METHODS FOR DETERMINING THE INVASIVE AND/OR METASTATIC POTENTIAL OF A TUMOUR

Abstract

The invention provides methods for determining tumour status in a subject comprising the steps of: (i) determining a quantitative value in a sample taken from a subject of a first biomarker selected from the group consisting of Ran, Ran binding protein 1, an active fragment of a Ran protein, a nucleic acid sequence encoding Ran, a nucleic acid sequence encoding Ran binding protein 1, a nucleic acid sequence encoding an active fragment of Ran and a nucleic acid sequence encoding an active fragment of Ran binding protein 1; (ii) comparing the quantitative value of the first biomarker in the sample with a selected pre-determined threshold value of the first biomarker; (iii) determining a quantitative value in a sample from the same subject of a second biomarker selected from the group consisting of MMP2, an active fragment of MMP2, a nucleic acid sequence encoding MMP2 and a nucleic acid sequence encoding an active fragment of MMP2; (iv) comparing the quantitative value of the second biomarker in the sample with a selected pre-determined threshold value of the second biomarker; wherein the quantitative values of the first marker and the second biomarkers in the sample as compared to their respective selected pre-determined threshold values indicate whether or not the tumour sample has invasive and/or metastatic potential.

Claims

1. A method for determining tumour status in a subject comprising the steps of: (i) determining a quantitative value in a sample taken from a subject of a first biomarker selected from the group consisting of Ran protein, Ran binding protein 1, an active fragment of a Ran protein, a nucleic acid sequence encoding Ran, a nucleic acid sequence encoding Ran binding protein 1, a nucleic acid sequence encoding an active fragment of Ran and a nucleic acid sequence encoding an active fragment of Ran binding protein 1; (ii) comparing the quantitative value of the first biomarker in the sample with a selected pre-determined threshold value of the first biomarker; (iii) determining a quantitative value in a sample from the same subject of a second biomarker selected from the group consisting of MMP2 protein, an active fragment of MMP2 protein, a nucleic acid sequence encoding MMP2 and a nucleic acid sequence encoding an active fragment of MMP2; (iv) comparing the quantitative value of the second biomarker in the sample with a selected pre-determined threshold value of the second biomarker; wherein the quantitative values of the first marker and the second biomarkers in the sample as compared to their respective selected pre-determined threshold values indicate whether or not the tumour sample has invasive and/or metastatic potential.

2. A method according to claim 1 wherein the sample obtained for determination of a qualitative value of the first biomarker is the same or different sample as that used to determine the quantiataive value for the second biomarker.

3. A method according to claim 1 wherein the sample is selected from the group comprising a solid tumour biopsy, a liquid biopsy, a tumour cell, circulating tumour cells in blood, circulating tumour cells in blood plasma and circulating levels of biomarkers in a body fluid.

4. A method according to claim 1 wherein the selected predetermined threshold value of the first biomarker is at least between 0.5 and 5.0% of the sample reference value of the first biomarker.

5. A method according to claim 4 wherein the selected predetermined threshold value of the first biomarker is at least 1.0% of the sample reference value of the first biomarker.

6. A method according to claim 1 wherein the selected predetermined threshold value of the second biomarker is at least between 1.0 and 10.0% of the sample reference value of the second biomarker.

7. A method according to claim 6 wherein the selected predetermined threshold value of the second biomarker is at least 5.0% of the sample reference value of the second biomarker.

8. A method according to claim 4 wherein the sample reference value is the number of cells expressing the first and/or second biomarkers in subject having an invasive and/or metastatic tumour.

9. A method according to claim 8 wherein the number of cells is assessed immunohisto/immunocyto-chemically or other similar platform.

10. A method according to claim 4 wherein the sample reference value is the level of expression of the first and/or second biomarkers in subject having an invasive and/or metastatic tumour.

11. A method according to claim 10 wherein the level of expression of the first and second markers is assessed by ELISA, immunoprecipitation or immunoblotting or other protein detection platform.

12. A method according to claim 1 wherein, when the quantitative value of the first marker in the sample is lower than the selected predetermined threshold value for the first marker and the quantitative value of the second marker in the sample is lower than the selected predetermined threshold value for the second marker indicates that the tumour does not have invasive and/or metastatic potential.

13. A method according to claim 1 wherein, when the quantitative value of the first marker in the sample is above the selected predetermined threshold value for the first marker and the quantitative value of the second marker in the sample is above the selected predetermined threshold value for the second marker indicates that the tumour has invasive and/or metastatic potential.

14. A method according to claim 1 wherein, when the quantitative value of the first marker in the sample is lower than the selected predetermined threshold value for the first marker and the quantitative value of the second marker in the sample is above the selected predetermined threshold value for the second marker indicates that the tumour may have invasive and/or metastatic potential and requires further monitoring.

15. A method according to claim 1 wherein, when the quantitative value of the first marker in the sample is above the selected predetermined threshold value for the first marker and the quantitative value of the second marker in the sample is lower than the selected predetermined threshold value for the second marker indicates that the tumour may have invasive and/or metastatic potential and requires further monitoring.

16. A method according to claim 1 wherein the tumour is selected from the group comprising human breast cancer, and, in particular, an oestrogen receptor positive and human epidermal growth factor receptor 2 negative breast cancer cell or a triple receptor negative breast cancer (TNBC) cell.

17. A method according to claim 1 wherein the tumour status includes, monitoring for metastasis following surgery and/or during chemotherapy or radiotherapy, stratification of a group of subjects with cancer and/or predicting probability of survival/metastatic potential of a subject.

18. A kit comprising a first reagent and a second reagent for assessing respectively levels of a first marker and a second marker in a tumour sample or a blood plasma sample, wherein the first marker is selected from the group consisting of Ran, Ran binding protein 1, an active fragment of a Ran protein, an active fragment of Ran binding protein 1, a nucleic acid sequence encoding Ran, a nucleic acid sequence encoding Ran binding protein 1, a nucleic acid sequence encoding an active fragment of Ran and a nucleic acid sequence encoding an active fragment of Ran binding protein 1; and the second marker is selected from the group consisting of MMP2, an active fragment of MMP2 protein, a nucleic acid sequence encoding MMP2 and a nucleic acid sequence encoding an active fragment of MMP2.

19. A kit according to claim 18 for use in a method for determining whether a tumour in a subject has invasive and/or metastatic potential.

20. Use of a kit according to claim 18 for determining whether a tumour in a subject has invasive and/or metastatic potential.

Description

[0090] The present invention is now described in more detail with reference to the following Examples and the accompanying drawings in which:

[0091] FIG. 1 is graph plotting the cumulative proportion of surviving patients against survival time as determined by a retrospective immunohistochemical study of sections of primary tumours taken from the 181 unselected human breast cancer patients—classified (a to d) according to positive and/or negative staining for Ran protein and for MMP2 protein;

[0092] FIG. 2 is a graph showing the extent of RAN expression in the blood plasma of 238 unselected breast cancer patients which went on to develop metastasis as compared to patients that did not go onto develop metastasis; and

[0093] FIG. 3 is a graph plotting the proportion of distance metastasis free survival (DMFS) against time as determined by retrospective Elisa assay of RAN levels (positive and negative) in the blood plasmas of the 238 cancer patients.

EXAMPLE 1

[0094] A retrospective statistical study was undertaken using (−/+) IHC staining of samples of 181 primary tumours from unselected breast cancer patients in order to determine the relationship between Ran, c-Met, c-Myc and MMP2.

[0095] The study was conducted in accordance with methods previously described for Ran (de Silva Rudland S. et al in in Am. J. Pathol. 2011, 79, 1061-1072 and Rudland P. S. et al in Am. J. Pathol. 2010, 176, 2935-2947). Briefly, patients received no adjuvant therapy including hormonal therapy and only patients with operable breast cancer (T1-4, NO-1) were included. Patient follow-up times ranged from 14.5 to 19.4 years (mean 16.4±0.1 years) with a mean±SE survival time of 9.0±0.5 years. Ethical approval was obtained from NRES Committee North West REC Ref 12/NW/0778, Protocol no. UoL000889, IRAS no 107845. Samples were preserved in neutral buffered formalin and embedded in paraffin wax as described previously (Rudland P. S. et al in Cancer Res. 2000, 60, 1595-1603).

Materials and Methods

[0096] IHC staining. Histological sections cut at 4 μm were mounted on slides, treated with 0.05% v/v H.sub.2O.sub.2 in methanol to inhibit endogenous peroxidase (Rudland P. S. et al, in Cancer Res. 2000, 60, 1595-1603) and incubated with the relevant primary and horseradish peroxidase labelled antibodies/polymers in kits (DAB) (Dako Ltd, Ely, UK), as described previously (de Silva Rudland S. et al, in Am. J. Pathol. 2011, 79, 1061-1072 and Ismail T. M. et al, in Cancer Res. 2017, 77, 780-789). Positive staining corresponded to an oxidised brown precipitate of diaminobenzidine (DAB). Slides were finally mounted in Glycergel mounting medium (Dako). Blocked antibodies prepared by mixing 1 mg/ml of the relevant blocking peptide/protein abolished this staining. Appropriate immune serum also yielded no staining. Western blots of breast cell lines verified the specifity of all antibodies used by yielding the appropriately-sized molecular weight bands on SDS—polyacrylamide gels.

[0097] IHC scoring analysis. IHC-stained sections were analysed and scored by two independent observers using light microscopy according to the percentage of stained carcinoma cells from 2 well separated sections of each specimen, 10 fields per section at 200× magnification and a minimum of 200 cells per field, as described previously (de Silva Rudland S. et al, in Am. J. Pathol. 2011, 79, 1061-1072 and Ismail T. M. et al, in Cancer Res. 2017, 77, 780-789).

[0098] Staining data analysis was performed using Excel (Microsoft, Redmond, Wash.), and SPSS version 22 (SPSS, Chicago, Ill.).

[0099] Staining for all proteins had already been separated into two categorical groups, a negative and positive group with a cut-off of either 1% or 5% of carcinoma cells staining, according to which cut-off yielded the more significant difference and greater relative risks: 1% cut-offs for Ran, cMyc, Ki67, CK5/6 and 5% cut-offs for cMet, MMP2, ERα, c-erbB-2, PgR (de Silva Rudland S. et al in Am. J. Pathol. 2011, 79, 1061-1072; Yuen H-F. et al in Clin. Cancer Res. 2012, 18, 380-391; Yuen H-F. et al in J. Natl. Cancer Inst. 2013, 105, 475-488; Yuen H-F. et al in Oncotarget 2016, 7, 75854-75864; and Ismail T. M. et al, in Cancer Res. 2017, 77, 780-789).

[0100] The association of staining for each protein separately in this set of patients was calculated from life tables constructed from survival data using Kaplan Meier plots and analysed by Wilcoxon (Gehan) statistics (Rudland P. S. et al in Cancer Res. 2000, 60, 1595-1603). Patients who died from causes other than cancer were censored. Unadjusted relative risk (RR) for survival with 95% confidence interval (95% CI) was calculated using Cox's univariate analysis (Rudland P. S. et al in Am. J. Pathol. 2010, 176, 2935-2947).

[0101] Association of IHC staining for Ran or MMP2 with other tumour variables was assessed by cross-tabulations using Fishers Exact test (2-sided) using either 1% or 5% cut-offs. For multiple comparisons the resultant P values were corrected by the Holm-Bonferroni formulae of I—(I-P).sup.n, where n is the number of tumour variables.

[0102] Binary Logistic Regression was used for calculation of the relative independent association (RA) of staining for one protein with the remaining proteins in the group. To determine if the association of patient survival with Ran, MMP2 etc. was significant within a group of proteins, Cox's multivariate analyses were performed on 181 patients, incomplete data arose mainly from lack of sampling (de Silva Rudland S. et al, in Am. J. Pathol. 2011, 79, 1061-1072).

Association of Individual Tumour Variables with Patient Survival Times

[0103] The relationship in human breast cancer of tumour variables including markers Ran, c-Met, c-Myc, MMP2, Ki67, Era, c-erbB-2 and CK5/6 with patient demise as a result of metastatic breast cancer was investigated as described above.

[0104] The results of the statistical analyses are shown in Table 1. As may be seen, the largest significant differences in relative risk (RR) within the present group of 181 patients were as follows: Ran (χ.sup.2=35.4, RR=14.9), cMet (χ.sup.2=32.9, RR=10.7), cMyc (χ.sup.2=40.3, RR=9.5), MMP-2 (χ.sup.2=64.8, RR=7.7) and CK5/6 (χ.sup.2=43.3, RR=5.6).

[0105] The differences in relative risk for ERα (χ.sup.2=1.24, RR=0.81), c-erbB-2 (χ.sup.2=1.93, RR=1.33) and Ki67 (χ.sup.2=1.6, RR=1.3) were not found to be significant within this group of patients—notwithstanding a report that in a larger patient group they significantly different (de Silva Rudland S. et al, in Am. J. Pathol. 2011, 79, 1061-1072).

[0106] In contrast to this report, the present group of patients showed a significant RR for nodal size (with or without the involvement of lymph nodes) of 2.3 (χ.sup.2=14.64, 1df, P<0.001). The tumour size and histological grade were not found to be significantly associated with patient survival.

Association of Ran and MMP2 with Other Tumour Variables

[0107] The association in human breast cancer of Ran with the other tumour variables and of MMP2 with other tumour variables was investigated as described above. The results are summarised in Table 2.

[0108] As may be seen, Ran was very significantly associated with c-Met (P=6.6×10.sup.−5), cMyc (P=4.4×10.sup.−5), MMP2 (P=5.7×10.sup.−6), and CK5/6 (P=5.5×10.sup.−5) but not at all associated with Ki67, ERα, c-erbB-2, tumour size and histological grade (P 0.34).

[0109] The association of Ran with TRNBC (P uncorrected=0.06) was of borderline significance and involved lymph nodes alone (P uncorrected=0.037). The most significant association of Ran with other tumour markers was found to be the association between Ran and MMP2.

[0110] MMP2 was strongly significantly associated with the same tumour markers as Ran viz., c-Met (P=6.4×10), c-Myc (P=1.8×10.sup.−7), CK5/6 (P=7.5×10.sup.−7) as well as with RAN itself (P=5.7×10.sup.−6) and not with the remainder of the tumour variables (P 0.35).

[0111] Note that there is no significant staining for Ran with staining for Ki67 which appears consistent with the fact that little increase in cell proliferation is observed in Ran transfected cells.

[0112] Further, the fact that staining for Ran, c-Met, c-Myc and MMP2 are all very significantly associated with staining for CK5/6 but not staining for Era or c-erbB-2 suggests that RAN c-Met, c-Myc and MMP2 occur mainly in the Basal Cell type of breast cancers.

[0113] This sub-group of breast cancers overlaps considerably with the triple receptor negative breast cancer (TRNBC) sub-group (de Silva Rudland S. et al, in Am. J. Pathol. 2011, 79, 1061-1072) which may explain the borderline association of Ran staining with triple receptor negative breast cancer.

TABLE-US-00001 TABLE 1 Association of tumour variables with patient survival times Cum. Median survival Tumour Patient survival at end variable.sup.a no Grouping (months) (%) χ.sup.2 b P .sup.b RR .sup.c 95% CI .sup.c Ran 181 − 228 93 35.44 <0.001 14.87   4.69-47.15 + 73.3 32 Met .sup.d 162 − 216 90 32.95 <0.001 10.71   4.30-26.64 + 59.6 26 cMyc 181 − 228 88 40.31 <0.001 9.49  4.13-21.80 + 58.9 29 MMP2 .sup.d 181 − 228 78 64.82 <0.001 7.70  4.67-12.70 + 48.4 10 Ki67 147 − 189.3 49 1.58 0.21 1.31 0.81-2.10 + 83.9 42 ERα .sup.d 179 − 103.9 46 1.24 0.265 0.81 0.53-1.25 + 228 51 c-erbB-2 .sup.d 176 − 185.0 49 1.93 0.165 1.33 0.82-2.15 + 59.8 44 CK5/6 177 − 228 72 43.29 <0.001 5.57 3.54-8.78 + 50.6 07 TRNBC.sup.e 164 − 216 55 0.37 0.545 1.23 0.72-2.11 + 156.1 45 Nodal 136 − 228 58 14.64 <0.001 2.32 1.43-3.77 status .sup.f + 59.1 35 Grade .sup.g 164 − 173.7 48 2.41 0.12 .sup. 1.4 .sup.g  .sup. 0.88-2.24 .sup.g (Gr1, 2) + 58.8 40 (Gr3) Tumour 175 − 216 51 1.02 0.31  .sup. 1.33 .sup.h  .sup. 0.82-2.16 .sup.h size .sup.h (T.sub.1, T.sub.2) + 80.0 39 (T.sub.3, T.sub.4) .sup.aNegative vs positive staining, 1% cut-off, except where stated. .sup.b χ.sup.2 and probability (P) were determined using generalised Wilcoxon (Gehan) statistics. .sup.c RR and 95% CI were determined using Cox's univariate analysis with 1 df. .sup.d Negative vs. positive staining, 5% cut-off. .sup.eTriple Receptor Negative Breast Cancer (TRNBC− vs TRNBC+). .sup.f No nodes vs. 1 or more nodes; P for 1 df. .sup.g Histological grade: 1, 2 vs 3; P for 1 df. RR for Grade 1 vs 2 = 2.74 (95% CI, 1.45-5.19), 1 vs 3 = 2.83 (1.42-5.66); all 1 df. .sup.h Tumour size <5 cm vs >5 cm in diameter, T.sub.1, T.sub.2 vs T.sub.3, T.sub.4; P for 1 df. RR for T.sub.1, T.sub.2 vs T.sub.3, T4; T.sub.1 vs T.sub.2 = 1.54 (95% CI, 0.67-3.58), T.sub.1 vs T.sub.3 = 1.76 (0.69-5-4.51), T.sub.1 vs T.sub.4 = 2.68 (0.86-8.32), all 1 df.

TABLE-US-00002 TABLE 2 Association of IHC staining for Ran and MMP2 with other tumour variables Tumour Patient Statistical significance.sup.c variable.sup.a no.sup.b RAN MMP2 Ran 181 — 5.7 × 10.sup.−6 cMet.sup.d 162 6.6 × 10.sup.−5 6.4 × 10.sup.−9 cMyc 181 4.4 × 10.sup.−5 1.8 × 10.sup.−7 MMP2.sup.d 181 5.7 × 10.sup.−6 — Ki67 147 0.94 0.50 ERα.sup.d 179 0.94 1.0 c-erbB-2.sup.d 176 1.0 0.85 CK5/6 177 5.5 × 10.sup.−5 7.5 × 10.sup.−7 TNRBC.sup.e 164 0.50.sup.f 0.97 Tumour size.sup.g 175 0.99 0.83 Grade.sup.h 164 0.99 1.0 Node.sup.i 136 0.34.sup.j 0.35.sup.j .sup.aNegative vs positive IHC staining for molecular variables using 1% cut-off, except where stated. .sup.bNumber of patients from original 181. .sup.cProbability P from Fisher's Exact test using the Holm-Bonferroni correction calculated as 1 − (1 − P).sup.n, where n = 11. .sup.dNegative vs positive staining for 5% cut-off. .sup.eTriple Receptor Negative Breast Cancer. .sup.fWithout Bonferroni correction P = 0.061. .sup.gTumour size <5 cm vs >5 cm in diameter. .sup.hHistological grade 1, 2 vs 3. .sup.iNo nodes vs 1 or more lymph nodes involved. .sup.jWithout Bonferroni correction P = 0.037 for Ran and 0.039 for MMP2.
Relative Independent Association of Ran, c-Met, c-Myc, MMP2 and Ki67

[0114] The probability of independent association in human breast cancer of Ran, c-Met, c-Myc and Ki67 with each other was investigated as described above.

[0115] The results are shown in Table 3. As may be seen, the relative independent association (RA) of Ran with cMet and with MMP2 was found to be strongest (RA=3.0 to 3.4), whilst the relative independent association of Ran with Ki67 was found not to be significant (RA=1.12, P=0.81).

TABLE-US-00003 TABLE 3 Probability of independent association of staining for Ran and other molecular markers Test.sup.a Other.sup.b variable variables Coeff β.sup.c SE of β.sup.c χ.sup.2 d P.sup.e RA.sup.f 95% CI.sup.f Ran cMet 1.221 0.536 5.185 0.023 3.39 1.19-9.69 cMyc 0.677 0.521 1.687 0.194 1.97 0.71-5.47 MMP2 1.099 0.628 3.064 0.080 3.00  0.88-10.27 Ki67 0.117 0.493 0.056 0.813 1.12 0.43-2.96 cMet Ran 1.228 0.525 5.462 0.019 3.41 1.22-9.56 cMyc 0.866 0.498 3.018 0.082 2.38 0.90-6.31 MMP2 2.067 0.566 13.327 <0.001 7.9  2.60-23.96 Ki67 0.488 0.484 1.015 0.314 1.63 0.63-4.21 cMyc Ran 0.697 0.515 1.833 0.176 2.01 0.73-5.50 cMet 0.852 0.504 2.856 0.091 2.34 0.87-6.30 MMP2 1.644 0.550 8.923 0.003 5.18  1.76-15.22 Ki67 0.079 0.469 0.029 0.866 1.08 0.43-2.72 MMP2 Ran 0.982 0.627 2.451 0.117 2.67 0.78-9.13 cMet 2.047 0.571 12.869 <0.001 7.74  2.53-23.70 cMyc 1.587 0.553 8.238 0.004 4.89  1.65-14.46 Ki67 0.305 0.468 0.424 0.515 1.36 0.54-3.40 .sup.aPrinciple IHC-staining variable for probability of association with other tumour variables using cut-offs defined in Tables 1, 2. .sup.bSets of other IHC-staining variables were included in binary Logistic Regression Analysis using cut-offs defined in Tables 1, 2 to separate positive and negative staining groups. .sup.cValue of coefficient β (Coeff β) with its standard error (SE) in binary Logistic Regression Analysis.

[0116] c-Met showed the strongest relative independent associations with Ran (RA=3.41, P=0.019) and with MMP2 (7.9, P<0.001).

[0117] The strongest relative independent association for c-Myc was with MMP2 and the strongest relative independent association of MMP2 was with cMet.

Association of Ran, cMet, cMyc and MMP2 with Patient Survival

[0118] The stainings for Ran, cMet, cMyc and MMP2 were investigated for relative independent association with patient survival times as described above.

[0119] The results are shown in Table 4. As may be seen, the set of stainings for Ran, c-Met, c-Myc and MMP2 showed a significant degree of independence (P≤0.036) with similar relative risks (RR) for patient demise of 3.1 fold to 3.7 fold. These RRs are considerably less than the 7 fold to 15 fold decreases shown in univariate analyses reported in Table 1.

[0120] The sets of stainings for Ran and c-Met and Ran and MMP2 showed a reduction in RR for patient demise as compared to Ran staining alone (from 14.9 fold to between 7.6 to 7.8 fold). The set of stainings for Ran and c-Myc showed a reduction in RR for patient demise as compared to Ran staining alone (from 14.9 fold to 9.8 fold).

TABLE-US-00004 TABLE 4 Summary of results for Cox's proportional hazards for cancer-related deaths Tumour variable.sup.a Coeff β.sup.b SE of β.sup.b χ.sup.2c P.sup.d RR.sup.e 95% CI.sup.e Set A Ran 1.305 0.622 4.398 0.036 3.69 1.09-12.49 cMet 1.153 0.503 5.257 0.022 3.17 1.18-8.49  cMyc 1.246 0.445 7.827 0.005 3.48 1.45-8.32  MMP2 1.127 0.310 13.225 <0.001 3.10 1.68-5.67  Set B Ran 2.025 0.594 11.600 0.001 7.57 2.36-24.28 cMet 1.985 0.470 17.879 <0.001 7.28 2.90-18.27 Set C Ran 2.286 0.592 14.918 <0.001 9.84 3.08-31.39 cMyc 1.873 0.427 19.252 <0.001 6.51 2.82-15.02 Set D Ran 2.056 0.600 11.727 0.001 7.82 2.41-25.36 MMP2 1.642 0.260 40.023 <0.001 5.17 3.11-8.59  .sup.aIn Set A comparisons were made between duration of survival time of patients with tumours stained for Ran, cMet, cMyc and MMP2; overall χ.sup.2 = 96.21, 4 df, P < 0.001. In Set B comparisons between patients with tumours stained for Ran and cMet; overall χ.sup.2 = 52.4, 2 df, P < 0.001. In Set C comparisons between patients with tumours stained for Ran and cMyc; overall χ.sup.2 = 60.6, 2 df, P < 0.001. In Set D comparisons between patients with tumours stained for Ran and MMP2; overall χ.sup.2 = 96.5, 2 df, P < 0.001. IHC cut-offs as described in Tables 1, 2. .sup.bValue of β coefficient (=log.sub.eRR) and standard error (SE) in Cox's multiple regression analysis. .sup.cCox's statistic χ.sup.2. .sup.dProbability P from Cox's statistic χ.sup.2, 1 df in each case. .sup.eRelative Risk (RR) for survival and 95% confidence interval (95% CI) from multivariate analysis.

[0121] Note that the stronger relative independent association between staining for MMP2, c-Met and c-Myc than staining for Ran suggests that the increase in MMP2 expression in tumours is not solely due to an increase in Ran but may arise from other signalling mechanisms.

[0122] Further, the partial nature of the confounding of RR for Ran suggests that other pathways not involving Ran are also involved in causing patient demise.

[0123] Note that the decline in RR for Ran staining with either c-Met staining or MMP2 staining is binary combination with Ran (49% and 48% respectively) was larger than the decline in RR for staining for Met or MMP2 staining in binary combination with Ran (32% for both) suggests that Met and MMP2 are more proximal members in this signalling pathway than Ran.

Association of Ran and MMP2 with Patient Survival Times

[0124] Table 5 and FIG. 1 shows cumulative proportions of surviving patients as a fraction of the total for each year (up to about 20 years) after presentation with carcinomas classified according to the following sets:

[0125] Set a (solid line): negatively stained for Ran (−ve) and for MMP2 (−ve); set b (dotted line): positively stained for Ran (+ve) and negatively stained for MMP2 (−ve) set c (dashed line): negatively stained for Ran (−ve) and positively stained for MMP2 (+ve); and set d (dashed and dotted line): positively stained for both Ran (+ve) and MMP2 (+ve).

[0126] As may be seen, in set a the median survival (ms) was greater than 228 months, the final cumulative survival (fcs) was 0.97. There were 39 censored observations (8 dead of other causes); in set b the ms was greater than 216 months and the fcs was 0.6. There were 44 censored observations (19 dead of other causes). In set c the ms was greater than 216 months and the fcs was 0.60. There were 3 censored observations (1 dead of other causes). In set d, the ms was 46.2 months and the fcs was 0.06. There were 8 censored observations (4 dead of other causes).

[0127] The statistical analysis of staining groups consisting of two of these sets shows that staining for Ran and for MMP2 can be synergistic and increase RR respectively from 17.1 fold and 23.1 fold to 82.1 fold (compare staining set a against staining set d). In terms of patient survival, the staining may show a reduction in patient survival after nearly 20 years respectively from 64% and 60% to only 6%.

TABLE-US-00005 TABLE 5 Difference in survival between staining sets Median Staining FIG. 1 survival .sup.d Cumulative Group.sup.a line χ.sup.2 b P .sup.b RR .sup.c 95% CI .sup.c (months) survival .sup.d Ran−/MMP2− a vs b 14.02 <0.001 17.11  2.30-127.56 >228 0.97 Ran+/MMP2− >216 0.64 Ran−/MMP2− a vs c 10.583 0.001 23.10  2.09-255.02 >228 0.97 Ran−/MMP2+ >216 0.60 Ran+/MMP2− b vs c 0.407 0.524 1.35 0.32-5.78  >216 0.64 Ran−/MMP2+ >216 0.60 Ran+/MMP2− b vs d 33.09 <0.001 4.80 2.88-7.99  >216 0.64 Ran+/MMP2+ 46.2 0.06 Ran−/MMP2+ c vs d 1.569 0.210 3.56 0.87-14.61 >216 0.60 Ran+/MMP2+ 46.2 0.06 Ran−/MMP2− a vs d 59.643 <0.001 82.11 11.34-594.64 >228 0.97 Ran+/MMP2+ 46.2 0.06 .sup.aImmunocytochemical staining class for MMP2 and Ran classified as staining (+; +ve) or not staining (−; −ve). .sup.b Wilcoxon statistic (χ.sup.2) and Probability (P) were determined using the generalised Wilcoxon (Gehan) test with 1 df.. .sup.c Relative risk (RR) and confidence interval (95% CI) were determined using Cox's univariate analysis with 1 df. .sup.d Median survival in months and cumulative proportion of patients surviving evaluated using life tables constructed from survival data.

[0128] A significant decrease in patient survival times is found for the doubly stained set d (Ran +ve/MMP2 +ve) over the singly-stained set b (Ran +ve/MMP2 −ve; P<0.001) which is not found for the doubly stained set d (Ran +ve/MMP2 +ve) over the singly stained set b (Ran −ve/MMP2 +ve; P=0.21).

[0129] This is particularly clear when cumulative proportion of surviving patients is plotted against survival time for each staining group. As may be seen from FIG. 1, noting that the number of patients presenting each year is set out below that figure, the curves corresponding to each staining group are highly significantly different from one another.

[0130] Note that Table 5 shows that when staining data for MMP2 is added to staining data for Ran in primary cancer cells, there is a significant decrease in patient survival times—but that when staining data for Ran is added to staining data for MMP2 there is no significant increase in patient survival times. So much supports the notion that the c-Met and MMP2 are more proximal members than Ran in the pathway leading to patient demise and is consistent with the order of the proteins that leads to an increase in metastatic properties of cultured cells.

TABLE-US-00006 TABLE 6 Sensitivity, Specificity, NPR and PPR in IHC assay for Ran and MIVIP2 alone and in combination Patients IHC Patients died from Total Staining Alive cancer Patients Sensitivity Specificity IHC Score at % % % (true +ve (true −ve Sub-Type Assay Diagnosis # alive # died # total rate) rate) PPR NPR All Ran <1% 42 44.7 3 3.4 45 24.9 44.7 93.3 (−ve) All Ran 2-5% 52 55.3 84 96.6 113 75.1 96.6 74.3 (+ve) All Ran All 94 100 87 100 181 100 ER+/HER− Ran <1% 23 51.1 1 1.9 24 24.7 51.1 95.8 (−ve) ER+/HER− Ran 2-5% 22 48.9 51 98.1 73 75.3 98.1 69.7 (+ve) ER+/HER− Ran All 45 100 52 100 97 100 Triple −VE Ran <1% 28 84.8 2 7.1 30 49.2 84.8 93.3 (−ve) Triple −VE Ran 2-5% 5 15.2 26 92.9 31 50.8 92.9 84.2 (+ve) Triple −VE Ran All 33 100 28 100 61 100 All MMP2 <2% 83 88.3 21 24.1 104 57.5 88.3 79.8 (−ve) All MMP2 2-5% 11 11.7 66 75.9 77 42.3 75.9 91.7 (+ve) All MMP2 All 94 100 87 100 181 100 All Ran/MMP2 −ve/−ve 39 83.0 1 1.5 40 32.0 83.0 97.5 All Ran/MMP2 +ve/+ve 8 17 64 98.5 72 68.0 98.5 88.8 All Ran/MMP2 All 47 100 65 100 112 100

[0131] The present study shows that the addition of staining data for MMP2 to staining data for Ran expression reveals a more accurate picture of the metastatic potential of human breast cancer cells.

[0132] In this study, when staining data for MMP2 expression is added to that for Ran expression, the RR for patient death is increased from the original 14.9 to 82.1 fold.

[0133] It will be seen that Example 1 provides an immunohistochemical assay based on Ran and MMP2 which is better correlated with patient survival as compared to immunohistochemical assay based on Ran alone (compare NPR of 97.5% with 93.3%). The improvement in NPR strongly suggests a corresponding improvement in assay of Ran and MMP2 levels within a tumour cell and blood plasma as compared to assay of Ran alone.

[0134] Table 6 above summarises the present inventors knowledge of the sensitivity, specificity, the negative percent response (NPR) and the positive percent response (PPR) for Ran expression and MMP2 expression in IHC assay as compared to Ran alone in selected human breast cancer sub-types (ER +ve/HER −ve (ER +/HER−) and triple negative (triple −VE)) as well as in unselected (all) human breast cancers.

EXAMPLE 2

[0135] A retrospective cohort study was undertaken of Ran levels in the blood plasma of 238 unselected human breast cancer patients (9236 cohort). The study utilised an Enzyme-Linked Immunosorbent Assay (ELISA) assay to determine the levels of Ran in blood plasma samples taken when the patients were first diagnosed with breast cancer.

[0136] Table 7 shows the mean Ran expression in patients that developed metastasis as compared to patients that did not not develop metastasis.

TABLE-US-00007 TABLE 7 Mean Ran expression with/without metastasis Patients Number Mean Ran expression Patients that did not go on to 177 1.33 develop metastasis Patients that went on to 61 2.08 develop metastasis

[0137] A statistical analysis was made on the determined levels of Ran against patient survival to determine a cut-off level for Ran which in combination with assay for MMP2 is considered likely to give a clinically useful test with NPR of about 98%.

[0138] FIG. 2 plots the variation in Ran expression in patients that went on to develop metastasis as compared to patients that did not develop metastasis in this cohort.

[0139] The blood plasma Ran levels were analysed at a series of cut-off levels at which levels of Ran in the blood plasma above a percentage integer were considered positive (+ve) viz. to indicate that a patient would go on to develop metastasis.

[0140] Table 8 shows a sensitivity analysis for Ran expression at different half-integer cut-off levels between 0.5 and 2.0.

[0141] As may be seen, the number of patients who are Ran +ve when the cut-off level is 0.5 is very high (at 216) having regard to the number of true positives (61, shown in Table 10). Furthermore, the sensitivity and NPR is high and the specificity acceptable but the number of patients who are Ran −ve is very low (at 30% of total true negative).

TABLE-US-00008 TABLE 8 Sensitivity analysis at different Ran expression cut-offs Ran expression Split % % cut-offs +ve/−ve sensitivity specificity PPV NPV 0.5 216/22 96.7 11.3 27.3 90.9 1 145/93 80.3 45.8 33.8 87.1 1.5  95/143 55.7 65.5 35.8 81.1 2  63/175 37.7 77.4 36.5 78.3

[0142] Although the cut-off level of 0.5 appears to offer a relatively safe test in that of the 22 patients who would not have undertaken chemotherapy, 90% (20) would not have developed metastasis, it is not that useful a test because the remaining 39 who would not have developed metastasis would have undertaken chemotherapy.

[0143] In choosing the optimal cut-off level, the sensitivity (% true positive) data was most important since it gave a measure of how many patients that went on to develop metastasis had a +ve RAN score. If the sensitivity is lower than about 95%, a larger number of patients that were scored as Ran −ve would not have undertaken chemotherapy and would have gone on to develop metastasis.

[0144] The specificity (% true negative) data was less important since it related to patients that did not go on to develop metastasis. If the specificity was low, say 60%, a large number of patients (40 out of 100) that would have scored Ran +ve but would not have gone on to develop metastasis. Although that is not necessarily a problem since the clinician would not alter treatment for patients who scored Ran +ve, a high selectivity is preferred.

[0145] In view of these considerations, it can be seen that setting the cut-off level at 2 leads to a poor test because that level is too high. Only 23 of the 63 patients who scored Ran +ve were true positives, the test having low sensitivity (37.7%) simply because a large number of true positives had values below 2 which meant that they were classed as negative.

TABLE-US-00009 TABLE 9 The 2 × 2 contingency table when Ran expression cut-off is equal to 1 Patients that did not go on to Patients that went on to develop metastasis develop metastasis Ran − ve 81 12 Ran + ve 96 49 Specificity = Sensitivity = 81/146 49/61

[0146] It appears that a cut-off level of 1 is the best this data set notwithstanding that it does not have NPR and sensitivity which are sufficiently high for the assay of Ran levels to offer a credible predictive test for all types of breast cancer.

[0147] Table 9 shows a 2×2 contingency table for Ran expression in this set of patients when the RAN expression cut-off is equal to 1.

[0148] FIG. 3 shows a plot of distance metastasis free survival (DMFS) of Ran positive patients and Ran negative patients against time. As may be seen, the proportion of Ran negative patients surviving over 5 years is higher than the proportion of Ran positive patients. However, the proportion of Ran negative patients surviving over 5 years appears to drop towards 50% over 7½ years and to drop below 50% towards 10 years.

[0149] Example 1 shows that the addition of a second biomarker (MMP2) to the RanDx IHC test on tumour samples significantly improves both NPR and % sensitivity and to a level that is equivalent or better than Oncotype and significantly that this is for all breast cancer sub-types.

[0150] Accordingly, it is expected that a blood plasma assay for both Ran expression and MMP2 expression will afford clinicians a useful and accurate predictive test of the probability of a patient developing metastasis.

[0151] As may be expected from the standard genomic test Oncotype, if the probability is low the clinician will not give the patient chemotherapy and if the probability is high the clinician will advise that the patient receives chemotherapy.

[0152] Therefore, the blood test may of itself remove the burden of chemotherapy from those patients that do not need it, saving the health service considerable expenditure and providing the patient a better quality of life.

[0153] It may also allow the clinician to monitor patients post-surgery or during chemotherapy treatment. Changes over time in the level of Ran and/or MMP2 in the patient's blood may be indicative of a change in their risk of developing metastasis from dormant cancer cells. An increase in Ran or MMP2 blood level may indicate a change in the risk and the clinician may then prescribe a course of chemotherapy with the intention of reducing risk. Such a test is not presently available from conventional tests.

[0154] Note that references herein to Ran are references to Ran protein and that references to RAN are references to a RAN gene unless the context demands otherwise. References to MMP2 are references to a MMP2 protein or, where the context demands, MMP2 gene.

[0155] Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0156] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

[0157] The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.