IL-6 −572C>G and CARD8 304T>A Genetic Polymorphisms are Associated with the Absolute Neutrophil Count in Patients with Hematological Malignancies Under Chemotherapy: An Application of Multilevel Models to a Preliminary Pharmacogenetic Study

Figures & data

Table 1 Multilevel Diagram

Name of Level	Subscript to Index the Level	Member Categories in Each Level	Variables Measured on Each Member of the Level
Patient	i = 1, …, 32	ID (1–32)	Diagnosisi, IL-6 Genotypei, IL-1β Genotypei, CARD8 Genotypei, Hospitali
Scheme	j = 1, …, 9	Hyper CVAD phase I^a, Hyper CVAD phase II^b, 15–30 induction^c, 15–30 consolidation^d, 3 + 7 (induction)^e, Cytarabine (consolidation), R BAC^f, FLAG – Ida^g, ARCHIMBAUD^h	No variables measured at this level
Cycle	k = 1, …, 6	Cycle (1–6)	No variables measured at this level
Time	t = 0, …, 50	Day (0–50)	ANCijkt, G-CSFijkt, G-CSFxDayijkt, G-CSFxHospitalijkt

Notes: The Multilevel diagram shows the organization of data, the lower level is contained or nested in the upper level, ie, every patient could have different schemes, every scheme could have different cycles, and in every cycle, there are several time measurements. The multilevel diagram also indicates what explanatory variables are measured at each level. ^aHyper CVAD phase I: cyclophosphamide, vincristine, doxorubicin, dexamethasone. ^bHyper CVAD phase II: methotrexate, cytarabine. ^c15–30 induction: dexamethasone, vincristine, daunorubicin, L-asparaginase. ^d15–30 consolidation: 6- mercaptopurine, cyclophosphamide, cytarabine, vincristine, L-asparaginase. ^e3 + 7 (induction): cytarabine, daunorubicin. ^fR BAC: rituximab, bendamustine, cytarabine. ^gFLAG – Ida: fludarabine, Ara-C (cytarabine), G-CSF, idarubicin. ^hARCHIMBAUD: mitoxantrone, cytarabine, etoposide. G-CSFxDay: Interaction between the administration of G-CSF and the time. G-CSFxHospital: Interaction between the administration of G-CSF and the hospital where it was administrated.

Abbreviations: IL6, Interleukin 6; CARD8, caspase recruitment domain family, member 8; ANC, absolute neutrophil count; G-CSF, granulocyte-colony stimulating factor.

Table 2 Absolute Neutrophil Count by Genotype and Therapeutic/Morbid Characteristics

	Total	IL6 GG	IL6 CC/CG	p – value	CARD8 AA	CARD8 TT/TA	p-value
ANC Mean ± SD	1726.1 ± 90.6	1299.3 ± 175.7	1798.2 + 108.1	0.038^I*	1424.3 ± 93.1	2059.8 ± 155.3	0.001^I*
ANC Median (IQR)	326 (0–1980)	207 (0–1420)	407 (0–2184)		146.5 (0–1980)	418 (0–1997)
Diagnosis, ANC Mean ± SD
ALL	1784.5 ± 3431.2	1530.0 ± 355.1	1842.4 ± 154.9	0.404^II	2207.2 ± 354.4	1660.0 ± 151.8	0.107^II
AML	1042.0 ± 2078.9	1139.5 ± 205.4	1015.0 ± 75.2	0.487^II	889.9 ± 75.1	1270.1 ± 145.1	0.012^II*
NHL	3183.1 ± 6112.9	2171.4 ± 459.1	3463.7 ± 396.2	0.103^II	2382.4 ± 210.1	3749.4 ± 534.8	0.039^II*
Scheme, ANC Mean ± SD
Hyper CVAD phase I^a	3348.9 ± 7001.1	2497.4 ± 1146.0	3434.4 ± 461.2	0.533^II	2041.3 ± 502.9	3743.8 ± 538.7	0.096^II
Hyper CVAD phase II^b	2177.7 ± 3887.4	2888.4 ± 647.4	2035.1 ± 199.7	0.109^II	2358.1 ± 396.7	2114.8 ± 229.7	0.5932^II
15 – 30 induction^c	450.1 ± 589.0	292.0 ± 64.1	586.0 ± 95.8	0.016^II*	445.1 ± 146.9	451.7 ± 65.9	0.963^II
15 – 30 consolidation^d	1436.3 ± 1886.8
3 + 7 (induction)^e	1095.8 ± 2778.1	1655.5 ± 500.0	894.1 ± 167.6	0.064^II	633.9 ± 143.3	1796.1 ± 392.3	0.002^II*
Cytarabine (consolidation)^f	1225.9 ± 1852.2	1019.0 ± 192.2	1275.8 ± 102.5	0.263^II	1216.0 ± 111.8	1239.6 ± 151.4	0.898^II
R BAC^g	2602.3 ± 2255.2	1340.6 ± 202.3	2857.4 ± 243.7	0.007^II*	2857.4 ± 243.7	1340.6 ± 202.3	0.007^II*
FLAG – Ida^h	585.4 ± 1545.9	372.7 ± 160.4	645.2 ± 166.3	0.404^II	296.4 ± 116.7	715.5 ± 186.8	0.150^II

Notes: *p-value <0.05. ^ITested using the Mann–Whitney test ^IITested using an independent samples t-test. ANC: absolute neutrophil count. ALL: acute lymphoblastic leukemia. AML: acute myeloblastic leukemia. NHL: non-Hodgkin’s lymphoma. ^aHyper CVAD phase I: cyclophosphamide, vincristine, doxorubicin, dexamethasone. ^bHyper CVAD phase II: methotrexate, cytarabine. ^c15 – 30 induction: dexamethasone, vincristine, daunorubicin, L-asparaginase. ^d15 – 30 consolidation: 6- mercaptopurine, cyclophosphamide, cytarabine, vincristine, L-asparaginase. ^e3 + 7 (induction): cytarabine, daunorubicin. ^fR BAC: rituximab, bendamustine, cytarabine. ^gFLAG – Ida: fludarabine, Ara-C, G-CSF, idarubicin. ^hARCHIMBAUD: mitoxantrone, cytarabine, etoposide.

Abbreviations: SD, standard deviation; IQR, interquartile range; IL6, interleukin 6; CARD8, caspase recruitment domain family, member 8.

Figure 1 Factors associated with Absolute Neutrophil Count (ANC).

Abbreviations: CARD8, caspase recruitment domain family member 8; IL-6, interleukin 6; G-CSF, granulocyte-colony stimulating factor; ANC, absolute neutrophil count; ALL, acute lymphoblastic leukemia; AML, acute myeloblastic leukemia; NHL, non-Hodgkin’s lymphoma; G-CSFxDay, interaction between the administration of G-CSF and the time; G-CSFxHospital, interaction between the administration of G-CSF and the hospital where it was administrated.

Figure 2 Relationship between protein function, studied polymorphic variants, and the effect in neutrophils survival.

Abbreviations: CARD8, caspase recruitment domain family member 8; IL-6, interleukin 6; G-CSF, granulocyte-colony stimulating factor; ANC, absolute neutrophil count; TLR2, toll-like receptor 2; NF-κB, nuclear factor kappa light chain enhancer of activated B cells.