Supplementary Table 1
Constitutionalcharacteristics of the cohort of young individuals (≤18 years) and adults (>50 years) which were used for molecular analysis of bone marrow biopsies
Diagnosis / Number of specimens analysed / Female/male individuals (n) / Age (years, median, range)ped-CML-MF1/2 / 8 / 4/4 / 15 (11-17)
ped-CML-MF0 / 8 / 2/6 / 14 (2-16)
ped-Control / 10 / 6/4 / 14.5 (1-18)
ad-CML-MF1/2 / 8 / 4/4 / 68.5 (52-75)
ad-CML-MF0 / 8 / 4/4 / 69 (59-78)
ad-Control / 8 / 3/5 / 61.5 (51-73)
Supplementary Table 2
Increased pro-platelet deposition in fibrotic stage ped-CML.
Diagnosis / PP0 / PP1 / PP2 / PP3ped-CML-MF1/2 / n=0/8 / n=0/8 / n=0/8 / n=8/8 (100%)
ped-CML-MF0
(1 case not analysed) / n=1/7 (14.5%) / n=1/7 (14.5%) / n=5/7
(71%) / n=0/7
ped-Control / n=8/10 (80%) / n=2/10 (PP0/1; 20%) / n=0/10 / n=0/10
ad-CML-MF1/2 / n=0/8 / n=2/8 (25%) / n=4/8 (50%) / n=2/8 (25%)
ad-CML-MF0 / n=2/8 (25%) / n=5/8 (62.5%) / n=1/8 (12.5%) / n=0/8
ad-Control / n=7/8 (87.5%) / n=1/8 (12.5%) / n=0/8 / n=0/8
Supplementary Table 3
Summary of expression profiles in children/adolescents with chronic stage myeloid neoplasms.
Gene (abbreviation) / ped-CML-MF0
(mean,
n = 8) / ped-CML-
MF1/2
(mean,
n=8) / ped-Control
(mean,
n =10) / K562
(mean of 3 sets) / BMF
(mean of 3 sets) / K562+
BMF
(mean of 3 sets)
ANGPT1 / 0.016 / 0.012 / 0.010 / 0.009 / 0.263 / 0.008
ANGPT2 / 0.003 / ND / 0.001 / 0.001 / 0.002 / 0.001
ANGPT4 / 0.000 / 0.007 / 0.005 / ND / 0.020 / 0.001
ANGPTL1 / ND / ND / ND / 0.001 / 0.020 / 0.001
ANGPTL4 / 0.006 / ND / 0.009 / 0.087 / 0.563 / 0.114
ARPC1B / 0.046 / 0.102 / 0.126 / 10.933 / 3.122 / 5.502
ARPC2 / 1.423 / 1.123 / 1.485 / 22.172 / 20.626 / 9.169
ARPC4 / 0.131 / 0.201 / 0.205 / 5.913 / 4.206 / 2.806
ARPC5 / 0.037 / 0.036 / 0.039 / 3.725 / 3.256 / 1.859
BACH1 / 0.028 / 0.003 / 0.028 / 2.991 / 0.900 / 1.151
BAI1 / 0.004 / 0.005 / 0.002 / ND / ND / 0.000
BCL2L1 / 0.215 / 0.171 / 0.738 / 13.733 / 3.563 / 7.690
BMP1 / 0.001 / 0.009 / 0.002 / 0.203 / 0.593 / 0.139
BMP2 / 0.011 / 0.017 / 0.030 / 0.033 / 0.032 / 0.023
BMP4 / 0.009 / 0.009 / 0.044 / 0.001 / 0.366 / 0.007
BMP6 / ND / 0.015 / ND / 0.009 / 0.294 / 0.011
BMP7 / 0.005 / 0.004 / ND / 0.009 / ND / 0.004
BMPR1B / ND / ND / ND / 0.001 / 0.017 / 0.001
BMPR2 / 0.007 / 0.005 / 0.010 / 0.679 / 0.416 / 0.400
CAV2 / 0.010 / 0.013 / 0.006 / 2.250 / 5.535 / 1.743
CCL5 / 0.119 / 0.239 / 0.194 / 0.130 / 0.004 / 0.127
CDC42 / 0.161 / 0.207 / 0.218 / 48.423 / 21.604 / 20.370
CDK1 / 0.002 / 0.002 / 0.005 / 6.017 / 0.176 / 1.839
COL1A2 / 0.086 / 0.139 / 0.252 / ND / 333.999 / 3.682
COL3A1 / 0.011 / 0.085 / 0.115 / ND / 79.144 / 1.981
COL4A1 / 0.004 / 0.021 / ND / ND / 18.034 / 0.663
CTGF / 0.081 / 0.081 / 0.240 / ND / 74.588 / 5.272
CXCL1 / 0.053 / 0.025 / 0.045 / 0.102 / 0.011 / 0.138
CXCL12 / 0.222 / 0.346 / 1.224 / ND / 38.206 / 0.215
CXCL13 / 0.006 / ND / ND / ND / ND / ND
CXCR4 / 0.242 / 0.222 / 0.212 / 0.002 / ND / 0.001
CXCR5 / 0.008 / ND / ND / 0.002 / ND / 0.001
EDN1 / 0.001 / 0.006 / ND / 0.005 / 0.158 / 0.018
EPHB4 / 0.022 / 0.013 / 0.019 / 0.486 / 0.361 / 0.407
FGF2 / 0.003 / 0.007 / 0.005 / 0.937 / 4.712 / 0.792
FGFR1 / 0.176 / 0.156 / 0.347 / 0.392 / 2.505 / 0.318
FLI1 / 0.123 / 0.203 / 0.122 / 0.003 / 0.002 / 0.003
FLT4 / 0.002 / 0.009 / 0.006 / 0.128 / 0.429 / 0.025
FIGF;PIR-FIGF / ND / ND / ND / 0.002 / 0.004 / 0.001
FOXP3 / 0.004 / ND / 0.005 / 0.018 / 0.005 / 0.012
GATA1 / 0.258 / 0.200 / 0.682 / 36.423 / ND / 20.994
GATA2 / 0.206 / 0.382 / 0.064 / 4.254 / 0.042 / 2.250
GP1BA / 0.015 / 0.068 / 0.020 / 0.000 / ND / 0.000
GREM1 / ND / ND / 0.002 / ND / 9.205 / 0.212
HGF / 0.017 / 0.021 / 0.009 / ND / 0.636 / 0.008
HIF1A / 0.300 / 0.368 / 0.329 / 2.005 / 42.435 / 1.734
IL6 / ND / 0.017 / ND / 0.420 / 2.801 / 1.101
IL8 / 0.016 / 0.036 / 0.019 / 3.854 / 0.029 / 2.575
ITGA2B / 0.876 / 3.064 / 0.340 / 0.031 / ND / 0.021
ITGB3 / 2.480 / 9.748 / 1.876 / 0.077 / 1.294 / 0.200
KDR / 0.007 / 0.012 / 0.012 / 0.004 / ND / 0.002
LECT1 / 0.001 / 0.001 / 0.006 / 0.008 / ND / 0.003
LOX / 0.008 / 0.018 / 0.048 / ND / 90.110 / 5.267
MDK / 0.026 / 0.004 / 0.004 / 2.394 / 2.274 / 0.967
MKL1 / 0.482 / 0.630 / 0.497 / 1.167 / 1.512 / 1.064
MMP1 / ND / ND / ND / 0.274 / 0.007 / 0.070
MMP2 / 0.034 / 0.057 / 0.148 / 0.003 / 0.013 / 0.003
MMP9 / 6.363 / 3.607 / 7.854 / 0.001 / 0.018 / 0.001
MMP11 / 0.002 / 0.003 / 0.002 / 0.028 / 13.602 / 0.414
MMP13 / 0.003 / 0.001 / 0.014 / 0.032 / 55.717 / 1.070
MMP14 / 0.021 / 0.032 / 0.088 / 0.008 / 0.003 / 0.002
MYH2 / 0.076 / 0.249 / 0.797 / ND / ND / ND
MYL2 / ND / 0.216 / 0.717 / 0.002 / 0.003 / ND
NFE2 / 0.033 / 0.047 / 0.081 / 6.579 / 0.145 / 3.901
NOG / ND / 0.011 / ND / 0.001 / 0.130 / 0.010
NOS3 / 0.001 / 0.002 / 0.002 / 0.011 / ND / 0.014
NOTCH4 / 0.015 / 0.015 / 0.025 / ND / ND / ND
NOX4 / ND / ND / 0.004 / ND / 0.026 / 0.001
NRP1 / 0.004 / 0.005 / 0.003 / 0.016 / 2.073 / 0.057
PDGFA / 0.001 / 0.004 / 0.002 / 0.105 / 1.601 / 0.140
PDGFRA / 0.007 / 0.012 / 0.017 / 0.020 / 5.748 / 0.145
PDPN / 0.099 / 0.060 / 0.287 / 0.003 / 0.064 / 0.001
PECAM1 / 0.355 / 0.407 / 0.454 / 0.057 / 0.042 / 0.003
PF4 / 0.094 / 0.149 / 0.092 / ND / ND / ND
PLAT / 0.003 / 0.036 / 0.023 / 0.105 / 0.608 / 0.118
PLAUR / 0.122 / 0.076 / 0.090 / 0.157 / 1.109 / 0.174
PLOD2 / 0.005 / 0.011 / 4.439 / 13.401 / 3.202
PTGS1 / 0.015 / 0.023 / 0.010 / 0.372 / 0.028 / 0.542
PTK2 / 0.037 / 0.062 / 0.038 / 2.547 / 0.561 / 1.375
PTRF / 0.023 / 0.054 / 0.075 / 1.327 / 11.743 / 1.061
RAB27B / 0.081 / 0.115 / 0.034 / 0.002 / 0.040 / 0.001
RAC1 / ND / ND / ND / 0.112 / 0.630 / 0.074
RAC2 / 0.056 / 0.057 / 0.081 / 1.927 / 0.444 / 1.384
RAC3 / 0.054 / 0.095 / 0.014 / 0.757 / 0.025 / 0.370
RANBP10 / 0.068 / ND / 0.088 / 0.138 / 0.030 / 0.077
RHOA / 2.253 / 1.921 / 1.738 / 24.452 / 47.371 / 14.644
RHOC / ND / 0.028 / 0.016 / 5.600 / 7.765 / 2.752
RHOD / 0.001 / ND / 0.025 / ND / 3.413 / 0.066
RHOH / 0.050 / 0.068 / 0.029 / 0.848 / 0.003 / 0.366
RHOJ / ND / 0.087 / 0.012 / ND / 0.347 / 0.013
RHOU / 0.156 / 0.210 / 0.132 / 0.014 / 0.008 / 0.007
RHOV / ND / ND / ND / 0.008 / 0.002 / 0.003
RND1 / ND / ND / 0.019 / 0.011 / 0.003 / 0.005
RND2 / ND / ND / ND / 0.288 / 0.025 / 0.171
RND3 / ND / ND / 0.006 / 0.007 / 2.278 / 0.500
RUNX1 / 0.324 / 0.262 / 0.283 / 2.352 / 2.082 / 1.207
S1PR1 / 0.005 / 0.004 / 0.009 / 0.192 / 0.326 / 0.153
SERPINE1 / 0.033 / 0.014 / ND / 0.277 / 60.509 / 5.322
SERPINF1 / 0.038 / 0.059 / 0.182 / 4.861 / 2.040 / 2.834
SMAD1 / 0.023 / 0.027 / 0.065 / 1.191 / 0.569 / 0.483
SMAD3 / 0.099 / 0.091 / 0.181 / 3.243 / 2.997 / 2.219
SMAD4 / 0.024 / 0.028 / 0.035 / 7.216 / 1.833 / 3.271
SMAD5 / 0.016 / 0.029 / 0.016 / 3.152 / 0.934 / 1.478
SRF / 0.056 / 0.090 / 0.067 / 2.902 / 2.935 / 1.786
TEK / 0.004 / 0.016 / 0.015 / 0.002 / 0.482 / 0.040
TGFB1 / 1.630 / 1.576 / 1.486 / 20.353 / 20.238 / 34.785
TGFB2 / 0.002 / ND / ND / 0.015 / 1.248 / 0.119
TGFB3 / 0.004 / 0.004 / ND / 0.047 / 0.110 / 0.029
TGFBR1 / 0.005 / 0.004 / 0.007 / 8.791 / 2.692 / 3.339
TGFBR2 / 0.065 / 0.037 / 0.094 / 0.835 / 1.384 / 0.402
THBS1 / 1.270 / 2.905 / 2.530 / 1.269 / 467.955 / 16.239
TIE 1 / 0.003 / 0.004 / 0.002 / 0.003 / 0.001 / 0.002
TIMP1 / 0.241 / 0.314 / 0.442 / 8.933 / 77.145 / 16.565
TIMP2 / 0.046 / 0.056 / 0.115 / 0.006 / 11.376 / 0.242
TNFAIP2 / 0.001 / 0.001 / 0.001 / 0.027 / 0.035 / 0.018
TUBB / 0.438 / 0.567 / 1.131 / 532.863 / 185.111 / 227.370
TYMP / 0.009 / 0.005 / 0.020 / ND / 0.096 / 0.007
VCAM1 / 0.020 / 0.011 / 0.068 / ND / 3.971 / 0.346
VEGFA / 0.305 / 0.238 / 0.379 / 14.328 / 15.944 / 6.485
VEGFC / 0.011 / 0.022 / 0.043 / 0.002 / 7.814 / 0.271
WAS / 1.271 / 1.101 / 0.754 / 0.117 / 0.006 / 0.060
WASF1 / ND / ND / ND / 0.373 / 0.150 / 0.305
WASF2 / 0.001 / ND / 0.012 / 0.460 / 0.137 / 0.243
WASF3 / ND / 0.003 / 0.004 / 0.002 / 0.070 / 0.002
WASL / 0.020 / 0.035 / 0.022 / 0.893 / 0.435 / 0.505
WIPF1 / 0.444 / 0.404 / 0.217 / 1.457 / 1.189 / 0.852
ZFPM1 / 0.376 / 0.466 / 0.335 / 0.613 / 0.135 / 0.558
miR-10a / 0.117 / 0.160 / 0.120 / NA / NA / NA
miR-146b / 0.992 / 1.332 / 0.616 / NA / NA / NA
Legend to Supplementary Table 1
Gomorii and trichrome staining were used for evaluation of fibrosis. All ped-CML and ad-CML were in chronic stage.
Legend to Supplementary Table 2
Protein expression analysis was performed with a ZytoChem-Plus HRP Polymer kit (Zytomed Systems, Berlin, Germany).Xylol-deparaffinized and re-hydrated FFPE sections (~2-3 μm) were pre-treated in an autoclave in a buffer solution (pH 6.0). Sections were then incubated with H2O2 (3% in 70% ethanol), followed by incubation with blocking solution and with monoclonal mouse antibodies (anti-THBS1, 1:25, clone 5B11; anti-MMP9, 1:200, clone 56-2A4, both Acris, Herford, DE). After visualisation of immunostaining with DAB-brown, sections were counterstained with hematoxylin. CD42b (monoclonal rabbit antibody, 1:50, SP219 clone, Medac, Wedel, DE) was stained on a Benchmark ULTRA (Ventana Medical Systems, Inc./Roche, Tucson, AZ, USA).
Legend to Supplementary Table 3
Mean values are given for pediatric study sub-groups and three independent cell culture experiments.Several factors were expressed at low relative expression levels or were not detectable (“ND”). MicroRNAs were analysed in bone marrow samples but not in cell cultures (not analysed; “NA”).The following human cell lines were used for co-culture experiments: K562 (DSMZ, Braunschweig, Germany) and primary bone marrow fibroblasts (kindly provided by Dr. Miriam Wittmann, University of Leeds, UK). Cells were incubated for 3-6 days (37°C, humidified atmosphere of 5% CO2)with RPMI 1640 (Gibco/Life Technologies, Darmstadt, Germany), 10% foetal calf serum (Gibco), 1% L-glutamine (PAA/GE Healthcare Life Sciences, San Francisco, CA, USA) and 1% penicillin/streptomycin (PAA). The following settings were used: i) bone marrow fibroblasts alone for 3 days (0.1x106 cells), ii) K562 alone for 3 days (2x106 cells), iii) fibroblasts and K562 in one cell culture dish (6 cm diameter, Nunc/Thermo Fisher Scientific, Braunschweig, Germany) for 3 days. RNA precipitation from cell lines was performed with TRIzol® (Invitrogen/Life Technologies, Darmstadt, Germany) and chloroform (Sigma-Aldrich, St. Louis, MO, USA).
Legend to Supplementary Figure 1
Overall survival in children with fibrotic stage CML
Data for survival analysis was available in a subfraction of 66 cases (44/56 ped-CML-MF0, 7/10 ped-CML-MF1/2). Survival analysis was performed with alog-rank (Mantel-Cox) test (Prism 5.0, GraphPad Software, San Diego, CA, USA). It has to be taken into account that this analysis is based on retrospective data, a small number of cases with fibrosis and different therapy modalities (e.g. duration of imatinib therapy, different indications and time intervals between diagnosis and stem cell transplantation). Any therapeutic intervention may change the natural course of progression or remission to fibrosis. In general, tyrosine kinase inhibitor (TKI) therapy was approved for children in 2003. Among 66 ped-CML patients, 31/66of cases were diagnosed between 1999 and 2002 and 35/66 between 2003 and 2012; among ped-CML-MF1/2, 4/10 cases were diagnosed in 2003 or later. One CML-MF1 case received stem cell transplantation (Tx), another developed secondary acute leukemia after 7 months and both died after 11 months. In ped-CML-MF0, 23 required Tx and 6/23 died after 5-94 months. One ped-CML MF0 patient died one month after diagnosis and had no Tx. Due to low numbers and limited data on TKI dosages, no meaningful multivariate analysis could be performed.
Legend to Supplementary Figure 2
Immunostaining of megakaryocytes showing significant correlation of CD42b and THBS1
Legend to Supplementary Figure 3
Aberrant megakaryocytes in CML with increased pro-platelet deposition in fibrotic stage cases
In children and adults without neoplastic hematemesis, megakaryocytes were located in the interstitial parts of the bone marrow and often next to vessels. No prominent clustering or endosteal localisation was noted. Pro-platelets were detectable and were usually small and round and did not form dense nets. In CML cases, the images show the expected increased quantity of megakaryocytic cells and the atypical clustering and endosteal translocation, both in children and adult patients. Pro-platelet deposition was present in megakaryocyte-rich areas. In addition, some adults with fibrotic and non-fibrotic CML showed areas with lower number of megakaryocytes but increased pro-platelet depositions (inserts). In adults, and in particular in children with fibrotic CML, pro-platelets appeared to form more dense nets. Occasionally, some enlarged megakaryocytes were detectable, but most were normal or small in diameter. The quantity of micro-megakaryocytes or enlarged cells appeared to be similar in children and adults and was not related to the presence or absence of fibrosis. Immunostaining was performed as indicated in the images. Original magnification in all images and inserts is x200.
Legend to Supplementary Figure 4
Expression of factors associated with fibrosis, cell mobilisation, vascularisation, stem cell niche formation as well as mRNA/protein translation regulators miR-10a and miR-146b
1