Dysregulated Class I histone deacetylases are indicators of poor prognosis in multiple myeloma

Figures & data

Figure 1. HDAC genes are overexpressed in human myeloma cell lines when compared to normal plasma cells Expression levels of HDAC in 14 human myeloma cell lines (HMCL) were compared to normal plasma cells (normal; n = 9 ). Class I HDAC (HDAC1, HDAC2, HDAC3, and HDAC8) were all significantly overexpressed. Among the class II HDAC, HDAC5 and HDAC10 were significantly overexpressed. Data are presented as mean±SEM. Significant differences were calculated with t-test utilizing GraphPad Prism 5.0d. p-values are indicated.

Table 1. HDAC1-2 (Class I HDAC) are overexpressed in all HMCL

		Primary translocations		Secondary translocations
Gene	Normal	t(4;14) (FGFR3 and MMSET)	t(11;14) (CYCLIN D1)	t(14;16) (C-MAF)	8q24 (c-MYC)
HDAC1	1.15±0.25	5.75±0.80****	7.73±2.04****	3.17±0.41***	6.67±0.95****
HDAC2	1.18±0.26	4.83±0.48****	5.13±0.88****	2.21±0.31**	4.33±0.39****
HDAC3	1.24±0.29	3.44±0.76**	4.00±0.71***	2.65±0.23**	3.68±0.44****
HDAC8	1.21±0.24	2.03±0.12**	2.29±0.36*	1.66±0.17	2.29±0.15***
HDAC5	1.36±0.32	5.41±0.70****	6.23±1.24***	3.15±0.54**	5.32±0.57****
HDAC7	1.30±0.33	2.57±0.31**	2.53±0.20*	1.61±0.17	2.68±0.31**
HDAC6	1.21±0.81	1.57±0.26	2.70±0.93	0.69±0.08*	1.82±0.37
HDAC10	1.12±0.17	2.76±0.26****	1.47±0.34	2.25±0.36*	2.53±0.22**
HDAC11	2.59±1.06	54.10±26.85*	7.85±2.73	1.77±0/36	9.93±3.37

HMCLs were grouped based on their translocation status [t(4;14), t(11;14), t(14;16) and translocations involving c-myc locus – 8q24] and expression levels of HDAC were compared to those in normal plasma cells. HMCL with t(4;14) had significantly higher levels of HDAC (8/11) followed by translocations involving c-myc locus (7/11). HMCLs with t(11;14) and t(14;16) had 6/11 HDACs significantly dysregulated. Significant differences were observed using t-test and GraphPad Prism Version 5.0d. Gray-filled boxes represent significant upregulation in expression. Asterisks represent p-values (* <0.05, **<0.01, ***<0.001, ****<0.0001).

Table 2. Patient characteristics-Gene expression cohort

MEDIAN AGE AT DIAGNOSIS	57 YEARS (31-91)
MALE : FEMALE	32 : 17
EARLY : ADVANCED	26 : 23
ISS STAGE AT DIAGNOSIS I : II : III	10 : 15 : 12

Table indicates characteristics of primary MM patient cohort utilized for assessment of HDAC1 -11 expression levels by quantitative RT-PCR.

Figure 2. HDAC levels are dysregulated in primary MM Expression levels of HDAC in primary MM (n = 55 ) were compared to normal plasma cells (normal; n = 9 ). All genes, except HDAC1 and HDAC11, were significantly altered. Data are presented as median with range. Significant differences were calculated utilizing Mann-Whitney test in GraphPad Prism 5.0d. P-values are indicated.

Figure 3. Increased HDAC levels correlate to poor prognosis Kaplan-Meir plots showing progression-free survival (PFS) in MM patients with high (>75th centile of MM patient cohort) compared to low (<75th centile) expression profiles. (A-E) Patients with high HDAC1, HDAC2, HDAC4, HDAC6, and HDAC11 expression depicted significantly shorter PFS days than patients with low levels of these genes. Significant differences and median survival days were calculated utilizing GraphPad Prism 5.0d. P-values are indicated.

Table 3. Immunohistochemistry for HDAC1 and HDAC6 on bone marrow trephines of MM patients

HDAC1	HDAC6
Total no. of samples: 97	Total no. of samples: 97
Assessable no. of samples: 79	Assessable no. of samples: 93
Proportions (no. of samples)	Proportions (no. of samples)
0 (1)	0 (2)
10 (1)	10 (3)
20 (10)	20 (3)
30 (5)	30 (4)
40 (4)	40 (5)
50 (19)	50 (9)
60 (5)	60 (10)
70 (6)	70 (9)
80 (15)	80 (17)
90 (12)	90 (18)
100 (1)	100 (13)

Table representing the split in the proportions of MM cells positive for HDAC1 and HDAC6 in the samples assessed.

Figure 4. Higher HDAC1 levels correlate with poor prognosis (A) Image depicting low staining of HDAC1 (20%) in MM cells; (B) Image depicting high proportion of HDAC1 staining (90%) in MM cells; (C) Kaplan-Meir survival plots for patients with 0–20%, 30–80%, and 90–100% proportion of HDAC1 staining, indicating patients with higher proportion of HDAC1 have shorter progression-free survival days; (D) Kaplan-Meir survival plots for patients with 0–20%, 30–80% and 90–100% proportion of HDAC1 staining indicating patients with higher proportion of HDAC1 have shorter overall survival days. Patients with >70% infiltration of MM cells was chosen for image capture. Significant differences and median survival days were calculated utilizing GraphPad Prism 5.0d and p-values are indicated.

Figure 5. The majority of patients show high HDAC6 staining in MM cells (A) Image depicting low proportion (30%) and weak staining intensity (+) of HDAC6 in MM cells; (B) Image depicting high proportion (90%) and strong staining intensity of HDAC6 in MM cells; (C) Graph representing the distribution of HDAC6 staining proportion and intensity in bone marrow trephines tested. Higher proportion of HDAC6 staining correlated to stronger staining intensity.