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01.12.2012 | Research | Ausgabe 1/2012 Open Access

Journal of Hematology & Oncology 1/2012

Cytogenetic and molecular predictors of response in patients with myeloid malignancies without del[5q] treated with lenalidomide

Zeitschrift:
Journal of Hematology & Oncology > Ausgabe 1/2012
Autoren:
Yuka Sugimoto, Mikkael A Sekeres, Hideki Makishima, Fabiola Traina, Valeria Visconte, Anna Jankowska, Andres Jerez, Hadrian Szpurka, Christine L O'Keefe, Kathryn Guinta, Manuel Afable, Ramon Tiu, Kathy L McGraw, Alan F List, Jaroslaw Maciejewski
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​1756-8722-5-4) contains supplementary material, which is available to authorized users.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

YS and JPM designed research, performed research, analyzed data, and wrote the paper. HM, FT, VV, AJ, AJ, HS, CLO, KG performed research. MA, RT, KRM and AFL analyzed data. All authors read and approved the final manuscript.

Background

Lenalidomide (LEN) is particularly effective in patients with myelodysplastic syndromes (MDS) and the del[5q] cytogenetic abnormality [13]. In MDS-003, the phase II registration trial of 148 lower-risk MDS patients with del[5q] with or without other karyotypic abnormalities, 67% achieved transfusion independence with a complete and partial cytogenetic response rate of 45% and 28%, respectively [2]. There was no significant association between karyotypic complexity and the frequency of a cytogenetic response. LEN also has activity in a proportion of MDS without del[5q] [4] and [5]. Transfusion-dependent MDS patients low- or Int-1 by the International Prognostic Scoring System (IPSS) without del[5q] achieved a 43% overall rate of hematologic improvement [4]. However, there were no significant differences in the rate of transfusion independence according to age, sex, FAB type, IPSS category, cytogenetic pattern, or early cytopenias. In higher risk (IPSS; Int-2, high) MDS patients with del[5q] with or without other karyotypic abnormalities, 27% achieved complete remission (CR), including 67% of patients with isolated del[5q], vs. 1/1 and 0/27 patients with one or more than one additional chromosomal abnormalities, respectively (P < .001) [3]. Recently, high-dose LEN therapy resulted in a 14% CR/partial response (PR) rate in AML patients with del[5q] [6], and a 30% CR/complete remission without complete recovery of all blood counts (CRi) rate in older AML patients without del[5q] [5]. To date, the presence of del[5q] with or without additional chromosomal abnormalities detected by metaphase cytogenetics (MC) remains the best prognostic factor for response to LEN. As patients without del[5q] can also show responses to LEN, identification of additional markers of response/resistance is of utmost importance. Clinically, cytogenetic abnormalities including cryptic deletions of 5q, along with certain other mutations, may constitute additional lesions predictive of response. For instance, the presence of TP53 mutations has been shown to be associated with poor prognosis in azacitidine-treated MDS patients [7], and in LEN-treated MDS or AML patients with del[5q] [8, 9].
The diagnostic yield of MC can be enhanced by application of fluorescence in situ hybridization (FISH) for targeted detection of chromosomal lesions including del[5q], as this technique is considered to be more sensitive and allow for detection of smaller clones [10]. Similarly, single nucleotide polymorphism array (SNP-A)-based karyotyping, due to its superb resolution, may allow for detection of previously cryptic unbalanced chromosomal defects [10] and [11]. Both techniques can be performed on interphase cells, and thereby do not require cell division.
In addition to mostly unbalanced cytogenetic defects, mutations of a number of genes, including TET2[12, 13], UTX[14], CBL[15], EZH2[1618], ASXL1[1921], TP53[7, 22, 23], RAS[24, 25], IDH1/2[26], and DNMT3A[27] have been implicated in the pathogenesis of MDS and may also modulate clinical features including responsiveness to LEN.
We examined a cohort of patients without del[5q] treated with LEN and explored the relationship between molecular features and clinical response to LEN.

Methods

Patients

Bone marrow (BM) and/or peripheral blood (PB) were collected from 755 patients with myeloid malignancies seen at Cleveland Clinic (CC) and H. Lee Moffitt Cancer Center between 2002 and 2010. First, a cohort of 122 patients, who were examined with all 3 cytogenetic methods (MC, FISH and SNP-A) on the same sample, was collected. Next, data from 42 patients with MDS (31; RA, 5; RARS, 13; RCMD, 1; RAEB-1, 4; RAEB-2, 7; MDS-U, 1), myeloproliferative neoplasms (MPN) (PMF, 2), MDS/MPN overlap syndrome (7; CMML-1, 2; CMML-2, 2; MDS/MPN-U, 3), or 2 secondary acute myeloid leukemia (sAML) without del[5q], who received LEN for at least 8 weeks, were collected retrospectively. The schedule and dosage of lenalidomide was primarily 10 mg/day (5 mg/day in a few cases) for 1-21 days, with a 28-day cycle. All bone marrow biopsies were reviewed and diagnoses confirmed at Cleveland Clinic and H. Lee Moffitt Cancer Center. Response to LEN was defined by the modified International Working Group (IWG) response criteria (2006) [28]. Informed consent for sample and clinical information collection was obtained according to protocols approved by the Cleveland Clinic or the H. Lee Moffitt Cancer Center IRBs.

Cytogenetic analysis

Cytogenetic analysis was performed on marrow aspirates and/or peripheral blood, in cases where bone marrow samples could not be obtained, according to standard methods (Figure 1). 20 metaphase spreads were examined per patient, if available. Chromosome preparations were G-banded using trypsin and Giemsa (GTG) and karyotypes were described according to ISCN [29].

Fluorescence in situ hybridization

FISH analysis was performed on cell pellets from unstimulated cytogenetic cultures. Thresholds for interpretation as a positive result were established for each probe at 3 standard deviations above the mean of 20 normal bone marrow samples. In 27 cases, FISH analysis was performed at an outside reference laboratory (Mayo Clinic) using the following dual color probe sets: 5p15.2 (normal range; 0-4%)/EGR1 (5q31) (0-6%), CEP7 (0-5%)/7q31 (0-7%), CEP8 (0-2%)/MYC (8q24) (0-2%) and 20q12 (0-5%)/20qter (0-5%). In 95 cases, FISH was performed at CC using three dual color probe sets (Abbott Molecular, Abbott Park, IL). The first probe set consisted of D5S23 and D5S721 (5p15.2) labeled in Spectrum Green (0-6%) and EGR1 (5q31) labeled in Spectrum Orange (0-6%). The second probe set consisted of the chromosome 7 centromere labeled in Spectrum Green (0-5%) and D7S486 (7q31) labeled in Spectrum Orange (0-7%). The third probe set consisted of the chromosome 8 centromere labeled in Spectrum Green (0-8%) and D20S108 (20q12) labeled in Spectrum Orange (0-4%).

DNA extraction

DNA was extracted from whole bone marrow using the ArchivePure Kit (5Prime, Gaithersburg, MD) per manufacturer's instructions. The concentration of the DNA was obtained using a ND-1000 spectrophotometer (NanoDrop, Wilmington, DE, USA). To study the germ line, T lymphocytes (CD3+) were isolated using RoboSep according to manufacturer's protocol (StemCell Technologies, Vancouver, BC, Canada).

Mutational analysis

Mutation screening was performed for genes known to be mutated in myeloid malignancies (TET2, UTX, CBL, EZH2, ASXL1, TP53, RAS, IDH1/2, and DNMT3A) in the cases for which DNA was available (N = 21). All of the coding exons for TET2, UTX, EZH2 and TP53 were screened as previously reported [1317] and [23]. Direct genomic sequencing of exons 8 and 9 of CBL, exon 12 of ASXL-1, exons 1 and 2 of N-RAS and K-RAS, exon 4 of IDH-1 and IDH-2, and exon 23 of DNMT3A was performed as previously described [15, 21, 26, 27, 30]. The reference sequence from UCSC Genome Browser was used to identify the position of each amino acid change listed in Table 1 (TET2, uc003hxk.2; EZH2, uc003wfb.1; ASXL-1, uc002wxs.1; KRAS, uc001rgp.1; DNMT-3, uc002rgc.1). In selected cases CD3+ cells were purified and used as controls to confirm the somatic status of mutations.
Table 1
Mutation analysis in the cohort of LEN patients
Case
Response
TET2
UTX
CBL
EZH2
ASXL1
TP53
N-RAS
K-RAS
IDH1
IDH2
DNMT3A
1
CR
WT
WT
WT
WT
WT
WT
WT
WT
WT
WT
WT
2
CR
WT
WT
WT
WT
L775fsX1
WT
WT
WT
WT
WT
WT
3
PR
WT
WT
WT
WT
WT
WT
WT
WT
WT
WT
WT
4
NR
WT
WT
WT
WT
WT
WT
WT
WT
WT
WT
WT
5
CR
WT
WT
WT
WT
E1102D
WT
WT
WT
WT
WT
R882H
6
PR
P1681fsX2
WT
WT
WT
P1277fsX2
WT
WT
WT
WT
WT
WT
7
CR
WT
WT
WT
WT
WT
WT
WT
WT
WT
WT
WT
8
HI
WT
WT
WT
WT
WT
WT
WT
WT
WT
WT
WT
10
CR
WT
WT
WT
WT
WT
WT
WT
WT
WT
WT
WT
11
HI
WT
WT
WT
WT
WT
WT
WT
L19F
WT
WT
WT
12
CR
V1417F
WT
WT
WT
WT
WT
WT
WT
WT
WT
WT
13
NR
WT
WT
WT
WT
WT
WT
WT
WT
WT
WT
WT
14
PR
WT
WT
WT
WT
WT
WT
WT
WT
WT
WT
WT
15
CR
T1978P
WT
WT
WT
WT
WT
WT
WT
WT
WT
WT
16
NR
WT
WT
WT
WT
S846N
WT
WT
WT
WT
WT
WT
17
NR
N1068fsX13
WT
WT
WT
WT
WT
WT
WT
WT
WT
WT
18
NR
WT
WT
WT
WT
WT
WT
WT
WT
WT
WT
WT
19
NR
P1962L
WT
WT
WT
WT
WT
WT
WT
WT
WT
WT
20
CR
WT
WT
WT
WT
WT
WT
WT
WT
WT
WT
R882H
21
HI
WT
WT
WT
T726X
WT
WT
WT
WT
WT
WT
WT
22
HI
WT
WT
WT
WT
WT
WT
WT
WT
WT
WT
WT
Abbreviation: CR complete response; PR partial response; HI hematologic improvement; WT wild type

SNP-A cytogenetics

Affymetrix Gene Chip Mapping 250 K Assay Kit or Genome-Wide Human SNP Assay Kit 6.0 (Affymetrix, Santa Clara, CA) was used for analysis of 52 and 70 samples with myeloid malignancies, respectively. Following Nsp I digestion (New England Biolabs, Ipswich, MA), fragmented DNA was ligated to adaptor using T4 ligase (New England Biolabs) followed by PCR amplification. The PCR product was hybridized to the array, processed with the Fluidic Station 450 and scanned using the Gene Chip Scanner 3000 (Affymetrix).

Biostatistical evaluation of SNP-A data

GeneChip Mapping 250 K Array data, signal intensity and SNP calls were determined using Gene Chip Genotyping Analysis Software Version 4.0 (GTYPE). Copy number and LOH were investigated using Copy Number Analyzer for Affymetrix GeneChip Mapping (CNAG v. 3.0). For Genome-Wide Human SNP Array 6.0, the genotype calls for each individual were determined by the Birdseed version 1 genotype-calling algorithm, embedded in the software included with the Affymetrix Genotyping Console 2.0 (Affymetrix).
For detection of lesions we used the following diagnostic algorithm: lesions identified by SNP-A were compared with the Database of Genomic Variants (http://​projects.​tcag.​ca/​variation/​) and our own internal control series to exclude known copy number variants (CNVs). In our internal control cohort, the largest area of copy neutral loss of heterozygosity (CN-LOH) we observed was 52.5 Mb and the average size of CN-LOH was 7.2 Mb. In addition, we observed that areas of LOH in controls were exclusively interstitial. Consequently, areas of LOH < 24.8 Mb (mean size ± 2SD) were excluded from analysis in the patient set. Deletions and gains of chromosomal material seen on metaphase karyograms and SNP-A samples that showed a concordantly normal karyotype by both MC and SNP-A testing were not further confirmed. When possible, all other remaining new defects were confirmed using paired analysis of CD3+ cells.

Statistical analysis

Demographic and baseline MDS disease characteristics of all patients were summarized descriptively, using medians and ranges. The response differences between 2 groups were compared using Fisher's exact test, with a two-sided alpha value of .05 denoting significance.

Results

Comparison between metaphase cytogenetics and other cytogenetic methods

We first identified a cohort of 122 patients for whom MC, FISH and SNP-A analyses were performed on the same sample, to evaluate the additional yield of more sensitive techniques for identifying del[5q]. In patients with MDS (N = 82), MDS/MPN (N = 13), AML (N = 23), and MPN (N = 4), the detection rate of del[5q] increased only marginally with the use of additional techniques, from 24% (MC + FISH), to 25% (MC + SNP-A), 25% (FISH + SNP-A) and 26% (all 3 methods) (Figure 1, Table 2). We also identified 3 cases with copy neutral loss of heterozygosity (CN-LOH) of 5q in this cohort using SNP-A. One region of CN-LOH was found in sAML with complex chromosomal abnormalities including del[5][q13q33] by MC, and the other 2 CN-LOH regions were detected in MDS cases with chromosomal abnormalities other than del[5q].
Table 2
Number and percentage of del[5q] detected using metaphase cytogenetics, FISH and SNP-A alone or in combination in myeloid malignancies (N = 122)
 
MC
FISH
SNP
MC+FISH
MC+SNP
FISH+SNP
MC+FISH+SNP
Number
24
27
25
29
30
31
32
Percentage
20%
22%
21%
24%
25%
25%
26%
MC metaphase cytogenetics; FISH fluorescence in situ hybridization; SNP single nucleotide polymorphism array-based karyotyping.

Clinical characteristics of non-del[5q] patients who had LEN therapy

Clinical characteristics of the patients with myeloid malignancies without del[5q] by MC and who received LEN are summarized in Table 3; 31 patients received LEN monotherapy (complete response [CR], N = 3; partial response [PR], N = 2; hematologic improvement [HI], N = 9; no response [NR], n = 13; not evaluated [NE], n = 4), and 11 patients received LEN/azacitidine (AZA) combination therapy (CR, N = 6; PR, N = 1; HI, N = 1; NR, N = 3). Only 1 patient had past history of Hodgkin's lymphoma and was suspected to have therapy-related MDS/MPN.
Table 3
Summary of clinical characteristics of patients without del[5q] on MC who received lenalidomide (N = 42)
Diagnosis (No. of Patients)
   
MDS
 
31
 
 
RA
 
5
 
RARS
 
13
 
RCMD
 
1
 
RAEB-1
 
4
 
RAEB-2
 
7
 
MDS-U
 
1
MDS/MPN
 
7
 
 
CMML-1
 
2
 
CMML-2
 
2
 
MDS/MPN-U
 
3
PMF
 
2
 
sAML
 
2
 
Age (years old)
   
 
Median (Range)
 
70 (46-83)
Sex (No. of Patients)
   
 
M
 
28
 
F
 
14
IPSS (No. of Patients)
   
 
LOW
 
12
 
INT-1
 
11
 
INT-2
 
7
 
HIGH
 
1
 
not indicated
 
11
Duration of MDS (months)
 
Median (Range)
 
15 (0-118)
Previous Therapies
 
Yes
 
27
 
No
 
15
Transfusion dependence (No. of Patients)
 
Yes
 
30
 
No
 
12
Neutropenia (< 1.5 × 109/μ) (No. of Patients)
 
Yes
 
5
 
No
 
37
Thrombocytopenia (< 100 × 109) (No. of Patients)
 
Yes
 
12
 
No
 
30
Therapy (No. of Patients)
   LEN (5-10 mg/day) alone
  
30
   LEN high dose (50 mg/day)
  
1
 
LEN/AZA
 
11
Duration of LEN therapy (months)
 
Median (Range)
 
5 (0-76)
Response to therapy (No. of Patients)
 
CR
 
9
 
PR
 
3
 
HI
 
10
 
NR
 
16
 
NE
 
4
Abbreviation: MDS myelodysplastic syndromes; RA refractory anemia; RARS refractory anemia with ring sideroblasts; RCMD refractory cytopenia with multilineage dysplasia; RAEB refractory anemia with excess blasts, MDS-U MDS unclassifiable; MDS/MPN MDS/myeloproliferative neoplasm; CMML chronic myelomonocytic leukemia; MDS/MPN-U MDS/MPN unclassifiable; PMF primary myelofibrosis; sAML secondary acute myeloid leukemia; M male; F female; LEN lenalidomide; LEN/AZA LEN/azacitidine; CR complete response; PR partial response; HI hematological improvement; NR no response; NE not evaluated.
By MC, 32 patients (76%) who received treatment with LEN showed a normal karyotype, 1 patient (2.4%) had no growth to their bone marrow sample, and 9 (21%) had an abnormal karyotype other than del[5q] (Table 4). However, the frequency of an abnormal karyotype was increased to 67% using FISH and SNP-A as karyotyping tools in patients receiving LEN without del[5q] by MC (Figure 2). Previously cryptic del[5q] was detected by both SNP-A and FISH in an additional 1/18 patients with normal MC (Case 19 in Table 4). Del[5q] was also revealed by FISH in 1 patient with unsuccessful MC (Case 14 in Table 4), but, due to the small size of the clone (8%), SNP-A was not able to detect this lesion.
Table 4
Patients characteristics who received LEN without del[5q] by MC (N = 42)
Case
Age (y.o.)
Sex
Diagnosis
IPSS
Therapy
Response
MC
FISH
SNP-A
  
1
73
M
RAEB-2
INT-2
LEN/AZA
CR
N
N
 
Gain
4q13.2
2
75
M
RAEB-1
INT-1
LEN/AZA
CR
N
trisomy 8
9%
Gain
8q11.1q11.21
          
UPD
11q14.1q21
3
62
M
MDS/MP N-U
INT-1
LEN/AZA
PR
N
N
 
UPD
1pterp32.3
          
UPD
3p21.31p21.1
4
68
M
RAEB-2
HIGH
LEN/AZA
NR
complex karyotype, including trisomy 8, del[7q], del[12], and del[20]
del[7q]
68%
Gain
8
        
del[20q]
60%
Loss
11p14.3p13
        
trisomy 8
41%
Loss
12p12.3p11.21
          
Loss
12q21.1q21.31
          
Loss
16q22.3q24.3
          
Loss
1p22.2p22.1
          
Loss
20q11.2q13.33
          
Loss
21q11.2q21.1
          
Loss
2q31.3q32.1
          
Loss
6q23.3
          
Gain
7q11.21q11.22
          
Loss
7q22.1q36.2
5
68
M
RAEB-2
INT-2
LEN/AZA
CR
47,XY,+8[6]
trisomy 8
9%
Gain
14q11.1q11.2
          
UPD
19p13.11p12
          
Gain
8p23.3q24.3
6
73
M
CMML-1
NE
LEN
PR
N
del[20q]
10%
UPD
9pterp22.2
7
67
M
PMF
NE
LEN
PR
N
N
 
Loss
11q23.3
8
66
M
RCMD
LOW
LEN
HI
46,XY,del(20)(q11q13)[2]/4 6,XY[18]
del[20q]
17%
Gain
8q11.1q11.23
9
69
M
RAEB-1
INT-2
LEN/AZA
CR
N
N
 
NE
 
10
64
M
MDS/MP N-U
NE
LEN
CR
N
N
 
N
 
11
79
F
CMML-2
NE
LEN/AZA
HI
47,XX,+8[20]
trisomy 8
42%
Gain
8
12
62
F
RAEB-2
INT-2
LEN/AZA
CR
N
N
 
N
 
13
62
F
RARS
INT-1
LEN
NR
46,XX,add(15)(p11.1),add(2 2)(p11.2)[3]/47,idem,+19[19].
del[7q]
11%
Gain
19
        
del[7]
6%
Gain
3q26.1
          
Gain
4p16.2
14
72
M
MDS/MPN-U
INT-2
LEN
PR
no growth
del[5q]
8%
Loss
20q11.1q13.12
        
del[20q]
35%
Loss
2p21p24.1
          
Loss
8q11.23q12.1
          
Gain
9p12pter
15
63
M
RAEB-2
NE
LEN/AZA
CR
N
N
 
Gain
12q24.32
          
Gain
8q11.1
16
81
F
RAEB-2
INT-2
LEN/AZA
NR
N
N
 
N
 
17
69
M
CMML-2
-
LEN/AZA
NR
N
NE
 
Loss
2p22.3
18
46
M
PMF
-
LEN
NR
balanced translocation at chromosomes 2 and 22
NE
 
UPD
14q31.3q32.33
          
Gain
9p24.3p11.1
19
70
F
sAML
-
LEN (High dose)
NR
N
del[5q]
33%
Loss
5q31.2
20
65
F
RARS
LOW
LEN
CR
N
NE
 
N
 
21
70
M
CMML-1
INT-1
LEN
HI
N
N
 
UPD
7q22.1qter
22
83
M
RARS
LOW
LEN
HI
N
N
 
N
 
23
83
M
RARS
INT-1
LEN
NR
N
NE
 
N
 
24
71
M
MDS-U
INT-1
LEN
NE
N
NE
 
Loss
15q14
25
69
M
RARS
LOW
LEN
NR
N
NE
 
N
 
26
76
M
RA
INT-1
LEN
NR
N
NE
 
Loss
3p22.3
27
68
M
RA
INT-1
LEN
NR
N
NE
 
Loss
21q21.2
          
Gain
3p14.1
          
Loss
11q14.3
28
59
M
RARS
LOW
LEN
NR
N
NE
 
N
 
29
73
F
RA
LOW
LEN
NR
N
NE
 
N
 
30
78
F
RARS
LOW
LEN
NE
N
NE
 
UPD
3q21.3qter
31
78
M
RAEB-1
INT-2
LEN
NR
47,XY,+19
NE
 
Gain
19
32
80
M
RAEB-1
INT-1
LEN
NE
N
NE
 
Loss
17q11.2
          
UPD
8p11.2qter
33
79
F
RARS
INT-1
LEN
HI
N
NE
 
N
 
34
73
F
RARS
LOW
LEN
HI
N
NE
 
Loss
18p11.32
35
78
F
sAML
-
LEN
NR
46,XX,t(3;3)
NE
 
Loss
3q26.1
36
80
M
RAEB-2
INT-2
LEN
NR
N
NE
 
Loss
22q13.2
37
69
M
RARS
LOW
LEN
HI
N
NE
 
N
 
38
59
F
RA
LOW
LEN
HI
N
NE
 
N
 
39
62
F
RARS
LOW
LEN
NE
N
NE
 
Gain
6p21.32
40
82
M
RARS
INT-1
LEN
NR
46,XY,del(20)(q11.2)
NE
 
Loss
20q11.2q13.2
41
53
M
RARS
LOW
LEN
HI
N
NE
N
  
42
56
F
RA
INT-1
LEN
HI
N
NE
 
Loss
9p21.2
          
Gain
1p21.1
Abbreviation: M male; F female; RAEB refractory anemia with excess blasts; MDS/MPN-U myelodysplastic syndromes/myeloproliferative neoplasm, unclassifiable; CMML chronic myelomonocytic leukemia; PMF primary myelofibrosis; RCMD refractory cytopenias with multilineage dysplasia; RARS refractory anemia with ring sideroblasts; sAML secondary acute myeloid leukemia; MDS-U, MDS unclassifiable; RA refractory anemia; LEN lenalidomide; AZA azacitidine; N normal; UPD uniparental disomy; NE not evaluated.

Impact of chromosomal abnormalities on response

In 27 patients who received LEN for more than 2 months, the overall response rate (ORR) was 52%, including 3 CR, 2 PR and 9 HI. Case 19, who was diagnosed as sAML with del[5q] by FISH and SNP-A only, was refractory to high-dose LEN. Case 14, a MDS/MPN unclassifiable (MDS/MPN-U) patient in whom a del[5q] clone was detected only by FISH due to the small size, had a sustained PR with transfusion independence. The ORR to LEN in patients with normal MC was 60%, vs. 17% for those with chromosomal aberrations by MC (p = .08); the addition of FISH or SNP-A did not improve the predictive value of normal cytogenetics (Table 5). We also analyzed 11 patients without del[5q] by MC who received combination therapy with AZA and LEN, for whom the ORR was 73% (6 CR, 1 PR, 1 HI). By MC, 8/11 patients had a normal karyogram and a response of 75%, compared to 3 patients with chromosomal lesions, 1 of whom did not respond. Similar to the results with LEN alone, inclusion of defects detected by SNP-A or FISH did not allow for better separation of responders based on normal cytogenetics by MC. The most frequent cytogenetic abnormality among the patients who received LEN was gain of chromosome 8 material (6/42), followed by the loss of chromosome 20 material (5/42). Patients with gain of chromosome 8 had high ORR to LEN (5 out of 6, 83%) and ORR in patients with chromosome 20 abnormalities was 3 out of 5. The response of patients with all other chromosomal abnormalities by MC, FISH or SNP-A was 44%. These findings indicate that responses tend to be more often observed in patients with gain of chromosome 8 material by either of all 3 karyotyping methods (p = .11), although 5 out of those 6 patients received combined LEN/AZA therapy.
Table 5
Cytogenetic categories and response to therapy in the cohort of LEN patients
A. All patient who received LEN for more than 3 months (N = 38)
 
 
Normal cytogenetic group
Abnormal cytogenetic group
p value
Categorized by MC only
64%
33%
0.07
Categorized by MC/FISH/SNP-A
64%
54%
0.4
B. Monotherapy (LEN only) patients (N = 27)
  
 
Normal cytogenetic group
Abnormal cytogenetic group
p value
Categorized by MC
60%
17%
0.08
only
   
Categorized by MC/FISH/SNP-A
64%
44%
0.27
C. Patients with combination therapy of AZA + LEN (N = 11)
 
 
Normal cytogenetic group
Abnormal cytogenetic group
p value
Categorized by MC only
75%
67%
0.85
Categorized by MC/FISH/SNP-A
67%
75%
0.85
LEN lenalidomide; MC metaphase cytogenetics; FISH fluorescence in situ hybridization; SNP-A single nucleotide polymorphism array karyotyping; AZA azacitidine.

Impact of mutational status on response

In 21 LEN-treated patients (11 patients with LEN only and 10 patients with LEN/AZA), somatic mutations were found in TET2 (N = 5), EZH2 (N = 1), ASXL1 (N = 4), K-RAS (N = 1), and DNMT3A (N = 2) in 11 patients. ASXL1 and DNMT3, or TET2 and ASXL1 mutations were each found in one patient, and each of these patients achieved CR with LEN/AZA or PR with LEN only. ORR was 73% in patients with any of investigated mutations and 70% in patients without a mutation (p = .36). For patients treated with LEN only, 3 out of 8 (38%) responders had mutations, and 1 out of 3 (33%) non-responders harbored mutations.

Discussion

Though the mechanism of action of lenalidomide has not been definitively determined, it purportedly works through inhibition of phosphatase activity in the common deleted region (CDR) of 5q that plays a key role in cell cycle regulation, through a defect in ribosomal protein function, via direct cytotoxic mechanisms in patients with the del (5q) cytogenetic abnormality, and supposedly through effects on the bone marrow microenvironment in patients who do not have this lesion, via abrogation of the effects of pro-apoptotic, pro-inflammatory cytokines [13]. Until now, additional markers of responsiveness to LEN beyond del[5q] have not been identified.
New cytogenetic tools such as FISH and SNP-A are likely to improve the diagnostic value of cytogenetic diagnostics [8, 9, 29]. We first assumed that we would detect previously unrecognized cases of del[5q] using these techniques. In our cohort of patients given LEN, del[5q] cases detected by FISH and/or SNP-A ranged from 2 out of all 42 cases (4.8%) of patients without del[5q] by MC, and 1 out of 32 cases (3.1%) with normal MC. These frequencies are similar to those reported in a previous study, in which 5.96% of cases without del[5q] by MC and 2.7% of those with normal karyotype by MC were found to be del[5q] by FISH [31]. These results suggest that FISH and SNP-A may marginally improve the detection rate of del[5q]. While the detection rate of cryptic del[5q] was only marginally enhanced with FISH and SNP-A, new karyotyping tools improved the detection rate of other chromosomal abnormalities in our cohort from 21% to 67% compared to MC. A previous study of 43 MDS patients suggested that the cytogenetic pattern correlates with hematologic response; 10 of 12 patients (83%) with del[5q] achieved sustained red blood cell transfusion independence, compared with 57% of those with a normal karyotype and 12% of those with other karyotypic abnormalities [1, 32]. Consequently, we hypothesized that improved detection of chromosomal abnormalities may better predict poor response to LEN. However, when more abnormalities were found using additional methods, no correlation with response to LEN or LEN/AZA therapy was detected.
We also speculated that we could recognize other chromosomal markers of response or refractoriness to LEN besides del[5q] using FISH and SNP-A. For example, trisomy 13 as the sole cytogenetic abnormality was reported to be possible good prognostic factor to LEN therapy [33], but was not detected in our LEN cohort. Instead, we found gain of chromosome 8 material to be predictive of response to LEN, although we acknowledge that 5 out of those 6 patients received combined LEN/AZA therapy [34, 35].
In addition to cytogenetic abnormalities, we also studied mutational status of a variety of genes as possible markers of response. For example, ASXL1 mutations in CMML [36] and DNMT3A in AML [27] were reported to be poor prognostic factors. We identified 2 patients with DNMT3A mutations in our cohort, both of them achieved CR with LEN or LEN/AZA therapy.
This cohort used in this study has several limitations, including a limited size and the inclusion of patients with heterogenous disease entities. But, we have been able to demonstrate that a normal karyotype and gain of chromosome 8 material was predictive of response to LEN, while additional testing by FISH or SNP-A is not useful for better prediction of response in non-del[5q] patients with myeloid malignancies.

Acknowledgements

This work was supported by in part by R01HL-082983, K24 HL-077522, and grants from AA&MDS International Foundation and Robert Duggan Charitable Fund (J.P.M).

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

YS and JPM designed research, performed research, analyzed data, and wrote the paper. HM, FT, VV, AJ, AJ, HS, CLO, KG performed research. MA, RT, KRM and AFL analyzed data. All authors read and approved the final manuscript.

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