Open Access

High IKZF1/3 protein expression is a favorable prognostic factor for survival of relapsed/refractory multiple myeloma patients treated with lenalidomide

  • Maryam Pourabdollah1,
  • Mohammad Bahmanyar1,
  • Eshetu G. Atenafu2,
  • Donna Reece3,
  • Jian Hou4 and
  • Hong Chang1, 5Email author
Journal of Hematology & Oncology20169:123

https://doi.org/10.1186/s13045-016-0354-2

Received: 16 September 2016

Accepted: 3 November 2016

Published: 21 November 2016

Abstract

The aim of this study is to assess nucleoprotein expression of IKZF1/3 in patients with relapsed/refractory multiple myeloma (MM) who received lenalidomide-based therapy and correlated them with their clinical outcomes. A total of 50 patients diagnosed with MM were entered in the study with the median follow-up of 86.4 months. By immunohistochemistry (IHC), IKZF1 and IKZF3 were expressed in 72 and 58% of the cases, respectively. IKZF1 and IKZF3 expressions were associated with longer median progression free survival (P = 0.0029 and P < 0.0001) and overall survival (P = 0.0014 and P < 0.0001). IKZF3 expression also appears predicted a favorable response to the lenalidomide-based therapy.

It has been demonstrated that lenalidomide causes selective degradation of IKZF1 (ikaros) and IKZF3 (aiolos) which are two essential transcription factors for myeloma cell proliferation [1, 2]. This anti-proliferative effect is mediated by downregulation of c-Myc and interferon regulatory factor 4 (IRF4) [3]. In particular, IKZF3 regulates expression of IRF4 which is linked with lenalidomide activity [46]. However, the clinical relevance of IKZF1/IKZF3 expressions in myeloma patients has not been established. Thus, we examined nuclear expression of IKZF1/3 and its correlation with clinical outcomes in patients with relapsed/refractory MM who received lenalidomide therapy.

A total of 50 patients diagnosed with MM in our institution were entered in the study. All had received lenalidomide-based therapy (lenalidomide plus dexamethasone) after relapse. The median follow-up after diagnosis was 7.2 years. The relevant clinical and laboratory features are summarized in Table 1.
Table 1

Clinical and laboratory features of MM patients treated with lenalidomide

Clinical feature

Total (n = 50)

IKZF1 high expression (n = 36)

IKZF1 low expression (n = 14)

P value

IKZF3 high expression (n = 29)

IKZF3 low expression (n = 21)

P value

Sex (M/F)

31/19

23/13

8/6

0.6590

19/10

12/9

0.5471

Age (year), median (range)

59(41–75)

57 (41–73)

59 (45–75)

0.6416

57 (41–69)

59 (44–75)

0.3105

International staging system, no. (%)

   

0.1785

  

0.1448

 I

24 (48)

14 (38.89)

10 (71.43)

 

11 (37.93)

13 (61.90)

 

 Il

18 (36)

14 (38.89)

4 (28.57)

 

11 (37.93)

7 (33.33)

 

 III

5 (10)

5 (13.89)

0 (0)

 

5 (17.24)

0 (0)

 

 NA

3 (6)

3 (8.33)

0 (0)

 

2 (6.90)

1 (4.76)

 

Hemoglobin concentration (g/L), median (range)

105 (76–147)

106 (76–147)

103 (86–132)

0.6027

104 (76–147)

107 (85–141)

0.3121

Calcium (mmol/L), median (range)

2.25 (1.98–2.57)

2.26 (1.98–2.57)

2.23 (2–2.55)

0.8162

2.25 (1.98–2.57)

2.25 (2–2.55)

0.7160

Creatinine (μmol/L), median (range)

76 (32–360)

86 (40–360)

67.58 (32–126)

0.0569

88 (57–360)

66 (32–126)

0.0072

Having lytic lesions, number of patients (%)

27 (54)

18 (50)

9 (64.29)

0.3628

12 (41.38)

15 (71.43)

0.0354

B2-microglobulin (mg/L)

3.08 (0.51–16.76)

3.50 (0.51–16.76)

2.96 (1.35–5.16)

0.5817

3.62 (0.51–16.76)

2.82 (1.07–5.16)

0.1293

Albumin (gr/L)

40.5 (28–47)

41 (28–47)

39 (29–44)

0.6151

41 (28–47)

40 (29–44)

0.9078

Prior therapies, no. (%)

 ≥3

23 (46)

15 (41.67)

8 (57.14)

0.3242

12 (41.38)

11 (52.38)

0.4411

 Thalidomide

29 (58)

22 (61.11)

7 (50)

0.4748

15 (51.72)

14 (66.67)

0.2907

 Bortezomib

21 (42)

14 (38.89)

7 (50)

0.4748

12 (41.38)

9(42.86)

0.9168

 ASCT

40 (80)

31 (86.11)

9 (64.29)

0.1180

25 (86.21)

15 (71.43)

0.2859

Response to lenalidomide plus dexamethasone, no. (%)

 Responsive

41 (82)

32 (88.89)

9 (64.29)

0.094

28 (96.55)

13 (61.90)

0.0025

 Non-responsive

9 (18)

4 (11.11)

5 (35.71)

 

1 (3.45)

8 (38.1)

 

Cytogenetics, no. (%)

del (13q)

   

1.0000

  

0.7243

 Positive

13 (26)

9 (25)

4 (28.57)

 

7 (24.14)

6 (28.57)

 

 Negative

37 (74)

27 (75)

10 (71.43)

 

22 (75.86)

15 (71.43)

 

del (17p)

   

0.1966

  

0.2552

 Positive

8 (16)

4 (11.11)

4 (28.57)

 

3 (10.34)

5 (23.81)

 

 Negative

42 (84)

32 (89)

10 (71.43)

 

26 (89.66)

16 (76.19)

 

t(4;14)

   

1.0000

  

0.7163

 Positive

9 (18)

7 (19.44)

2 (14.29)

 

6 (20.69)

3 (14.29)

 

 Negative

41 (82)

29 (80.56)

12 (85.71)

 

23 (79.31)

18(85.71)

 

amp(1q21)

   

0.1228

  

0.1283

 Positive

20 (40)

12 (33.33)

8 (57.14)

 

9 (31.03)

11 (52.38)

 

 Negative

30 (60)

24 (66.67)

6 (42.86)

 

20(68.97)

10 (47.62)

 
CD138 and IKZF1/3 immunohistochemical (IHC) staining were performed on the bone marrow aspiration/biopsy specimens taken before starting lenalidomide. CD138 positive myeloma cell aggregates (Additional file 1: Figure S1B) were examined for IKZF1/3 expression (Additional file 1: Figure S1C, D). H-score method (range 0–300) according to staining intensity and percentage of myeloma cells was applied. The median H-scores for IKZF1 and IKZF3 were 150 and 200, respectively, and high or low expression was based on above or below the median H-score (Fig. 1a).
Fig. 1

Expression of IKZF1 and IKZF3 proteins in tumor cells, measured by H-score (a) and their correlation with clinical response (b). Progression free survival (PFS) and overall survival (OS) in relation to IKZF1 nuclear expression (c, d), and in relation to IKZF3 nuclear expression (e, f) detected by IHC, respectively

Of the 50 MM cases, IKZF1 and IKZF3 were expressed in 36 (72%) and 29 (58%) cases, respectively. Twenty-eight of 29 cases (97%) with high IKZF3 expression also showed IKZF1 positivity (P < 0.0001). High IKZF3 (P = 0.0025), but not IKZF1 expression (P = 0.094) was strongly correlated with clinical response (Fig. 1b). Patients with high IKZF1 or IKZF3 expression showed longer PFS (median 22.6 vs. 6.3 months, P = 0.0029; or 43.2 vs. 3.7 months; P < 0.0001) and OS (median 44.3 vs. 12.1 months, P = 0.0014; or 47 vs. 12.1 months; P < 0.0001) (Fig. 1c–f), respectively. In addition, the group with both high IKZF1/3 expression was associated with longer PFS (median 31.8 vs. 3.9 months, P < 0.0001) and OS (median OS 58.7 vs. 12.1 months, P < 0.0001), whereas the group with both low expression was correlated with shorter PFS (median 4.1 vs. 26.8 months, P = 0.0003) and OS (median 12.1 vs. 46.2 months, P = 0.0002). Of note, high IKZF3 expression appeared associated with higher creatinine but with less lytic lesion, and by multivariable analysis, high IKZF3 expression remained an independent poor risk factor for PFS (P < 0.0001) and OS (P < 0.0001) after adjusting these two covariates. There was no significant association between IKZF1/3 protein expression and other clinical or biological risk factors (Table 1).

Previous studies have indicated controversial results about the relationship between Ikaros expression level and resistance to lenalidomide. Lu et al. [7] found that some MM cell lines with higher expressions of IKZF1 or IKZF3 showed resistance to the drug; in contrast, Zhu et al. [8] showed that low IKZF1 transcript levels were correlated with poor response to IMiDs. They also found that higher IKZF1 but not IKZF3 gene expression was associated with better OS. Our study demonstrates that expression of IKZF1/3 proteins (especially IKZF3) is correlated with better outcome in refractory MM patients treated with lenalidomide. A possible explanation for this observation is that in the presence of high IKZF1/3 levels, myeloma cells are more dependent on IKZF-associated signaling for proliferation. Particularly, IKZF3 is linked to plasma cell development and lenalidomide efficacy as IKZF3 is specifically required for the generation of long-lived plasma cells and it has been shown to be reduced by lenalidomide [9, 10].

To the best of our knowledge, this is the first report to show a correlation between IKZF1/3 protein expressions and clinical outcomes in refractory MM treated with lenalidomide. However, this study has limitations as it is retrospective with limited sample size. Nevertheless, as paraffin IHC is routinely available, robust, and inexpensive, if confirmed in a larger prospective study, IKZF1/3 (especially IKZF3) immunostaining can be readily adopted in clinical practice for prediction of drug response and clinical outcomes in MM patients receiving lenalidomide therapy.

Declarations

Acknowledgements

Not applicable.

Funding

The study was supported in part by the grants from Leukemia and Lymphoma Research Society of Canada (LLSC), Cancer Research Society (CRS) and Shanghai Science and Technology International Collaboration Project (No. 15410710300). The funding agencies played no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.

Availability of data and materials

Please contact author for data requests.

Authors’ contributions

MP and MB carried out the experiments. MP drafted the manuscript. EA performed the statistical analysis. JH participated in design the study. DR contributed to patient clinical data and sample collections. HC conceived of the study, participated in its design and coordination, and helped to draft the manuscript. All authors read and approved the final manuscript.

Competing interests

D.R. is a consultant/advisory role for Celgene. All remaining authors declare no competing interests.

Consent for publication

NA.

Ethics approval and consent to participate

The study was approved by Research Ethic Board of University Health Network (UHN): Identification of Novel Prognostic Markers in Multiple Myeloma. 05-0573-TE.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Department of Laboratory Medicine and Pathobiology, University of Toronto
(2)
Department of Biostatistics, University of Toronto
(3)
Department of Hematology and Medical Oncology, University of Toronto
(4)
Department of Hematology, Shanghai Chang Zheng Hospital
(5)
Department of Laboratory Hematology, University Health Network

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© The Author(s). 2016

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