasible. Supporting this is the observation that all patients with MLL-PTD who received a transplant (other than those with therapy-related myeloid neoplasms) were still alive at the conclusion of this study. For those patients who developed MLL-PTD AML, in cases where 7+3 induction chemotherapy was not successful, complete response was achievable in most cases treated with FLAG reinduction therapy. The presence of additional FLT3-ITD mutation may possibly predict a better response to 7+3 induction therapy; however, the sample population is too small to draw a definitive conclusion. Co-occurrence of FLT3-ITD and MLL-PTD has been reported previously in AML, though the significance of this finding on outcome or response is currently unknown. 15

6 The potential use of demethylating agents and histone deacetylase inhibitors, as has been proposed for cases of MLL-PTD AML, 3,9 is one area of further exploration. Of note, de novo MLL-PTD AML patients, which exclude patients with underlying MDS, showed the best outcomes of patients in this study, possibly due to early transplantation, and may have better prognosis than has been previously reported. In summary, our findings suggest that MLL-PTD MDS presents as high grade disease with excess blasts and typically normal. Testing for MLL-PTD might be considered in all patients with MDS, and its discovery could potentially warrant consideration for early transplantation. Because the size of our sample is limited, additional studies on a larger cohort of patients to further define the features of this subgroup and delineate appropriate therapy are needed.

7 Acknowledgments Not applicable. Authorship Contributions Contribution: S.M.C. and L.S. designed the study and collected and analyzed the data. P.B. also analyzed the data. S.M.C., R.D., P.B., and L.S. wrote and/or edited the manuscript. Conflict-of-interest disclosure The authors declare no competing financial interests.

8 References 1. Schnittger S, Kinkelin U, Schoch C, et al. Screening for MLL tandem duplication in 387 unselected patients with AML identify a prognostically unfavorable subset of AML. Leukemia. 2000;14(5): Döhner K, Tobis K, Ulrich R, et al. Prognostic Significance of Partial Tandem Duplications of the MLL Gene in Adult Patients 16 to 60 Years Old With Acute Myeloid Leukemia and Normal Cytogenetics: A Study of the Acute Myeloid Leukemia Study Group Ulm. J Clin Oncol. 2002;20(15): Whitman SP, Liu S, Vukosavljevic T, et al. The MLL partial tandem duplication: evidence for recessive gain-of-function in acute myeloid leukemia identifies a novel patient subgroup for molecular-targeted therapy. Blood. 2005;106(1): Steudel C, Wermke M, Schaich M, et al. Comparative analysis of MLL partial tandem duplication and FLT3 internal tandem duplication mutations in 956 adult patients with acute myeloid leukemia. Genes Chromosomes Cancer. 2003;37(3): Strout MP, Marcucci G, Bloomfield CD, Caligiuri MA. The partial tandem duplication of ALL1 (MLL) is consistently generated by Alu-mediated homologous recombination in acute myeloid leukemia. Proc Natl Acad Sci U S A. 1998;95(5): Shiah HS, Kuo YY, Tang JL, et al. Clinical and biological implications of partial tandem duplication of the MLL gene in acute myeloid leukemia without chromosomal abnormalities at 11q23. Leukemia. 2002;16(2): Caligiuri MA, Strout MP, Lawrence D, et al. Rearrangement of ALL1 (MLL) in Acute Myeloid Leukemia with Normal Cytogenetics. Cancer Res. 1998;58(1): Schichman SA, Caligiuri MA, Gu Y, et al. ALL-1 partial duplication in acute leukemia. Proc Natl Acad Sci U S A. 1994;91(13): Kühn MWM, Hadler MJ, Daigle SR, et al. MLL partial tandem duplication leukemia cells are sensitive to small molecule DOT1L inhibition. Haematologica. 2015;100(5):e190-e Dicker F, Haferlach C, Sundermann J, et al. Mutation analysis for RUNX1, MLL-PTD, FLT3-ITD, NPM1 and NRAS in 269 patients with MDS or secondary AML. Leukemia. 2010;24(8): Arber DA, Orazi A, Hasserjian R, et al. The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia. Blood. 2016;127(20): Fernandez HF, Sun Z, Yao X, et al. Anthracycline Dose Intensification in Acute Myeloid Leukemia. N Engl J Med. 2009;361(13): Jackson G, Taylor P, Smith GM, et al. A multicentre, open, non-comparative phase II study of a combination of fludarabine phosphate, cytarabine and granulocyte colonystimulating factor in relapsed and refractory acute myeloid leukaemia and de novo refractory anaemia with excess of blasts in transformation. Br J Haematol. 2001;112(1): Greenberg PL, Tuechler H, Schanz J, et al. Revised international prognostic scoring system for myelodysplastic syndromes. Blood. 2012;120(12): Kihara R, Nagata Y, Kiyoi H, et al. Comprehensive analysis of genetic alterations and their prognostic impacts in adult acute myeloid leukemia patients. Leukemia. 2014;28(8):

9 Table 1. Clinicopathologic characteristics of MLL-PTD cases Age at diagnosis, years Sex Disease category at diagnosis MLL-PTD Cytogenetics Molecular analysis results Prognostic score and category Therapy OS, months IPSS-R IPSS-RA Supportive Hypomethylating agent Induction Transplant 7+3 FLAG Alive at end of study MLL-PTD MDS Patient M MDS-MLD exons 2-10; 24 kb Normal Patient F MDS-EB-1 exons 2-8 on one allele; 347 kb including entire gene on other allele Normal Patient M MDS-EB-2 exons 2-5; 11 kb Normal Negative for JAK2 V617F mutation. 2 low 0.8 very low X Negative for FLT3 ITD and D835, IDH1/2 mutations. Negative for FLT3 ITD and D835 mutation. 6 high 5.97 high X high 4.94 high X X X Patient M MDS-EB-2 exons 2-10; 17 kb Normal MLL-PTD MDS with subsequent AML transformation Patient M MDS-EB-2 exons 2-10; 21 kb Normal Negative for FLT3 ITD and D835, and CEPBA mutations. Negative for FLT3 ITD and D835, CEPBA, IDH1/2, NPM1 mutations. 7 very high 6.64 very high X intermediate 4.4 intermediate X (failed) X Patient M MDS-EB-2 exons 2-10; 33 kb Gain of 4 Positive for IDH2 R172K. Negative for FLT3 ITD and D835, CEPBA, IDH1, NPM1, and KIT mutations. 6.5 very high 6.46 very high X X 1.81 MLL-PTD therapy-related MDS (t-mds) Patient 7 (Later transformed to AML) 70.2 F t-mds MDS-EB-1 exons 2-10; 17 kb Normal Negative for FLT3 ITD and D835, CEPBA, IDH1/2, NPM1 mutations. 5 high 5.01 high X X Patient 8 (Later transformed to AML) 64.9 F t-mds-mld exons 2-8; 16 kb Normal Positive for FLT3 D835 and IDH1 R132F. Negative for FLT3 ITD and IDH2 mutations. 3.5 intermediate 3.33 intermediate X (failed) 2.30 MLL-PTD de novo AML Patient F AML exons 2-8; 16 kb Inadequate Negative for FLT3 ITD and D835, CEPBA, IDH1/2, NPM1 mutations. X (failed) X X X Patient M AML exons 2-10; 21 kb Normal Patient F AML exons 2-8; 17 kb Normal Negative for FLT3 ITD and D835, CEPBA, IDH1/2, NPM1 mutations. Positive for FLT3 ITD and D835. Negative for CEBPA, IDH 1/2, NPM1, KIT, BCR-ABL, PML-RARa. X (failed) X X X X X X Patient F AML exons 2-10; size unspecified Trisomy 11 Positive for FLT3 ITD. Negative for CEPBA, IDH 1/2, NPM1 mutations. MLL-PTD detected at AML transformation from MPN or MDS/MPN X X X Patient 13 (History of PMF) 67.6 M PMF exons 3-5; 9 kb Normal Positive for FLT3 ITD. Negative for FLT3 D835, IDH 1/2 mutation. X 0.66 Patient 14 (History of CMML) 60.3 M CMML exons 2-8; 16 kb Normal Negative for FLT3 ITD and D835. Negative for IDH 1/2 and JAK2 V617F mutation. X (residual CMML) X X PTD indicates partial tandem duplication ; OS, overall survival; MDS, myelodysplastic syndrome; ITD, internal tandem duplication; AML, acute myeloid leukemia; PMF, primary myelofibrosis; and CMML, chronic myelomonocytic leukemia

10 Figure 1. MLL-PTD MDS presents with lower absolute neutrophil and platelet counts and displays worse prognosis than high risk non-mll-ptd MDS. (A) Hemoglobin, absolute neutrophil count, platelet count, and bone marrow blast percentage were compared between MLL-PTD MDS and non-mll-ptd MDS with high-very high IPSS-RA score [mean value and standard error of the mean are depicted; *(p=0.012) **(p=0.046)]. (B) Survival analysis comparing MLL-PTD MDS to non-mll-ptd MDS based on IPSS-RA prognostic group (p<0.0001). (C) Survival analysis comparing MLL-PTD MDS to high or very high risk non- MLL-PTD MDS based on IPSS-RA score (p=0.002). (D) Survival analysis comparing MLL- PTD MDS to non-mll-ptd MDS with poor-very poor cytogenetic and good cytogenetic groups (p=0.027). (E) Survival analysis comparing MLL-PTD MDS, transformed MLL-PTD MDS, and de novo MLL-PTD AML cases (p=0.043).

11

12 Supplemental Methods: Patients: This study was approved by the Institutional Review Board at the University of Michigan. Clinical and pathology records were obtained through retrospective review of the electronic medical chart. Prior history of chemotherapy or radiation was noted, along with laboratory values at diagnosis [hemoglobin, absolute neutrophil count (ANC), platelet count, bone marrow blast percentage] (Table S1-S2). The clinical and treatment course were also recorded (transformation to AML, transplant status, chemotherapeutic regimen used). Subtype of MDS (WHO 2016 criteria) 1 at the time of diagnosis as well as flow cytometric immunophenotyping results were also catalogued. Conventional cytogenetics/ results were also recorded. A prognostic score was calculated using the revised International Prognostic Scoring System without and with age adjustment (IPSS-R and IPSS-RA, respectively). 2 Overall Survival (OS) was calculated from the diagnosis date to date of death, censoring for patients alive at the time of study completion. The control cohort (Table S2) was comprised of patients with low (n=3), intermediate (n=5), high (n= 13), very high (n=17) IPSS-RA scores. There were 12 patients with complex of > 3 abnormalities, 11 patients with other abnormalities, and 15 patients with normal. In terms of pathologic diagnosis, 27 patients had MDS with excess blasts and 11 had MDS with multilineage dysplasia. Cytogenetics and cytogenomic array: MLL-PTD was detected by whole genome Affymetrix CytoScan array analysis in all the cases as described previously. 3 All cases with available were stratified according to the Comprehensive Cytogenetic Scoring System of the Revised International Prognostic Scoring System. Molecular analysis: Mutational analysis of NPM1, FLT3, JAK2 V617F and CEPBA were performed as previously described. 4-8 KIT and IDH1/2 mutation analysis was done by Sanger sequencing.

13 Statistical analysis: Statistical analysis was performed with survival curve analysis using log rank (Mantle-Cox) test and unpaired t test with Welch s correction.

14 Supplemental Tables: Table S1. Laboratory values at diagnosis for MLL-PTD patients Hgb, g/dl ANC, x10 9 /L Plts, x10 9 /L BM blasts, % Patient Patient Patient Patient Patient Patient Patient Patient

15 Table S2. Control cohort characteristics of non-mll-ptd MDS patients Indicates alive at conclusion of study Age at dx, years Sex Disease category at diagnosis Hgb, g/dl ANC, x10 9 /L Plts, x10 9 /L Bone marrow blasts, % Cytogenetic Karyotype IPSS-R IPSS- RA Overall survival, months 64.8 M MDS-MLD M MDS-EB ,XY,t(1;12)(q21;q24.1),-3,add(3)(p10),der(5)t(3;5)(p13;q23),-6,-7,+11, der(11)t(3;11)(q21;q13)[15]/46,xy[5] 6 High 5.9 High ,XY,add(3)(p12),der(4)t(4;12)(q27;q12),add(5)(q13),add(7)(p11.2),add(11) (p11.2),-12,del(13)(q12q22),-14,- 17,del(20)(q11.2q13.1),add(22)(q13)[cp20] 9 Very high 8.99 Very High F MDS-MLD ,XX,-7[3]/46,idem,+mar[5]/46,XX[14] 6.5 Very high 6.52 Very High M MDS-EB ,XY,+8[18]/46,XY[2] 5.5 High 5.44 High M MDS-MLD ,X,-Y,-7,+mar[15]/45,X,-Y[5]. 3 Low 3.24 Intermediate M MDS-EB F MDS-EB ,XY,der(3)t(3;16)(p12;q13),der(5)t(3;5)(p12;q13),- 12,der(13)t(12;13)(q12;q34), der(17)t(17;20)(p13;p11.2),-20,idic(22)(p11.2)[15]/ 44,sl,+del(12)(q11.2)[1]/ 43,sl,idic(7)(q36)[1]/ 44,sl,+idic(22)[1]/ 46,XY[2] 9 Very high 9.08 Very High ,X,idic(X)(q13)[2]/47,sl,+idic(X)[9]/47,sl,+mar[2]/46,X,del(X)(q13q28)[1]/46, XX[6] 5.5 High 5.62 High F MDS-EB ,XX,del(5)(q13q33)[12]/46,XX[8] 5 High 5.19 High M MDS-EB F MDS-EB ,XY,del(5)(q22q33),der(17;20)(q10;p10)[2]/46,sl,del(7)(q11.2q36),+8[5]/44, sl,-7[5]/85-94,slx2,add(11)(q12)x2,+1-2mar,4-11dmin[cp4]/46,xy[2]/46,sl,del(2)(p21p25),add(11)(q12),- 21,+2mar[1]/45,sdl2,+X[1] 7.5 Very high 7.81 Very High ,XX,del(3)(q26q27),-5,add(7)(q11.2*),add(8)(q24),add(12)(p13)[2]/43,sl,- 12,add(17)(p13),add(20)(q11.2),-21[16]/43,sdl,- add(7)(q11.2*),+add(7)(q11.2**)[2] Very high 7.44 Very High M MDS-EB ,XY,t(1;21)(p36.3;q22)[17]/46,XY[3] 5 High 4.83 High F MDS-EB M MDS-EB ,XX,del(2)(q31q37),idic(5)(q11.2),del(6)(p21p25),del(7)(q11.2q36),del(12)( p11.2p13),dic(20;21)(p13;p11.2)[2] /45,sl,-del(7)(q11.2q36),-7,+r[4] /46,XX[14] 7.5 Very High 7.44 Very High ,XY,add(3)(p12),der(4)t(4;12)(q27;q12),add(5)(q13),add(7)(p11.2),add(11) (p11.2),-12,del(13)(q12q22),-14,-17,del(20)(q11.2q13.1),add(22)(q13)[cp20] 8 Very high 7.97 Very High 3.8

16 67.5 M MDS-EB ,XY,+8[18]/46,XY[2] 5.5 High 5.44 High M MDS-EB ,XY,del(3)(p21),del(5)(q31q35),- 7,add(9)(q34),inv(12)(p13q13),add(17)(p13),-18,-20,-21,-22, +1-4mar[cp10]/46,XY[10] 6.5 Very high 6.24 Very High M MDS-EB ,XY,del(3)(q12q24)[20] 6.5 Very high 6.37 Very High M MDS-EB ,XY,+11[5]/46,XY[15] 6.5 Very high 6.46 Very High M MDS-EB ,X,-Y,-7,+mar[18]/46,XY[2] 4 Intermediate 4.21 Intermediate ,XY,t(1;3)(p13;q21),del(4)(q21q25),del(11)(q14q24),+19,del(20)(q11.2q M MDS-MLD )[4] 7.5 Very high 7.27 Very High M MDS-RS- MLD ,XY,der(2)ins(2;6)(q23;p24p12)add(2)(q23),der(3)t(3;12)(p24;p13),add(4)( q12),der(5;22)(p10;q10),- 6,der(12)t(3;12)t(?6;12)(q12;q24),der(19)dup(19)(q13.1q13.4)add(19)(q13.4), +mar[cp17]/43-44,sl,add(x)(p11.2),-der(2)ins(2;6)add(2),+add(2)(q32),- der(3)t(3;12),+3,+6,-7,- der(12)t(3;12)t(6;12),+add(12)(p13),add(19)(p13),add(?21)(p11.2),+del(?22)( q11.2q13),-mar[cp3] 6.5 Very high 6.5 Very High M MDS-EB ,XY[19]/47,XY,+19[1] 5.5 High 5.74 High M MDS-EB ,XY,-7,del(20)(q11.2q13.3)[7] 7 Very high 6.49 Very High F MDS-EB ,XX,del(1)(p22p36.1),del(2)(p24),del(3)(p21),- 5,der(6)t(1;6)(p13;q25),der(7)add(7)(p21)del(7)(q32q36),+8,+8,+8,del(11)(q2 2q23),- 12,+13,del(13)(q12q14)x2,add(16)(q11.2),add(17)(p13),add(19)(p13),-21,+0-2mar[cp20] 9.5 Very high 9.51 Very High M MDS-MLD ,XY[20] 2 Low 1.68 Low M MDS-MLD ,XY[20] 3.5 Intermediate 3.4 Intermediate M MDS-MLD ,XY[20] 2.5 Low 1.98 Low F MDS-MLD ,XX[20] 5.5 High 5.6 High M MDS-EB ,XY[20] 3.5 Intermediate 3.8 Intermediate F MDS-EB ,XX[20] 5 High 5.08 High (with 59.1 M MDS-EB Auer rod) 46,XY[20] 5 High 4.73 High F MDS-MLD ,XX[20] 2.5 Low 2.65 Low F MDS-EB ,XX[20] 6 High 5.79 High F MDS-EB ,XX[20] 4 Intermediate 3.73 Intermediate M MDS-EB ,XY[20] 7 Very High 7.25 Very High M MDS-EB ,XY[20] 5.5 High 5.28 High M MDS-EB ,XY[20] 5.5 High 5.26 High M MDS-MLD ,XY[20] 6.5 Very High 6.62 Very High M MDS-EB ,XY[20] 6 High 6.25 Very High 10.8

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