CD8 T CELL SUBSETS AS A BIOMARKER FOR PREDICTING NON-FUSION AFTER SPINAL FUSION SURGERY

Abstract

The present invention relates to 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. The invention further relates to a system for predicting the probability of having or developing a non-fusion.

Claims

1. Method for predicting the probability of having or developing a non-fusion, particularly before undergoing a spinal fusion surgery or after a spinal fusion surgery, 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.

2. Method for monitoring the course of treatment of a non-fusion in a patient, particularly after spinal fusion surgery, 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 said patient.

3. 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, particularly a non-fusion between two or more vertebrae.

4. The method according to claim 1, wherein said sample is a blood sample.

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

6. The method according to claim 1, wherein a 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 large population with normal fusion after bone fusion is assigned to a patient having an elevated probability for having or developing non-fusion after spinal fusion surgery.

7. The method according to claim 1, wherein a sample exhibiting a fraction of CD8+CD57+CD28 cells referred to the total sum of all CD8+ cell in said sample of at least 22.7% more particular of at least 33.1%, is assigned to a patient having an elevated probability for having or developing non-fusion after spinal fusion surgery, and/or a sample exhibiting a summed fraction of CD8+CD57+ cells and CD8+CD28 cells referred to the total sum of all CD8+ cells in said sample of at least 85.6% is assigned to a patient having an elevated probability for having or developing non-fusion after spinal fusion surgery, and/or a sample exhibiting a fraction of CD8+CD57+ cells referred to the total sum of all CD8+ cells in said sample of at least 24.4%, more particular of at least 37.6%, is assigned to a patient having an elevated probability for having or developing non-fusion after spinal fusion surgery, and/or a sample exhibiting a fraction of CD8+CD28 cells referred to the total sum of all CD8+ cells in said sample of at least 28.0%, more particular of at least 42.9%, is assigned to a patient having an elevated probability for having or developing non-fusion after spinal fusion.

8. A system for predicting the probability of having or developing a non-fusion, particularly before undergoing a spinal fusion surgery or after a spinal fusion surgery, and/or for monitoring the course of treatment of a non-fusion of a patient, particularly after spinal fusion surgery, comprising a device for determination the frequency of a cell population in a sample obtained from a patient programmed microprocessor, wherein said microprocessor is configured to run a method according to claim 1.

9. Use of a combination of antibodies for predicting the probability of having or developing a non-fusion, particularly before undergoing a spinal fusion surgery after a spinal fusion surgery, wherein said combination comprises an anti-CD8-antibody and an anti-CD57-antibody, or an anti-CD8-antibody and an anti-CD28 antibody, or an anti-CD8-antibody, an anti-CD28-antibody and an anti-CD57-antibody, and wherein said antibodies are suitable for fluorescence based flow cytometry.

10. A bone morphogenetic protein for use in a method for preventing or treating non-fusion after spinal bone fusion surgery, wherein said bone morphogenetic protein is administered during or after undergoing a spinal fusion surgery or a revision surgery after failed spinal fusion to a patient that has an elevated probability of developing or having a non-fusion after spinal bone fusion surgery.

11. The bone morphogenetic protein for use in a method for treating non-fusion after spinal bone fusion surgery in a patient having an elevated probability of non-fusion after spinal bone fusion surgery according to claim 10, wherein said bone morphogenetic protein is selected from BMP2, or BMP7,

12. The bone morphogenetic protein for use in a method for treating non-fusion after spinal bone fusion surgery in a patient having an elevated probability of non-fusion after spinal bone fusion surgery according to claim 10, wherein said elevated probability of developing or having a non-fusion after spinal bone fusion surgery have been determined by a method according to claim 1.

Description

SHORT DESCRIPTION OF THE FIGURES

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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%)

[0058] 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%)

[0059] 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

[0060] 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.

[0061] Table 1 shows the characterization and blood parameters of the patients involved in the retrospective study (spinal fusion and spinal non-fusion patients)

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

[0062] Table 2: shows the blood parameters and time to diagnosis of spinal non-fusion at the initial and revision surgery for non-fusion patients.

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

[0063] 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.

[0064] Time Dependent Criteria:

[0065] 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)

[0066] Radiological+Clinical Criteria:

[0067] 1) no bridging of the trabecular bone connecting the two vertebral bodies,

[0068] 2) angular motion in excess of 5 degrees

[0069] 3) sagittal translation in excess of 3 mm

[0070] 4) radiolucencies that involve more than half of the interfaces between the dowels and the host vertebral end plates

[0071] 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).

[0072] 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.

[0073] 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.