Toward personalized therapy for chronic lymphocytic leukemia

Figures & data

Table 1. Clinical characteristics of patients

Initials	PZ	RJ
Age/sex	65/M	54/M
Rai	I B	II A
Blood count (before treatment) WBC Hb PLT	174.63 x 103/μl 13.7 g/dl 193.0 x 10^3/μl	126.27 x 103/μl 11.1 g/dl 136.0 x 10^3/μl
Blood count (assessment of response) WBC Hb PLT	3.00 x 10^3/μl 11.6 g/dl 108 x 10^3/μl	1.87 x 10^3/μl 13.5 g/dl 83 x 10^3/μl
IgHV mutational status	Mutated	Unmutated
CD 38	20%	21%
β2-microglobulin (mg/L)	4.20	8.78
LDH (U/L) (before treatment)	225	260
Cytogenetics	del 11q, del 13q	del 11q
Lymph nodes diameter (before treatment)	Cervical region 2 × 1 cm Axillary region 3 × 4 cm Inquinal region 2 × 2 cm	Cervical region 3 cm Axillary region 3 cm Inquinal region 3 cm
Lymph nodes diameter (during assessment of response)	Complete regression of peripheral and central lymph nodes	Complete regression of peripheral lymph nodes and < 50% regression of central lymph nodes
Spleen and liver (before treatment)	Not enlarged	Spleen enlarged to 8 cm below the left costal margin; Liver enlarged to 4 cm below the left costal margin;
Spleen and liver (during assessment of response)	Not enlarged	Spleen decreased to 2 cm below the left costal margin (CT = 15 cm); Liver enlarged to 3 cm below the left costal margin;
Treatment response*	CR MRD+	NR

The cut-off values for CD38 was 30%.

No p53 mutation.

* RCC treatment response after 6 cycles.

Figure 1. Viability of CLL cells (A), differential scanning calorimetry profiles of nuclear fraction (B) and changes in expression of apoptosis-related proteins (Mcl-1, Bcl-2, procaspase-3, Noxa) in PBMC lysates (C), isolated from blood of patients PZ and RJ, incubated for 48 h with and without RCM combination. Immunoblot analysis of PBMCs after their exposure to drug combination were analyzed by alkaline phosphatase method in the presence of appropriate antisera. Protein lysates (50 μg) were loaded into 12.5% polyacrylamide gels; β-actin was used as loading control; Cp^ex - excess heat capacity.

Figure 2. Microarray analysis indicating changes in expression of 89 apoptosis-related genes in two CLL samples following immunotherapy (top); boxplots of the DDCT values of samples from patients PZ and RJ (bottom). The rows and the columns represent individual genes and mRNA samples, respectively. The relative level of gene expression is depicted according to the color scale shown below the matrix. The accuracy of reading in the presented experiments is illustrated by boxplots of the signal (signal DDCT ~-logRQ).

Table 2. Alterations in selected gene expression in two CLL cell samples (patients: PZ and RJ) after 2 wk of RCC therapy illustrated as fold change and ΔΔCt values in reference to control samples (before therapy)

Gene description	PZ		RJ
		ΔΔCt	RQ	ΔΔCt	RQ
NOXA (Phorbol-12-myristate-13-acetate-induced protein 1; proapoptotic)	0.44	1.28	-4.39	161.35
CASP3 (Caspase 3; proapoptotic)	-0.32	2.75	-0.43	3.08
BCL2 (B-cell CLL/lymphoma 2; antiapoptotic)	-0.35	2.84	-3.54	69.60
MCL1 (Myeloid cell leukemia sequence 1; antiapoptotic)	-0.78	4.35	-1.45	8.60
PUMA (P53 upregulated modulator of apoptosis; proapoptotic)	-0.19	2.42	-0.44	3.13
BCL2A1 (BCL2-related protein A1; antiapoptotic)	-0.72	4.11	-1.30	7.36
APAF1 (apoptotic protease activating factor 1; proapoptotic)	-0.76	4.29	-0.41	3.01
ESRRBL1 (IFT57-intraflagellar transport 57 homolog; proapoptotic)	0.47	1.24	-3.01	40.58

ΔΔCt, a value of the difference in expression of a gene between two examined groups (before and after RCC treatment); RQ, fold change (fold difference); RCC, rituximab + cladribine + cyclophosphamide