CD8 T cell subsets as a biomarker for predicting non-fusion after spinal fusion surgery

Abstract

Provided herein is a method for predicting the probability of having or developing a non-fusion, wherein said method comprises determining the frequency of a subpopulation of CD8+ T cells selected from CD8+CD57+, CD8+CD28− and CD8+CD57+CD28− in a sample obtained from a patient. Also provided herein is a system for predicting the probability of having or developing a non-fusion.

Claims

1. A method for treating a human patient that has an elevated probability of having or developing a non-fusion before undergoing a spinal fusion surgery or after a spinal fusion surgery, wherein said method comprises detecting the frequency of a subpopulation of CD8+ T cells selected from the group consisting of CD8+CD57+, CD8+CD28− and CD8+CD57+CD28− in a blood sample obtained from a human patient, thereby determining if the human patient has an elevated probability for having or developing non-fusion, wherein: a blood sample exhibiting an at least two-fold higher frequency of said subpopulation of CD8+ T cell when compared to a standard value determined for a population with normal fusion after bone fusion is assigned to a human patient having an elevated probability for having or developing non-fusion after spinal fusion surgery, and/or a blood sample exhibiting a fraction of CD8+CD57+CD28− cells referred to the total sum of all CD8+ cell in said blood sample of at least 22.7% is assigned to a human patient having an elevated probability for having or developing non-fusion after spinal fusion surgery, and/or a blood sample exhibiting a summed fraction of CD8+CD57+ cells and CD8+CD28− cells referred to the total sum of all CD8+ cells in said blood sample of at least 85.6% is assigned to a human patient having an elevated probability for having or developing non-fusion after spinal fusion surgery, and/or a blood sample exhibiting a fraction of CD8+CD57+ cells referred to the total sum of all CD8+ cells in said blood sample of at least 24.4% is assigned to a human patient having an elevated probability for having or developing non-fusion after spinal fusion surgery, and/or a blood sample exhibiting a fraction of CD8+CD28− cells referred to the total sum of all CD8+ cells in said blood sample of at least 28.0% is assigned to a human patient having an elevated probability for having or developing non-fusion after spinal fusion; and administering to the human patient determined to have an elevated probability for having or developing non-fusion after spinal fusion, a bone morphogenic protein selected from the group consisting of BMP-2 and BMP-7.

2. The method according to claim 1, wherein said non-fusion is a non-fusion between two or more bones or bone segments naturally not connected by osseous tissue.

3. The method according to claim 1, wherein said blood sample is a blood sample that has been obtained before, during or after spinal fusion surgery from said human patient.

4. The method according to claim 1, wherein a blood sample exhibiting a fraction of CD8+CD57+CD28− cells referred to the total sum of all CD8+ cell in said blood sample of at least 33.1%, is assigned to a human patient having an elevated probability for having or developing non-fusion after spinal fusion surgery.

5. The method according to claim 1, wherein a blood sample exhibiting a fraction of CD8+CD57+ cells referred to the total sum of all CD8+ cells in said blood sample of at least 37.6%, is assigned to a human patient having an elevated probability for having or developing non-fusion after spinal fusion surgery.

6. The method according to claim 1, wherein a blood sample exhibiting a fraction of CD8+CD28− cells referred to the total sum of all CD8+ cells in said blood sample of at least 42.9%, is assigned to a human patient having an elevated probability for having or developing non-fusion after spinal fusion.

7. The method according to claim 1, wherein said non-fusion is a non-fusion between two or more vertebrae.

Description

SHORT DESCRIPTION OF THE FIGURES

(1) FIG. 1 shows the results of flow cytometric analyses of the circulating immune cell subset CD45+CD3+CD8+CD57+ T cells in the peripheral blood of patients with spinal non-fusion and normal fusion. Spinal non-fusion patients (healing class 2) showed a significantly higher frequency of CD45+CD3+CD8+CD57+ T cells (CD57+ in % of CD45+CD3+CD8+ T cells) compared to normal healing patients (healing class 1) indicates a dramatic increase of terminally differentiated CD8+ effector cells (TEMRA CD8+). *=P<0.001; Mann-Whitney U test.

(2) FIG. 2 Results of flow cytometric analyses of the circulating immune cell subset CD45+CD3+CD8+CD28− T cells in the peripheral blood of patients with spinal non-fusion and normal fusion. Spinal non-fusion patients (healing class 2) showed a significantly higher frequency of CD45+CD3+CD8+CD28− T cells (CD28− in % of CD45+CD3+CD8+ T cells) compared to normal healing patients (healing class 1) indicates a dramatic increase of terminally differentiated CD8+ effector cells (TEMRA CD8+). *=P<0.001; Mann-Whitney U test.

(3) FIG. 3 shows the results of flow cytometric analyses of the circulating immune cell subset CD45+CD3+CD8+CD57+28− T cells in the peripheral blood of patients with spinal non-fusion and normal fusion. Spinal non-fusion patients (healing class 2) showed a significantly higher frequency of CD45+CD3+CD8+CD57+28− T cells (CD57CD28−+ in % of CD45+CD3+CD8+ T cells) compared to normal healing patients (healing class 1) indicates a dramatic increase of terminally differentiated CD8+ effector cells (TEMRA CD8+). *=P<0.001; Mann-Whitney U test.

(4) FIG. 4 illustrates the receiver operating characteristics (ROC) based on the summation of the expression of CD57+ in % of CD45+CD3+CD8+ T cells and CD28− in % of CD45+CD3+CD8+ T cells. The ROC analysis showed a high sensitivity (76%) as well as specificity (96%) in the detection of the affected patients based on their blood levels using a cut-off level of 85.6% with an area under the curve (AUC): 0.886 and p<0.001.

(5) FIG. 5 illustrates the receiver operating characteristic (ROC) based on the expression of CD57+CD28− in % of CD45+CD3+CD8+ T cells with an area under the curve (AUC): 0.859 and p<0.001. The ROC analysis showed a high sensitivity (90%) as well as a specificity (71%) in the detection of the affected patients based on their blood levels using a cut-off level of 22.7%. A cut-off level of 33.1% showed a sensitivity of 71% with a higher specificity (96%)

(6) FIG. 6 illustrates the receiver operating characteristics (ROC) based on the expression of CD57+ in % of CD45+CD3+CD8+ T cells with an area under the curve (AUC): 0.863 and p<0.001. The ROC analysis showed a high sensitivity (90%) as well as a specificity (61%) in the detection of the affected patients based on their blood levels using a cut-off level of 24.4%. A cut-off level of 37.6% showed a sensitivity of 71% with a higher specificity (96%)

(7) FIG. 7 illustrates the receiver operating characteristics (ROC) based on the expression of CD28− in % of CD45+CD3+CD8+ T cells with an area under the curve (AUC): 0.876 and p<0.001. The ROC analysis showed a high sensitivity (90%) as well as a specificity (66%) in the detection of the affected patients based on their blood levels using a cut-off level of 28.0%. A cut-off level of 42.9% showed a sensitivity of 76% with a higher specificity (91%)

EXAMPLES

(8) In order to define novel biomarkers for predicting the individual risk and to identify novel therapeutic targets, the inventors included 44 patients that received a spinal bone fusion surgery with secured vertebral body fusion results into a retrospective study. These patients were classified in patients with normal vertebral body fusion (n=23) and patients with pseudarthrosis/non-fusion (n=21), based on their CT-scan results.

(9) Table 1 shows the characterization and blood parameters of the patients involved in the retrospective study (spinal fusion and spinal non-fusion patients)

(10) TABLE-US-00001 Spinal non- Spinal fusion p- Parameter fusion (n = 21) (n = 23) value Age (y) 70.7 ± 8.1  63.7 ± 10.1 0.01 Sex 6 male/ 10 male/ 0.3 15 female 13 female (28.6%)/ (43.5%)/ (71.4%) (56.5%) Time to revision or 21.7 ± 12.7 27.0 ± 10.3 0.1 revision free Minimum: 8 Minimum: 12 (month) Maximum: 47 Maximum: 57 Height (m) 1.62 ± 0.1  1.66 ± 0.1  0.3 Weight (kg) 76.9 ± 10.7 79.6 ± 13.7 0.5 BMI 29.1 ± 3.2  29.3 ± 6.4  0.9 Leucocytes (/nL) 8.5 ± 3.9 6.7 ± 2.0 0.06 Haemoglobin (g/L) 13.2 ± 1.9  14.2 ± 1.3  0.06 Haematocrite (%) 0.39 ± 0.05 0.41 ± 0.03 0.07 Erythrocytes (/pL) 4.4 ± 0.6 4.6 ± 0.5 0.2 Thrombocytes (/nL) 271.5 ± 77.8  253.0 ± 53.0  0.3 Sodium (mmol/L) 139.0 ± 3.9  139.0 ± 2.9  0.9 Potassium (mmol/L) 4.2 ± 0.4 4.1 ± 0.3 0.6 Calcium (mmol/L) 2.4 ± 0.1 2.3 ± 0.1 0.4 Glucose (mg/dL) 102.3 ± 24.1  109.1 ± 41.8  0.5 Creatinin (mg/dL) 0.88 ± 0.2  0.81 ± 0.2  0.3 Urea (mg/dL) 32.8 ± 9.2  32.4 ± 11.3 0.9 Albumin (g/L) 42.9 ± 9.3  44.5 ± 2.7  0.4 GPT (U/L) 24.5 ± 19.9 28.4 ± 16.0 0.5 GOT (U/L) 26.5 ± 14.2 31.0 ± 28.8 0.5 CRP (mg/L) 0.9 ± 1.1 4.0 ± 6.0 0.03

(11) Table 2: shows the blood parameters and time to diagnosis of spinal non-fusion at the initial and revision surgery for non-fusion patients.

(12) TABLE-US-00002 Spinal non- fusion Initial Spinal non-fusion p- Parameter Surgery Revision Surgery value Time to diagnose  8 month = n = 2 = 9.5% non-fusion (month) 10 month = n = 2 = 9.5% 11 month = n = 2 = 9.5%  12 month = n = 3 = 14.3% 13 month = n = 1 = 4.8% 18 month = n = 1 = 4.8% 19 month = n = 1 = 4.8% 24 month = n = 1 = 4.8% 26 month = n = 1 = 4.8% 31 month = n = 1 = 4.8% 33 month = n = 1 = 4.8% 36 month = n = 2 = 9.5% 40 month = n = 2 = 9.5% 47 month = n = 1 = 4.8% Revision performed — 15/21 (71.4%) — Spongiosa + BMP-2 — 8/15(53.3%) — Spongiosa 7/15 (46.7%) — Leucocytes (/nL) 8.5 ± 3.9  6.5 ± 1.6 0.9 Haemoglobin (g/L) 13.2 ± 1.9  13.9 ± 1.9 0.6 Haematocrite (%) 0.39 ± 0.05  0.41 ± 0.06 0.8 Erythrocytes (/pL) 4.4 ± 0.6  4.4 ± 0.7 0.7 Thrombocytes (/nL) 271.5 ± 77.8  261.4 ± 68.1 0.5 Sodium (mmol/L) 139.0 ± 3.9  137.7 ± 4.4  0.4 Potassium (mmol/L) 4.2 ± 0.4  4.2 ± 0.4 0.7 Calcium (mmol/L) 2.4 ± 0.1  2.4 ± 0.1 0.5 Glucose (mg/dL) 102.3 ± 24.1  106.4 ± 23.8 0.3 Creatinin (mg/dL) 0.88 ± 0.2  0.87 ± 0.2 0.7 Urea (mg/dL) 32.8 ± 9.2  36.5 ± 9.1 0.3 Albumin (g/L) 42.9 ± 9.3  43.8 ± 2.9 0.4 GPT (U/L) 24.5 ± 19.9 19.6 ± 9.7 0.2 GOT (U/L) 26.5 ± 14.2 22.7 ± 4.9 0.2 CRP (mg/L) 0.9 ± 1.1  4.4 ± 9.5 0.01

(13) As established in literature and routinely used in clinics, there are time dependent and radiological criteria for the classification of a non-fusion. To fulfil the definition of a non-fusion process, the patients have to meet one or more of the following criteria.

(14) Time Dependent Criteria:

(15) An incomplete vertebral body fusion after at least 24 post-operative weeks based on the presence of scar tissue formation in the intervertebral zone. (Zenya Ito et al., SPINE Volume 35, Number 21, pp E1101-E1105; 2010)

(16) Radiological+Clinical Criteria: 1) no bridging of the trabecular bone connecting the two vertebral bodies, 2) angular motion in excess of 5 degrees 3) sagittal translation in excess of 3 mm 4) radiolucencies that involve more than half of the interfaces between the dowels and the host vertebral end plates

(17) Based on this former classification, the non-fusion patients showed differences in immune and inflammatory parameters in peripheral blood. Particularly, the spinal non-fusion was strongly associated with a significantly enhanced frequency (>2 fold) of terminally differentiated CD8+ effector T cells expressing the phenotype CD45+CD3+CD8+CD28− and/or CD45+CD3+CD8+CD57+(CD8+ TEMRA).

(18) Accordingly, patients with enhanced levels of CD8+ TEMRA cells, because of a personal history of chronic immunostimulation (e.g. by persistent and frequently reactivating viruses like CMV/EBV), have a worse vertebral body fusion or quality following spinal fusion surgery as a result of an overwhelming inflammation that inhibits normal vertebral body fusion.

(19) The data presented herein demonstrate that the individual immune profile (frequency of CD8+ TEMRA) is a reliable biomarker for predicting impaired bone fusion patients allowing early interventions with known and established methods.