Revisiting Autoimmunity in Chronic Lymphocytic Leukemia: Prognostic Value of Positive Direct Antiglobulin Test in a Retrospective Study and Literature Review

Figures & data

Table 1 Clinical and Laboratory Characteristics of Patients with CLL Based on DAT Status

Patient Characteristics		DAT-Positive (N = 36)	DAT-Negative (N = 84)	P-value
Age (years) mean ± SD		60.9 ± 12.2	59.8 ± 12.7	0.7
Male		15 (41.7%)	49 (58.3%)	0.1
Performance stage ECOG (0–1)		23 (63.9%)	67 (79.8%)	0.07
B-symptoms		19 (54.3%)	44 (68.8%)	0.2
Rai stage	Stage 0–2	9 (25%)	39 (46.4%)	0.03*
	Stage 3–4	27 (75%)	45 (54.8%)
HCV (Positive cases)		8 (22.2%)	13 (15.5%)	0.4
White blood cells × 10^9/L (median, IQR)		141 (191)	65 (99.5)	0.1
Hemoglobin gm/dl (mean ± SD)		8.4 ± 2.5	9.7 ± 2.4	0.006*
Platelets × 10^9/L (median, IQR)		137.5 (116.3)	141 (99)	0.8
Lactate dehydrogenase U/L (median, IQR)		529 (402)	391 (233.5)	0.4
Albumin mg/dl (mean ± SD)		36.8 ± 4.7	37.5 ± 5.7	0.3

Notes: *Statistically significant at <0.05; chi-square test was used.

Abbreviations: SD, standard deviation; ECOG, Eastern cooperative oncology group; HCV, hepatitis C virus; IQR, interquartile range; LDH, lactate dehydrogenase; DAT, direct antiglobulin test.

Table 2 Associations of CD38 and ZAP-70 with DAT

	DAT-Positive (N = 26)	DAT-Negative (N = 64)	p-value
CD38 positive cases (n, %)	8 (30.8%)	17 (26.6%)	0.7
ZAP-70 positive cases (n, %)	9 (34.9%)	23 (35.9%)	0.9

Note: Chi-square test was used.

Abbreviation: DAT, direct antiglobulin test.

Table 3 Proportions of the Therapy Lines in DAT-Positive and DAT-Negative Cases

	DAT-Negative (n = 79)	DAT-Positive (n = 34)	p-value
Chlorambucil	24 (30.4%)	11 (32.4%)	0.6
Fludarabine-based	13 (16.5%)	8 (23.5%)
CVP-R	42 (53.2%)	15 (44.1%)

Abbreviation: CVP-R, cyclophosphamide, vincristine, prednisone, and rituximab.

Table 4 Response Assessment-Based DAT

	DAT-Positive (N = 34)	DAT-Negative (N = 79)	P-value
CR	3 (8.8%)	13 (16.5%)	0.03
PR	13 (38.2%)	47 (59.5%)
Progression	18 (53%)	19 (24%)
Death	5 (14.7%)	8 (10.1%)	0.5

Abbreviations: CR, complete response; PR, partial response.

Table 5 Multivariate Analysis of Overall Response to the First-Line Therapy

	Regression Coefficient	p-value	OR
Positive DAT	−1.1	0.013	0.3
ECOG >2	−1.4	0.02	0.2
Age in years	−0.006	0.9	0.9

Abbreviations: OR, odds ratio; DAT, direct antiglobulin test; ECOG, Eastern cooperative oncology group.

Figure 1 Kaplan–Meier estimates of overall survival in CLL patients according to DAT status (P = 0.2).

Figure 2 Kaplan–Meier estimates of PFS in CLL patients according to DAT status (P = 0.004). Median PFS of positive and negative cases: 40.9 and 84 months, respectively.

Table 6 Univariate Cox Regression Hazard Analysis of Possible Predictors of PFS

Variables	HR	95% CI	P-value
DAT-positive	2.5	1.3–4.6	0.005
Advanced Rai stage	1	0.5–1.9	0.5
Zap-70 positive	1.9	1–4.3	0.07
CD38 positive	2.5	1.2–5.3	0.02
Age	0.97	0.95–0.99	0.05
Fludarabine-based chemotherapy as first-line treatment	1.2	0.5–3.1	0.7

Abbreviations: HR, hazard ratio; CI, confidence interval; DAT, direct antiglobulin.

Figure 3 Kaplan–Meier estimates of PFS in DAT-positive CLL patients according to AIHA development (P = 0.9).

Table 7 Studies About the Prognostic Impact of a Positive DAT on CLL Outcomes

Study	CLL	DAT-Positive N (%)	AIHA N (%)	CLL Patients with DAT Positive Characteristics
Atef et al; 2019^Citation25	101	28 (27.7%)	20 (19.8)	Higher WBC, lower HB, lower PLT
				Positive CD38
				Positive ZAP-70
				Less BM infiltration
Demir and Ekinci; 2017^Citation28	192	8 (4%) +DAT (n = 4)	3 (2.5)	Older age (P = 0.036),
				Advanced disease stage (P = 0.004)
				Received more first-line treatments (P = 0.003)
Visentin et al; 2013^Citation29	795	27 (3.4%)	27 (3.8)	Autoimmune disease more in females, advanced disease stage and 11q deletion by FISH
				Autoimmune diseases have better survival compared to other CLL complications as major infections or secondary cancers
Quinquenel et al; 2015^Citation8	378	56 (14.8%)		In Binet stage A correlated only with IgHV unmutated status (p < 0.001)
				In Binet stage A, positive DAT had a significantly shorter OS, regardless of their IGHV mutational status
				By multivariate analysis, a positive DAT was found to be an independent adverse prognostic factor for OS.
Abbas et al; 2015^Citation13	60	14 (23.3%)	14 (23.3)	Advanced Rai stage (P<0.01)
				Anemia (P<0.001)
Ricci et al; 2013^Citation14	146*	20 (14%)	8 (5.5) #	Higher β2-microglobulin
				Shorter treatment free survival (TFS)
Shvidel et al; 2013^Citation23	1477	93 (6.3%)	80 (5.4)^#	66 patients with DAT positive have no clinical or laboratory signs of hemolysis at time of CLL diagnosis
				Shorter OS compared to DAT negative CLL (P =0.001)
Moreno et al; 2010^Citation10	960	49 (5%)	50 (5.2)^#	Higher WBC, shorter lymphocyte doubling time
				Higher β2 microglobulin
				Higher CD38
				Advanced (Binet stage C) disease 2^nd to autoimmune mechanism had better survival vs Advanced stage2^nd to a massive bone marrow infiltration (P = 0.02).
Xu et al; 2009^Citation18	123	34 (27.6%)	12 (9.8)^#	Advanced Binet stage (p < 0.001),
				Higher level of serum lactate dehydrogenase (LDH) (p = 0.003)
				Higher β2 microglobulin (p = 0.011),
				Higher IgVH un mutated status (p < 0.001),
				Positive ZAP-70 (p = 0.012), and
				Trisomy 12 cytogenetic aberration (p = 0.004)
Ricci et al; 2009^Citation22**	158	42 (26.6%)	8 (5.5)^#	Higher β2 microglobulin (P=0.011)
				Higher IgVH un mutated status (P=0.0014).
				Higher LDH (P=0.051).
				Positive ZAP-70 (P=0.083) and
				More frequency of deletion in 17p13, 11q22 or 6q21 (P=0.064),
Zent et al; 2008^Citation24	1750	41 (2.3%)	41 (2.3)	Survival from onset of cytopenia was significantly better for patients with autoimmune mechanism (median 9.1 years) compared to patients with BM failure (median 4.4 years, p < 0.001)
Dearden et al; 2008^Citation12	637	89 (14%)	89 (14)	Less Overall response (66% vs 81%, P = 0.004)
				Less likely to have had a good (complete or nodular partial) response (18% vs 41%, P <0.001)
				Lower 5-year PFS (9% vs 18%; P=0.001)
Duek et al; 2006^Citation30	964	62 (6.4%)	55 (5.7)	Higher atypical prolymphocytes
				Higher CD38
				Higher β2-microglobulin
Barcellini et al; 2006^Citation9	3150 (18 center) 194 had AIC	126/3150 (4%)	129 (4)	Advanced stage (P<0.001).
				Older age (OR 3.43, CI 1.22–9.63)
				First line therapy (OR 15.62, CI 5.00–48.82)
				Second line therapy (OR 48.64, CI 10.00–239.19)
				Multivariate analysis showed that Fludarabine as a second line did not show difference in RR
		126/194 (66%)
Kyasa et al; 2003^Citation21	132	16 (18%)	6 (4.5)^#	No CLL/SLL patients had DAT-negative hemolytic anemia
				10 DAT positive CLL/SLL patients, did not have AIHA, 2 had ITP.
Mauro et al; 2000^Citation20	1203	52 (4.3%)	45 (3.7)^#	More LC > 60 x 10(9)/L (P <0.00001)
				Age above 65 years (P <0.01)
				Male gender (P <0.01)

Notes: *Untreated CLL; **Abstract ASH 2009; #studies that not all DAT positive patients suffer from AIHA.

Abbreviations: WBC, white blood cells; Hb, hemoglobin; PLT, platelet; LC, lymphocytic count; LDH, lactate dehydrogenase; ZAP, zeta-associated protein; IgVH, immunoglobulin variable heavy chain; PFS, progression-free survival; OS, overall survival; DAT, direct antiglobulin.

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