Supplemental Table 1. Detailed Clinical Summary of 26Patients with Flt3m and Rasm in Lower

Supplemental Table 1. Detailed clinical summary of 26patients with FLT3m and RASm in lower risk MDS.

Pt / Age/Sex / Mut / How* / WHO / IPSS / BMBL / BM BL at acquisition / Cytogenetics / Other mut / Tx† / Timing‡ / SCT / TTT (Month) / Details
1 / 50M / ITD
KRAS (G13D) / -/+ / RA / Low / 0 / 25 / Diploid / Yes / Tx / No / 53 / Progressed after thalidomide, R115777, and 5-azacitidine. Transformed to AML with both FLT3-ITD and KRAS acquisition. sAML was refractory to FA, IA and MGCD103. Died with fungal pneumonia.
2 / 46M / ITD / -/+ / RA / Int-1 / 2 / 67 / Diploid / Yes / Tx / No / 6 / Rapidly transformed to sAML with FLT3-ITD acquisition. Achieved CR after FA+GO. Relapsed and received CEP-701 for 1 cycle with progression. Refractory to subsequent cytotoxic therapies and died because of leukemia progression.
3 / 64F / ITD / -/+ / RAEB-1 / Int-1 / 9 / 71 / Diploid / Yes / Tx / No / 4 / Rapidly progressed to sAML with FLT3-ITD acquisition. Received induction with IA and achieved CR. Lost follow up and cause of death unknown.
4 / 84M / ITD / -/+ / RCMD / Low / 4 / 94 / Diploid / Yes / Post / No / 21 / Received supportive care initially but transformed to sAML. Received 9 cycles of decitabine after transformation and acquired FLT3-ITD when disease progressed. Eventually died from pneumonia.
5 / 73M / ITD
NRAS (G12D) / -/+ / RAEB-1 / Int-1 / 6 / 6 / Diploid / Yes / Pre / No / 36 / Received decitabine for 12 cycles and 5-azacitidine with valproic acid for 5 cycles. Transformed to sAML with FLT3-ITD and NRAS acquisition. Lost follow up and died from unknown cause.
6 / 61M / ITD / -/+ / RAEB-1 / Int-1 / 7 / 10 / Diploid / Yes / Pre / No / 9 / Received 7 cycles of decitabine and valproic acid and transformed to sAML with FLT3-ITD acquisition. sAML was refractory to IA and CIA.
7 / 64F / ITD / -/+ / RA / Int-1 / 3 / 68 / Diploid / NPM1 / Yes / Tx / No / 11 / Received vorinostat without response. Transformed to sAML with FLT3-ITD and NPM1 acquisition. sAML was refractory to decitabine+GO. Died from sepsis and GI bleeding.
8 / 52M / ITD / -/+ / RAEB-1 / Int-1 / 6 / 49 / Diploid / NPM1 / Yes / Tx / MUD / 11 / Received 7 cycles of decitabine + valproic acid and underwent MUD transplant. Relapsed with sAML 2 months post-transplant with FLT3-ITD and NPM1 acquisition. Shortly after receiving salvage therapy with IA, died from sepsis.
9 / 71M / ITD / -/+ / MDS-U / Int-1 / 7 / 65 / Inv(3)(q21q26.2) / Yes / Tx / No / 9 / Received 5-azacitidine for 3 cycles but progressed to sAML with FLT3-ITD acquisition. Received FA but died from infection.
10 / 82M / D835 / -/+ / RA / Int-1 / 4 / 30 / del 11(q13q23) / Yes / Tx / No / 4 / Shortly after the diagnosis, transformed to sAML with FLT3-D835 acquisition. Achieved CR after IA induction. Lost follow up and died from unknown cause.
11 / 67M / D835
NRAS (G12A) / -/+ / RAEB-1 / Int-1 / 9 / 18 / dup(2)(p15p23) / Yes / Pre / No / 22 / Had long term response to oral 5-azacitidine trial. Acquired FLT3-D835 and NRAS mutation with increasing blast and then transformed to sAML. sAML was refractory to clofarabine and cytarabine, ON1910, and FA.
12 / 77F / NRAS (G13R) / -/+ / RA / Int-1 / 2 / 70 / Diploid / Yes / Tx / No / 23 / Had long term response to decitabine for more than 4 years. Eventually transformed to sAML with NRAS acquisition. Refractory to salvage therapy and died from pneumonia.
13 / 79F / NRAS (G12S) / -/+ / RAEB-1 / Int-1 / 9 / 17 / Diploid / Yes / Tx / No / 67 / Had long term response to Oplelvekin. Transformed to sAML with NRAS acquisition. Patient opted not to receive treatment and died.
14 / 63M / KRAS (G12A) / -/+ / RARS / Low / 1 / 69 / Diploid / Yes / Tx / No / 49 / Received R115777, decitabine, 5-azacitidine. Eventually progressed to sAML with KRAS acquisition. Refractory to salvage therapies and died from leukemia progression.
15 / 77M / KRAS (G12D) / -/+ / RAEB-1 / Int-1 / 6 / 5 / Trisomy 8 / No / NA / No / NA / Initially was treated conservatively. Acquired KRAS mutation and the disease became proliferative with increased white blood cells and immature myelocytes in bone marrow. Received 1 cycle of 5-azacitidine. Lost follow up and died from unknown cause.
16 / 60M / KRAS (G12A) / -/+ / RA / Low / 1 / 25 / t(3:12)(q26.2;p13), -7, +r / Yes / Tx / No / 36 / Received 12 cycles of 5-azacitadine with initial good response. Transformed to sAML with KRAS acquisition. sAML was refractory to IA and received GSK 1120212 (MEK inhibitor) and had reduction of BM BL however lost response after 3 cycles and died.
17 / 72M / KRAS (G12A) / -/+ / RCMD / Int-1 / 2 / 12 / Monosomy 7, der(18;21)(q10;q10),+21 / Yes / Pre / No / 46 / Received 5 cycles of 5-azacitidine but the disease became proliferative with increased blast and white blood cell count and KRAS acquisition was noted. Subsequently transformed to sAML. Patient was not able to receive salvage therapy due to poor performance status and died from leukemia progression.
18 / 61F / NRAS (G12D) / -/+ / RA / Low / 1 / 51 / Diploid / Yes / Tx / No / 17 / Initially was treated conservatively but transformed to sAML with NRAS acquisition. Disease was refractory to clofarabine and FA. Died from leukemia progression.
19 / 55F / NRAS (G13V) / -/+ / RA / Int-1 / 2 / 3 / Trisomy 8 / No / NA / MUD / NA / Originally with NRAS mutation. Received decitabine for few cycles and underwent MUD stem cell transplant. Post-transplant course was complicated with GvHD and infectious complication and died 5 months post-transplant.
20 / 60F / NRAS (G13R) / -/+ / RA / Int-1 / 4 / 46 / Monosomy 7 / Yes / Tx / No / 42 / Received decitabine and valproic acid, oral sapacitabine. Transformed to sAML with NRAS acquisition. Disease was refractory to FAI and cytarabine vosaroxin. Received MSC1936369B (MEK inhibitor) with stable disease. Still alive at last follow up.
21 / 53M / ITD / +/+ / RAEB-1 / Int-1 / 7 / NA / Diploid / No / NA / No / NA / Originally with FLT3-ITD RAEB. Had long term response to decitabine. Died from unexplained neurological deterioration.
22 / 58M / ITD / +/+ / RAEB-1 / Int-1 / 8 / NA / Diploid / Yes / NA / MRD / 2 / Rapidly transformed to sAML after the MDS diagnosis. Originally had FLT3-ITD. Achieved CR after IA induction and underwent MRD transplant. Maintaining remission for more than 4 years and still alive.
23 / 72F / D835 / +/+ / RAEB-1 / Int-1 / 8 / NA / Diploid / No / NA / No / 23 / Originally with FLT-D835 mutation. Had long term response to decitabine. Lost follow up and died from unknown cause.
24 / 28M / D835 / +/+ / RAEB-1 / Int-1 / 8 / NA / Diploid / No / NA / MUD / 98 / Originally with FLT3-D835 mutation. Received 2 cycles of IA and achieved CR. Underwent MUD transplant with sustained remission for more than 9 years. Still alive.
25 / 50F / KRAS (G12D) / +/+ / RA / Low / 1 / NA / Diploid / No / NA / No / NA / Originally with KRAS mutation. Treated with supportive care only. Died after cardiac arrest from unknown cause.
26 / 61M / NRAS (G12C) / +/+ / RAEB-1 / Int-1 / 9 / NA / Del 20q / No / NA / No / NA / Originally with NRAS mutation. Received 2 cycles of 5-azacitidine with stable disease. Lost follow up and died from unknown cause.

NOTE: Case 1-20 is patient who acquired FLT3m/RASm. Case 21-26 is patients who original had FLT3m/RASm.

* +/+ indicates that mutation was detected at time of MDS diagnosis, -/+ indicates that mutation was acquired afterwards.

† Tx indicates Transformation to AML

‡ Timing of mutationacquisition, Pre= before transformation, Tx = at time of transformation, Post = post transformation

FLT3m indicates FLT3 molecular alterations, RASm; RAS mutations, WHO; World Health Organization classification, IPSS; International Prognostic Scoring System, SCT; stem cell transplant, ITD; FLT3 internal tandem duplication, D835; codon D835 mutation of FLT3, RA; refractory anemia, RAEB; refractory anemia with excess blasts, RCMD; refractory cytopenia with multilineage dysplasia, MDS-U; MDS unclassified, MRD; matched related donor transplant, MUD; matched unrelated donor transplant, CR; complete remission, FA; fludarabine and cytarabine, IA; idarubicin and cytarabine, GO; gemtuzumab ozogamicin, FAI; fludarabine, cytarabine and idarubicin, GvHD; graft versus host disease.

Supplemental Table 2. Patients characteristics at time of diagnosis between patients with FLT3m at diagnosis (+/+), later acquisition (-/+), and without alterations

Total / FLT3m +/+ / FLT3m -/+ / None / P-value‡
Characteristics / N=278 (%) / N=4 (%)* / N= 11 (%)† / N=263 (%)
Median Age (range) / 64 (23-91) / 56 (28-72) / 64 (46-84) / 64 (23-91) / 0.78
Female. no. (%) / 107 (39) / 1 / 2 / 104 / 0.21
WHO (%) / RA / 71 (26) / 0 / 4 / 68 / 0.99
RAEB-1,2 / 102 (37) / 4 / 5 / 92 / 0.19
RARS / 18 (7) / 0 / 0 / 18 / 0.99
RCMD / 77 (28) / 0 / 1 / 76 / 0.19
MDS-U / 6 (2) / 0 / 1 / 5 / 0.22
5q- / 4 (1) / 0 / 0 / 4 / 0.99
IPSS / Low / 72 (26) / 1 / 2 / 69 / 0.73
Int-1 / 206 (74) / 3 / 9 / 194
Cytogenetics / Diploid / 179 (64) / 4 / 8 / 167 / 0.75
Aberrant / 99 (36) / 0 / 3 / 96
RAS mutation** / 14 (5) / 0 / 3 / 11 / 0.014
Median HGB, g/dL (range) / 10.1 (5-15) / 9.2 (9.0-10.0) / 9.8 (8-12) / 10.1 (5-15) / 0.31
Median PLT count, 109/L (range) / 92 (3-805) / 29.5 (24.0-62.0) / 52 (23-150) / 95 (3-805) / 0.21
Median WBC, 109/L (range) / 3.2 (1.0-159) / 4.7 (2.0-15.0) / 2.7 (1.0-27.0) / 3.2 (1.0-159) / 0.63
Median BM BL, %(range) / 3 (0-10) / 8 (7-8) / 6 (0-9) / 3 (0-10) / 0.035
Transformation to AML, no. (%) / 74 (27) / 1 (25) / 11 (100) / 62 (24) / <0.001
SCT (%) / 49 (18) / 2 (50) / 0 (0) / 47 (18) / 0.22

ITD indicates internal tandem duplication; TKD, tyrosine kinase domain mutation;SE, standard error; WHO, World Health Organization classification; RA, refractory anemia; RAEB, refractory anemia with excess blasts; RARS, refractory anemia with ring sideroblasts; RCMD, refractory cytopenia with multilineage dysplasia; MDS-U, myelodysplastic syndrome, unclassified; 5q-, 5q minus syndrome; IPSS, International Prognostic Scoring System; Int-1, intermediate-1; WBC, white blood cell count; SCT,stem cell transplantation.

*FLT3-ITD = 2, FLT3-TKD = 2.

† FLT3-ITD = 9, FLT3-TKD = 2.

‡P value is based on FLT3m -/+ group vs. none.

** Including both detected at time of diagnosis and acquired later.

Supplemental Table 3. Patients characteristics at time of diagnosis between patients with RASm at diagnosis (+/+), later acquisition (-/+), and without mutations

Total / RASm +/+ / RASm -/+ / None / P-value‡
Characteristics / N=278 (%) / N=2 (%)* / N= 12 (%)† / N=264 (%)
Median Age (range) / 64 (23-91) / 56 (50-61) / 65 (50-79) / 64 (23-91) / 0.55
Female. no. (%) / 107 (39) / 1 / 2 / 104 / 0.21
WHO (%) / RA / 71 (26) / 1 / 6 / 65 / 0.18
RAEB-1,2 / 102 (37) / 1 / 4 / 96 / 0.71
RARS / 18 (7) / 0 / 1 / 17 / 0.56
RCMD / 77 (28) / 0 / 1 / 76 / 0.19
MDS-U / 6 (2) / 0 / 0 / 6 / 0.99
5q- / 4 (1) / 0 / 0 / 4 / 0.99
IPSS / Low / 72 (26) / 1 / 4 / 67 / 0.48
Int-1 / 206 (74) / 1 / 7 / 198
Cytogenetics / Diploid / 179 (64) / 1 / 6 / 172 / 0.28
Aberrant / 99 (36) / 1 / 6 / 92
FLT3 alteration** / 15 (5) / 0 / 3 / 12 / 0.021
Median HGB, g/dL (range) / 10.1 (5-15) / 10.7 (9.0-12.0) / 10.4 (10-13) / 10.1 (5-15) / 0.16
Median PLT count, 109/L (range) / 92 (3-805) / 62 (6.0-118.0) / 108 (38-465) / 91 (3-805) / 0.57
Median WBC, 109/L (range) / 3.2 (1.0-159) / 7.3 (4.0-11.0) / 2.7 (1.0-9.0) / 3.2 (1.0-159) / 0.16
Median BM BL, %(range) / 3 (0-10) / 5 (1-9) / 2 (0-9) / 3 (0-10) / 0.77
Transformation to AML, no. (%) / 74 (27) / 0 / 10 (83) / 64 (24) / <0.001
SCT (%) / 49 (18) / 0 / 1 (8) / 48 (18) / 0.70

*NRAS = 1, KRAS = 1.

† NRAS = 7, KRAS= 5.

‡P value is based on RASm -/+ group vs. none.

** Including both detected at time of diagnosis and acquired later.

Supplemental Table 4. Univariate Cox proportional hazards analysis for TFS and OS.

TFS / OS
Variable / HR / P-value / HR / P-value
Age (>60 vs. ≤60) / 1.71 / 0.001 / 1.80 / 0.0004
Sex (Male vs. Female) / 1.28 / 0.11 / 1.26 / 0.14
Cytogenetics (Diploid vs. No) / 1.03 / 0.83 / 0.97 / 0.86
Cytogenetics (High risk vs. No)† / 1.25 / 0.238 / 1.31 / 0.32
IPSS (Int-1 vs. Low) / 1.48 / 0.02 / 1.63 / 0.006
MDALR model (Cat 1,2 vs. 3) / 1.79 / <0.0001 / 1.96 / <0.0001
FLT3m/RASm (Yes vs. No)* / 3.94 / <0.0001 / 3.35 / <0.0001
Initial BM Blast (>5 vs 0-5) / 1.64 / 0.003 / 1.57 / 0.007
WBC (<1.0 vs ≥1.0) / 0.99 / 0.99 / 1.13 / 0.86
PLT (<100 vs ≥100) / 1.32 / 0.05 / 1.31 / 0.07
HGB (<10 vs ≥ 10) / 1.53 / 0.003 / 1.66 / 0.001

HR, hazard ratio; IPSS, International Prognostic Scoring System; MDALR model, MD Anderson Lower Risk model; WBC, white blood cell count; BM Blast, bone marrow blast; PLT, platelet; HGB, hemoglobin.

*variable was fitted as a time-dependent covariate.

†High risk cytogenetics were monosomy 7, del 7q, complex, 3q abnormalities.

SupplementalTable 5. Multivariable Cox model for TFS and OS.

TFS / OS
Variable / P / HR / 95% CI / P / HR / 95% CI
Lower / Upper / Lower / Upper
Age (>60 vs. ≤60) / 0.02 / 1.64 / 1.09 / 2.47 / 0.006 / 1.81 / 1.19 / 2.77
BM Blast (>5 vs 0-5) / 0.05 / 1.46 / 0.99 / 2.01 / 0.32 / 1.20 / 0.84 / 1.72
HGB (<10 vs ≥ 10) / 0.03 / 1.46 / 1.04 / 2.04 / 0.03 / 1.45 / 1.03 / 2.04
IPSS (Int-1 vs. Low) / 0.69 / 1.08 / 0.73 / 1.66 / 0.36 / 1.21 / 0.81 / 1.80
MDALR model (Cat 3 vs. 1 and 2) / 0.64 / 1.10 / 0.73 / 1.66 / 0.51 / 1.15 / 0.76 / 1.73
FLT3m/RASm (Yes vs. No)* / 0.001 / 2.59 / 1.49 / 4.51 / <0.001 / 3.26 / 2.02 / 5.27

P, P value; HR, hazard ratio; 95% CI, 95% confidence interval; IPSS, International Prognostic Scoring System; MDALR model, MD Anderson Lower Risk model; WBC, white blood cell count; BM Blast, bone marrow blast; HGB, hemoglobin; FLT3m/RASm, FLT3 molecular alteration and/or RAS mutations

* variable was fitted as a time-dependent covariate.

Supplemental Table 6. Multivariate Cox proportional hazards analysis for TFS and OS when FLT3m and RASm are analyzed as a separate covariate.

TFS / OS
Variable / HR / P-value / HR / P-value
Age (>60 vs. ≤60) / 1.56 / 0.03 / 1.66 / 0.02
IPSS (Int-1 vs. Low) / 1.05 / 0.82 / 1.15 / 0.49
MDALR model (cat 3 vs. cat 1, 2) / 1.16 / 0.46 / 1.23 / 0.32
FLT3m (Yes vs. No)* / 2.53 / 0.008 / 2.33 / 0.009
RASm (Yes vs. No)* / 2.51 / 0.006 / 3.11 / <0.0001
Initial BM Blast (>5 vs 0-5) / 1.46 / 0.033 / 1.33 / 0.12
HGB (<10 vs ≥ 10) / 1.41 / 0.045 / 1.40 / 0.05

HR, hazard ratio; IPSS, International Prognostic Scoring System; BM Blast, bone marrow blast; HGB, hemoglobin.

*variable was fitted as a time-dependent covariate.