Abstract

Patients and methods

Patients and treatment

The diagnosis of CLL was made according to standard criteria,¹³ and staged according to the Rai staging system.¹³ In all, there were 44 men and 12 women, with a median age of 64.5 (range: 37–91) years. The median lymphocyte count was 17×10⁹/l. Apart from two patients with an uncertain Rai stage at diagnosis, there were 22 (40.7%) and 32 (59.3%) patients with Rai stage >2 and 2, respectively (table 1​1).). Treatment would be given for B symptoms, symptomatic organomegaly, extreme lymphocytosis, immune cytopenia or a rapid rise in lymphocyte count. The treatment included prednisolone, fludarabine or chlorambucil, or combination chemotherapy such as COPP (cyclophosphamide, vincristine, prednisolone and procabazine), CVP (cyclophosphamide, vincristine and prednisolone) or FND (fludarabine, mitoxantrone and dexamethasone).¹⁴

Table 1Demographic data of the patients

	n	%
Patients	56	100
Sex
Men	44	78.6
Women	12	21.4
Median age (years)	37–91 (64.4)
Presenting lymphocyte count (×109/l)	2–236 (17.5)
Rai stage
>2	22	40.7
2	32	59.3
Unknown	2
Karyotypic abnormalities	39
del 13q14	3	7.7
Trisomy 12	5	12.8
Complex	11	28.2
Normal	20	51.3

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High‐molecular‐weight genomic DNA was isolated by standard protocols from diagnostic bone marrow aspirates of 56 patients with CLL and 12 normal bone marrow donors in addition to DNA from the peripheral blood of 12 healthy blood donors from the Hong Kong Red Cross Association.¹⁵ Cytogenetic data were available in 39 patients.¹⁶ Previous studies showed that trisomy 12 in CLL is associated with atypical morphology, progressive disease and poor survival, whereas del(13q) seems to indicate a good prognosis.¹⁷ Therefore, in this study, patients with poor‐risk cytogenetic aberrations were those with trisomy 12 and complex abnormalities, and patients with standard‐risk cytogenetic aberration included those with normal karyotype and isolated deletion of 13q14.

Methylation‐specific polymerase chain reaction

The methylation‐specific polymerase chain reaction (MSP) for gene promoter methylation was carried out as described in detail previously.^7,17 Briefly, treatment of DNA with bisulphite for conversion of unmethylated, but not methylated, cytosine to uracil was carried out with a commercially available kit (CpGenome DNA modification kit, Intergen, New York, New York, USA) according to the manufacturer's instructions. Table 2​2 shows the primers for the methylated (M‐MSP) and unmethylated (U‐MSP) gene promoter regions for p15, p16, p18, Rb, p21, p27 and p57.¹⁸ DNA from eight normal donors was used as negative control, whereas methylated‐control DNA (CpGenome Universal Methylated DNA, Intergen) was used as positive control in all the experiments. MSP was carried out in a thermal cycler (9700, PE Biosystems, Foster City, California, USA). The polymerase chain reaction (PCR) mixture contained 50 ng of bisulphite‐treated DNA, 0.2 mM deoxynucleoside triphosphates, 2 mM magnesium chloride, 10 pmol of each primer, 1 × PCR buffer II and 2.5 units of AmpliTaq Gold (Perkin‐Elmer Biosystems, Wellesley, Massachusetts, USA) in a final volume of 50 μl.

Table 2MSP primers and PCR conditions

Gene	Forward primer (5′–3′)	Reverse primer (5′–3′)	Tm and cycles	Size in bp	Locus	Reference
p15
M‐MSP	TGAGGATTTCG CGACGCGTTC	CGTACAATAA CCGAACGAC CGATCG	63°C/35	162	9p21	15
U‐MSP	TGAGGATTTTG TGATGTGTTT	CATACAATAA CCAAACAAC CAATCA		162
p16
M‐MSP	TTA TTA GAG GGT GGG GCG GAT CGC	GAC CCC GAA CCG CGA CCG TAA	65°C/35	150	9p21	15
U‐MSP	TTA TTA GAG GGT GGG GTG GAT TGT	CAA CCC CAA ACC ACA ACC ATA A		151
p18
M‐MSP	TTATCGAATTG TTATTTTCGTT CG	CGTCTCGCCG AAAAAATAA TC	64°C/35	93	1p32	15
U‐MSP	GGGTTATTGAA TTGTTATTTTT GTTTG	CATCCATCTC ACCAAAAAAA TAATC		95
RB
M‐MSP	GGGAGTTTCGC GGACGTGAC	ACGTCGAAAC ACGCCCCG	66°C/35	152	13q14	15
U‐MSP	GGGAGTTTTGT GGATGTGAT	ACATCAAAAC ACACCCCA		152
p21
M‐MSP	TTG GGC GCG GAT TCG TC	CTA AAC CGC CGA CCC GA	62°C/35	100	6p21	25
U‐MSP	TTA GTT TTT TGT GGA GTT G	CTC AAC TCT AAA CCA CAA		120
p27
M‐MSP	AAG AGG CGA GTT AGC GT	AAA ACG CCG CCG AAC GA	66°C/35	195	12p13	25
U‐MSP	ATG GAA GAG GTG AGT TAG T	AAA ACC CCA ATT AAA ACA		212
p57
M‐MSP	TCG GTT AGG TTT GAG CGA GC	TAC GTA TAC GAA AAA CGC GAC GAC	62°C/35	137	11p15	26
U‐MSP	TTG GTT AGG TTT GAG TGA GTG A	TCT ACA TAT ACA AAA AAC ACA ACA A		139		—

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PCR, polymerase chain reaction; M, methylated; U, unmethylated; MSP, methylation‐specific PCR.

Results

Controls

None of the seven genes tested were methylated in 12 normal bone marrow and 12 normal peripheral blood samples. The positive and negative controls showed the expected MSP results (normal DNA: U‐MSP positive/M‐MSP negative; methylated DNA: U‐MSP negative/M‐MSP positive; ​positive;figsfigs 1A and 2A​2A).). Authenticity of MSP was confirmed by sequencing of samples from the M‐MSP products from methylated positive control (​(figsfigs 1B and 2B​2B).

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Figure 1(A) Methylated methylation‐specific polymerase chain reaction (M‐MSP) and unmethylated methylation‐specific poylmerase chain reaction (U‐MSP) of p15 in primary chronic lymphocytic leukaemia (CLL) samples. B, reagent blank; N, normal marrow DNA controls; P, methylated positive control; patients 1, 2, 3 and 6, primary CLL marrow samples. (B) Sequence of positive control M‐MSP product and (C) sequence of M‐MSP of the primary CLL marrow sample. Wildtype (WT) cytosine (C) residues that remained unchanged in methylated CpG are coloured blue and underlined, whereas those that were changed to thymidine (T) are coloured red and underlined. U‐MSP shows that the methylated control (M) was totally methylated.

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Figure 2(A) Methylated methylation‐specific polymerase chain reaction (M‐MSP) and unmethylated methylation‐specific polymerase chain reaction (U‐MSP) of p57 in primary chronic lymphocytic leukaemia (CLL) samples. B, reagent blank; N, normal marrow DNA controls; P, methylated positive control; patients 8, 9, 10 and 19, primary CLL marrow samples. (B) Sequence of positive control M‐MSP amplification. (C) M‐MSP sequence of primary CLL marrow sample. Wildtype (WT) cytosine and (C) residues that remained unchanged in methylated CpG were coloured blue and underlined, whereas those that were changed to thymidine (T) are coloured red and underlined. U‐MSP shows that the methylated control (M) was totally methylated.

MSP in primary CLL marrow samples

In the INK4 family, methylation of p15 and p16 occurred in 20 (35.7%) and 8 (14.3%) patients, respectively (table 3​3).). Of them, 5 (8.9%) patients with CLL harboured concurrent methylation of both p15 and p16. In contrast, p18 and Rb are unmethylated. In the CIP or KIP family, apart from infrequent methylation of p57 in 4 (7.1%) patients, methylation of p21 and p27 are absent. Authenticity of MSP was confirmed by sequencing samples from the M‐MSP products from methylated samples (​(figsfigs 1C and 2C​2C).

Table 3Association between gene methylation and demographic characteristics

	p15		p	p16		p	p57		p
	Unmethylated	Methylated		Unmethylated	Methylated		Unmethylated	Methylated
Median age	63.5	68.5	0.19	64.5	65.5	0.72	64.5	67.5	0.47
Median lymphocyte count	17.0	21.5	0.25	16.0	64.5	0.016	16.0	31.5	0.17
Sex
Men	30	14	0.31	38	6	0.99	41	3	0.99
Women	6	6		10	2		11	1
Rai stage
2	21	10	0.77	27	5	0.99	30	2	0.99
>2	13	9		19	3		20	2
Poor‐risk karyotype
Yes	15	8	0.74	21	2	0.63	21	2	0.50
No	9	7		13	3		16	0

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Association between gene methylation and age and lymphocyte count was analysed by the Mann–Whitney test, and association with sex, Rai stage and presence of poor‐risk karyotype was analysed by the χ² or Fisher's exact test.

Association of gene methylation with demographic data and overall survival

No association was found between p15 methylation and age (p=0.19) and diagnostic Rai stage (p=0.77), presence of poor‐risk karyotype (p=0.74) and median lymphocyte count at presentation. No correlation was observed between p16 methylation and sex (p=0.99), Rai stage (p=0.99), poor‐risk karyotype (p=0.63) and age (p=0.72), but p16 methylation was associated with a high median lymphocyte count at diagnosis (p=0.016). Similarly, no correlation was identified between p57 methylation and sex (p=0.99), Rai stage (p=0.99), poor‐risk karyotype (p=0.50), age (p=0.47) and presenting lymphocyte count (p=0.17). Projected 5‐year overall survival for patients with and without p15 methylation was 66% and 59%, respectively (p=0.75; fig 3​3).

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Figure 3Overall survival (OS) for patients (A) with and without advanced Rai stage disease, (B) poor‐risk karyotype and (C) p15 methylation.

Discussion

Ideally, MSP status of the neoplastic lymphocytes will only be elucidated if marrow cells have been sorted for CD5 and CD23 dually positive cells. Here, MSP of the genes has been first validated in normal control DNA (both normal bone marrow and normal peripheral blood DNA) by showing a lack of methylation and further verified by sequencing. This confirms that gene methylation is an aberration that does not exist in normal cells, be it marrow or peripheral blood cells. Given that methylation detected by MSP is a positive signal with a high sensitivity (up to 1×10⁵ for p15 gene),⁷ our results are still valid without sorting for lymphocytes. The presence of amplification in U‐MSP in some methylated primary samples was due to the presence of the unmethylated gene in normal marrow cells.

Although global hypomethylation has been first shown in CLL,¹⁹ a recent genome‐wide methylation study showed that 2.5–8% of the CpG islands are aberrantly methylated in a non‐random manner in CLL.²⁰ Moreover, although CLL has conventionally been described to be a disease with impaired apoptosis, the role for cell cycle dysregulation may be less important. However, a subpopulation of cycling B cells,²¹ especially those in the pseudofollicles, may be important in disease progression.

Although there are recent reports of p53 and ZAP‐70 gene methylation in CLL, with the inherent effect on prognosis,^22,23 data on methylation of p15 and p16 in CLL are surprisingly scanty. Two studies reporting on the frequency of p15 and p16 methylation in Caucasian patients were available. One study showed infrequent p15 (5% of patients) and p16 (9%) methylation, whereas another showed frequent methylation of p15 (50%) and less frequently of p16 (17%), in Caucasian patients with CLL.^24,25 Our study on Chinese patients for methylation of p15 and p16 showed a rate of 37.5% and 14.3%, respectively, which is comparable to that in the Caucasians. It is noteworthy that deletion or mutation associated with p15 and p16 is extremely rare in CLL.²⁶ Therefore, methylation of p15 and p16 may be the major mechanism of p15 and p16 gene inactivation in CLL. Compared with the high frequency (>70%) of p15 methylation in acute leukaemia,^7,8 the lower frequency of p15 methylation in CLL suggested a less important role for cell cycle dysregulation in CLL. Interestingly, a similar rate of p15 methylation has also been reported in multiple myeloma, a disease also characterised by a mature B cell phenotype and impaired apoptosis instead of cell proliferation.⁹ Our data also showed that p16 methylation was associated with a higher leucocyte count at presentation (64.5×10⁹/l in patients with methylated p16 and 16.0×10⁹/l in patients with unmethylated p16; p=0.016), suggesting the activation of the cell cycle.

A tumour suppressor property of p18 gene has been demonstrated in mice.²⁷ However, p18 methylation was absent in our patients in this and other studies.^25,26 A literature review indicates that p18 methylation is probably not targeted in cell cycle dysregulation in CLL.^28,29 Although 13q deletions are common in CLL,³⁰Rb is not associated with the minimally deleted region, and thus genes other than Rb are targeted.³⁰ Our results showed that Rb was not methylated in CLL.

We have also shown the infrequent p57, and absence of p21 and p27, methylation in CLL. p21^{CIP1/WAF1/CDKN1A} is a tumour suppressor gene inducible by wildtype p53 in the presence of DNA damage, leading to cell cycle arrest. This is the first report of the absence of p21 methylation in CLL. p21 methylation has been shown to be rare in other lymphoid malignancies.³¹ Infrequent p27^KIP1/CDKN1B methylation has been reported to occur in solid cancers,³² but has not been studied in haematological cancers. However, recent studies showed that cellular p27 level may be down regulated by post‐translational ubiquitination and proteasomal degradation.³³ Moreover, functional inactivation of p27 may also be mediated by the phosphorylation of the nuclear localisation domain, resulting in the cytoplasmic mislocalisation of p27, thus precluding p27 from its negative regulation of CDK₂.³³ Therefore, although p27 methylation may not have an important role in its inactivation, post‐translational modification by ubiquitination or phosphorylation may still be important in oncogenesis. Therefore, our data suggest that epigenetic inactivation of the cell cycle control primarily targets the CDK4/INK4 complex and does not require additional inactivation of the CDK2/CIP/KIP. Indeed, a recent study showed that CDK2 (target of inhibition by KIP proteins), but not CDK4, is dispensable for cell proliferation,³⁴ underscoring the importance of CDK4 in cell proliferation and thus the potential importance of INK4 targeting by methylation.

In summary, p15 and, less frequently, p16 of the INK4 family, instead of the CIP or KIP families, are targets of methylation in CLL, and thus might be important for its pathogenesis, posing potentially important targets for 5‐azacytidine demethylation.

Acknowledgements

Abbreviations

Footnotes

References