WO2015061844A1 - Assay to stratify cancer patients - Google Patents

Abstract

The present invention discloses assays to stratify progressive B-cell chronic lymphocytic leukemia (CLL) from stable or stable- progressive forms. An antibody microarray is provided which is specific for 18 or more of following the 27 CD antigens differentially abundant in progressive CLL: CD11a, CD11b, CD11c, CD18, CD19, CD20, (two epitopes), CD21, CD22, CD23, CD24, CD25, CD38, CD40,CD43, CD45, CD45RA, CD52, CD69, CD81, CD84, CD98, CD102, CD148, CD180, CD196 and CD270.

Description

ASSAY TO STRATIFY CANCER PATIENTS

FILING DATA

[00013 This application is associated with, and claims priority from Australian Provisional Patent Application No. 2013904183, filed on 30 October 2013. entitled "Assay to stratify cancer patients", the entire contents of which, are incorporated herein by reference.

BACKGROUND

FIELD

[00023 The present specification teaches an assay for stratifying subjects with chronic lymphocytic leukemia and a device useful for same.

DESCRIPTION OF RELATED ART

[0003] Bibliographic details of the publications referred to by author in this specification are collected alphabetically at the end of the description.

[00043 Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that thi prior art form part of the common general knowledge in any country.

[00053 B-cell chronic lymphocytic leukaemia (CLL) is characterized by a dynamic balance between proliferating cells in lymphoid organs such as lymph nodes and spleen, and restin cells in peripheral blood resistant to apoptosis. Heterogeneity with variable clinical courses is a hallmark of CLL (Shanafelt et al (2004) Blood 70J:1201-12.1.0). Some patients live with stable disease for many years without intervention, while 20-30% follow an aggressive course, sometimes with rapid progression requiring treatment (Kay et aL (2002) Blood 100:1 110-11.11 ). Indicators of poor prognosis include advanced Rai (Rai et al (1975) Shod 46:219-234) or Binet (Bi.net et al (1977) Cancer 40:855-864) stage, mini utated immunoglobulin variable heavy-chain genes (IGVH) [Damle et al (1999) Blood 94:1840-1847; Hamblin et al (1999) Blood 94:1848-1854], B-ceil expression of the cytoplasmic tyrosine kinase zeta-associated protei 70 (ZAP-70) [Orchard et al. (2004) Lancet 363: 105-111 J, and genomic abnormalities, particularly deletions in the 17p and 1 Iq chromosomal regions (Dohner et al. (2000) ,V Engl J Med 343:1910-1916), However, IGVH mutation testing is not widely available and assays for ZAP-70 levels are inconsistent (Letestu et al, (2006) Cytometry Part B-CHmcal Cytometry 70S: 309-314).

[00061 CLL is characterized by a unique immunophenotype of CD5⁺. CD 19* CD23⁺, with CD20 (dim/weak), CD22 (dim/weak), CD79b (dim/weak) and FMC7 (negative), surface immunoglobulin (sig, weak), often IgM with or without IgD (Matutes et al (2010) Best Pracl Res Clin Haematol 23:3-20). B-cell expression of CD5 and CD23 is essentially diagnostic of CLL. Currently, flow cytometry is widely used to identify clonal cell populations and t determine- the levels of prognostic antigens such as CD38 and CD49d, with simultaneous analysis of up to 10 CD antigens from a single flow cytometry analysis tube. However, the relatively limited panel of CD antigens possible to be analyzed by a flow cytometric analysis (5-10 CD antigen) does not enable unsupervised hierarchical, clustering of samples with similar patterns of CD antigens .

[0007] The DotScan (Trademark) CD antibody mieroarray overcomes this limitation for surface antigen profiling, providin very extensive immunophenotypes of any type of human ceil (Belov et al (2001) Cancer Res 67:4483-4489). When a relatively purified clonal population is present, the surface profiles provide very extensive phenotypes that can be used as signatures for cancer subtypes (Belov et al (2006) Br J Haematol 735:184- 197). Using this methodology, a study by Wierda et al (2005) Blood /0ό:826 profiled 101 CLL samples from the peripheral blood of patients using DotScan (Trademark) CD antibody ieroarrays (Version 1, 82 CD antibodies) and correlated CD antigens expressed with IGVH genes mutational status and ZAP-70 levels. For the un-mutated IGVH genes group, increased binding on DotScan (Trademark) was noted for CD2, CD9, Cl la. CD 1.3, C 22, CD25, CD38, CD86 and CD95. For the ZAP-70* group, increased binding was noted for CD2, CD 11a, CD38, CD49d, CD79b, CD95, and Kappa light chain. However, there was not a distinct difference in surface profiles between good prognosis (mutated^' IGVH genes or ZAP-70^") and poor prognosis (un-mutated IGVH genes or ZAP-TO*). In a subsequent study, 143 CLL samples were profiled using DotScan [Trademark] (Versio 2, 122 CD antibodies). Three clusters were identified using hierarchical clustering, but these clusters did no correlate with the clinical subtypes, stable (S), slo -progressive (SP) or progressive (P) CLL, or other prognostic parameters. There is a need to be able to distinguish between P and S/SP form's to enable triaging and appropriate treatment: of patients at risk of death.

SUMMARY

[0008] The present specification enables an assay to stratify chronic lymphocytic leukemia (CLL) in a patient on the basis of whether it is a stable (S) or slow-progressive (SP) form of the disease, or whether it is a. progressive (P) form of the disease. Early knowledge of the form of CLL enables selection of appropriate therapeutic intervention based on the likely aggressiveness or otherwise of the disease. For example, P forms of CLL may require immediate chemotherapy whereas S/P forms of the disease may simply require regular observation.

[0009] The assay comprises contacting B-cells or a sample comprising B -cells with an array of immunoglobulins immobilized to a solid support,

[0010] The array comprises discrete sites or regions of inuiiunoglobulins immobilized on a planar solid support.

[0011] The immunoglobulins at each site are homogeneous with respect to binding specificity to a particular cluster of differentiation (CD) antigen on the surface of B -cells or a positive or negative control. Whilst the immunoglobulins are homogeneous with respect to the CD antigen, the immunoglobulins may comprise a mixture of epitope specificities on the CD antigen,

[00123 In use, the B-cells are contacted with the immunoglobulin array for a time and under conditions sufficient for B-cells to be captured based on the level of expression of particulai- CD antigens to which the immunoglobulins at each discrete site bind. The CD antigens are selected to discriminate and differentiate between P and S/SP forms of CLL, Hence, the assay discriminates -cells from CLL patients on the signature of expression of. CD antigens wherein the relative abundance of bindin of B-cells to the discrete immunoglobulin sites of a microarray determines if the CLL is of a P form or an. S/SP form. [0013] In a embodiment, the CD antigens comprise at least 18 CD antigens selected from CDl la, CDl lb, CDl lc, CD18, CD1 , CD20 (2 epitopes), CD2L CD22, CD23. CB24, CD25, CD38, CD40, CD43, CD45, CD45RA, CD52, CD69, CD8 CD84, CD98, CD 102, CD148, CD180, CD196 and CD270. The array may comprise immunoglobulins to any of 18, 19, 20, 21 , 22, 23, 24, 25, 26 or 27 of the above CD antigens. Other CD antigens may be added or substituted based on clinical evidence of a signature of a P or S/SP form of CLL.

[0014] In an embodiment, the number of specified CD antigens is selected to provide an accuracy of at least 70%, a sensitivity of at least 75% and a specificity of at least 60%,

[0015] The present specification further enables an assay device comprising a carrier stage adapted to receive the solid support comprising the array of immunoglobulins and a scanner comprising a light source for illuminating the array of immunoglobulins on the solid support, A drive means moves the carrier stage such that successive portions of the solid support are illuminated. A digital camer system is disposed so that it captures successive portions of light ray which emerge from the solid support at an angle offset relative to the light rays from the light source. The resulting images are then reconstructed into an image of the array of immunoglobulins and cells bound thereto,

[0016] Generally, the device detects fluorescence after staining with fluoreseently labeled antibodies.

[0017] Enabled herein is a method of treatment of a patient with CLL wherein the patient is stratified into having a P or S/SP form of CLL and then a therapeutic protocol selected. Further taught herein is a method for monitoring the progress of treatment of a P stratified .or S/SP stratified CLL patient based on screening for maintenance or changes in. the CD expression signature associated with the P and S/SP forms of CLL. BRIEF DESCRIPTION OF THE FIGURES

[0018] Some figures contain color representations or entities, Color photographs are available from the Patentee upon request or from an appropriate Patent Office. A fee may be imposed if obtained from a Patent Office.

[0019] Figure 1 is a representation of DotSean (Trademark) binding patterns for normal peripheral blood mononuclear cells compared with a sample of progressive (P) CLL. (A) Key indicating locations of antibodies i the microarray with numbers referring to CD antigens. Abbreviations are: , , immunoglobulin light chains; slg, surface immunoglobulin; AMIGA, adhesion molecule that interacts with CXADR Antigen 1; CXCR7, chemokme receptor type 7; CLEC4A, C -type lectin domain family 4 member A; TNFRSF6B, tumour necrosis factor receptor super-family member 6B; LGALS3, Galeetin-3; IL-23R, interleukin 23 receptor; LTBR, lymphotoxin beta receptor: Hsp9G, heat shock protein 90; HLA-G, human leukocyte antigen G; Mabthera, humanized anti- CD20; CD20(H299), CD20 from clone H299. Alignment dots around the microarray consist of a mixture of CD44 and CD29 antibodies. (B) Dot pattern for normal, peripheral blood mononuclear cells purified with Fieoli-Hipaque, (C) dot pattern for progressive (P) CLL cells purified as above then fractionated using CD 19 antibody-magnetic beads. Cell capture and optical scanning are described in the Examples.

[00203 Figure 2 is a representation of statistical analysis of DotSean (Trademark) profiles for samples of CLL. (A) Unsupervised hierarchical clusterin (Euclidean distance, mean centred data) of 126 CD antigens for 100 CD 19+ CLL samples. CD antibodies (56) whose intensities were <2, and 12 isotype control antibodies were eliminated for this analysis. S, stable; SP, slow-progressive; P, progressive; Unk, unknown. (B) Supervised clustering (Euclidean distance, mean centred data) for the 27 differentially abundant CD antigens identified between the 3 clinical groups (S, SP and P). Samples with unknown CLL subtype (n - 4) were eliminated before clustering. The tables under the 2 clusters indicate the proportions of samples of each subtype for all the classified CLL samples (n = 96), The levels of abundance are quantified from 1 (red) to 126 (blue) on an 8-bit pixel greyness scale from 1-256. The color bar above the heat map represents the subtype of CLL as determined by established pathology criteria (Table 1); the color key for subtypes is on the left; red is progressive (P) CLL. The color bars beneath th heat map are the results from fluorescence in situ hybridization (FISH) for the corresponding patient; not all CLL samples were subjected to FISH. Green, no abnonnalities; blue, 13q~; orange, l lq-; pink, 12+; light pink, 12+, l lq-; purple, 13q-, 17p-; light purple, 13q-_s l lq-; and red, 12+, 13q-,

DET AILED DESCRIPTION

[0021] Throughout this specification, unless the context require otherwise, the word "comprise" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or method step or group of elements or integers or method steps hut not the exclusion of any element or integer or method ste or group of elements or integer or method steps,

[0022] As used in the subject specification, the singular forms "a", "an" and "the" include plural aspects unless the context, dearly dictates otherwise. Thus, for example, reference to "a CD antigen" include a single CD antigen, as well as two or more CD antigens; reference to "an antibody" includes a single antibody, as well as two or more antibodies; reference to "the disclosure" includes a single and multiple aspects taught b the disclosure; and so forth. Aspects taught and enabled herein are encompassed by the term "invention". All such aspects are enabled within the width of the present invention.

[0023] Enabled herein is an assay and assay device useful in the stratification of chronic lymphocytic leukemia (CLL) int progressive (P) and stable (S)/slo -progressive (SP) [S/SP] forms. A. single assa device is provided which comprises a solid support with an array of immunoglobulin molecules immobilized to discrete sites or regions of the solid support wherein the immunoglobulins in each discrete region are homogeneou with respect to binding specificity to a single CD antigen on a B-cel.1. The immunoglobulins may bind to different epitopes on the one CD antigen. Nevertheless, B-cells will bind to the discrete region on the solid support, It is the relative abundance of B-eell binding which gives a signature- indicative of a P or S/SP form of CLL. The CD antigens are selected on the basis of a clinically significant correlation between CD expression and one or other of P or S/S forms of CLL. In an embodiment, the B-cells are derived from a biologieal sample selected from whole blood, urine, lymph fluid, purulent discharge and a fraction of an of the above.

[0024] In an embodiment, the array comprises immunoglobulin which bind to at least 18 CD antigens selected from the List comprising GDI la, CDllb, CDl le, CD .18, CD19, CD20 (2 epitopes), CD2L CD22, CD23, CD24, CD25, CD38, CD40, CD43, CD45, CD45RA; CD52, CD69, CD81 , CD84, CD98, CD 102» CD148, CD 180, CD196 and CD270.

[00253 Reference to "at least. 18" includes 18, 19, 20, 21, 22, 23, 24, 25, 26 and 27. More or less tha the 27 CD antigens listed above may be selected based on clinical data. Furthermore, othe CD antigens may substitute for the listed CD antigens.

[0026] The assay enabled herein measures the relative abundance of binding of B-celLs from the patient to be stratified to the immunoglobulins in the discrete regions of the solid support. In an embodiment, the single assay enables simultaneous binding of B-cells to the immunoglobulin discrete regions. It is the relative scale of the pattern whic distinguishes a P form of CLL from an S/SP form. The pattern is a differential abundance of at least 18 CD antigens which distinguishes between P and S/SP patients,

[0027] Accordingly, taught herein is an assay to stratify a patient wit CLL int a P form or an S/SP form of CLL, the method comprising contacting B -cells from the patient with an arra of immunoglobulins immobilized to discrete regions on a solid support wherein the immunoglobulins in each discrete region are homogeneous with respect to their specificity to a CD antigen on a B-cell or a control wherein the array of immunoglobulins is specific for at least 18 CD antigens selected to enable a differential abundance of binding to the CD antigens which distinguishes between a P tbrm and an. S/SP form of CLL wherein a P patient exhibits greater binding to the at least 18 CD antigens compared to an S/SP patient. In an embodiment, the immunoglobulins are specific for at least 18 of CDl la, CDl lb, CDllc, CD 18, CD1.9, CD20 (2 epitopes), CD21, CD22, CD23, CD24, CD25, CD38, CD40, CD43, CD45, CD45RA, CD52, CD.69, CD 1, CD84, CD98, CD102, CD148, CD 180, CD 196 and CD270, However, one or more of these CD antigens may be substituted for by other CD antigens where clinical data support a correlation between expression o non-expression and a P form or S/SP form of CLL. [0028] Enabled herein i an assay to stratify a patient with CLL into a P form or an S/SP form of CLL, the method comprising contacting B -cells from the patient wi h an array of immunoglobulins immobilized to discrete regions on a solid support wherein the immunoglobulins in each discrete region are homogeneous with respect to thei specificity to a CD antigen on a B-ce!! or a control wherein the array of immunoglobulins is specific for at least 18 CD antigens selected from the list comprising CD 1.1 a, CD l ib, CDl lc, CD18, CD19, CD20 (2 epitopes), CD2L CD22, CD23, CD24_? CD25, CD38, CD40, CD43, CD45, CD45RA, CD52, CD69, CD8L CD84, CD98, CD102, CD148, CD180, CD196 and CD270 wherein the differential abundance of binding to the CD antigens distinguishes between a P form and an S/SP form of CLL wherein a P patient exhibits greater binding to the at least 18 CD of the antigens compared, to an S/SP patient,

[0029] in an embodiment, at least 22 CD antigens are chosen,

[0030] Hence, accordingly, taught herein is an assay to stratify a patient with CLL into a P form or an S/SP form of CLL, the method comprising contacting B-cells from the patient with an array of immunoglobulins immobilized to discrete regions on a solid support wherein the immunoglobulins in each discrete region are homogeneous with respect to their specificity to a CD antigen on a B-cell or a control wherein the array of immunoglobulins is specific for at least 22. CD antigens selected from the list comprising CDUa, CDl lb, CDl lc, CD18, CD 19, CD20 (2 epitopes), CD21, CD22, CD23, CD24, CD25, CD38, CD4CL CD43, CD4.5, CD45RA, CD52, CD69, CD81, CD84, CD98, .CD 102, CD148, CD18G, CD 196 and CD270 wherein the differential abundance of binding to the CD antigens distinguishes between a P form and an S/SP form of CLL wherei a P patient exhibits greater binding to the at. least 22 CD antigens compared to an S/SP patient,

[0031] In an embodiment, at least 25 CD antigens are chosen.

[0032] Hence, enabled herein is an assay to stratify a patient with CLL into a. P form or an S/SP form of CLL, the method comprising contacting B -cells from the patient with an array of immunoglobulins immobilized to discrete regions on a solid support wherein the immunoglobulins in each discrete region are homogeneous with respect to thei specificity to CD antigen or a B-cell or a control surface antigen wherein the array of immunoglobulins is specific for at least 25 CD antigens selected from the list comprising CDl la, CD! l b, CD 11c, CD 18, CD1.9, CD20 (2 epitopes), CD21, CD22, CD23, CD24, CD25, CD38, CD40. CD43, CD45, CD45RA, CD52, CD69, CD81, CD84, CD98, CD102, CD148, CD 180, CD 196 and CD270 wherein the differential abundance of binding to the CD antigens distinguishes betwee a P form and an S/SP form of CLL wherein a P patient exhibits greater binding to the at least 25 CD antigens compared to an S/SP patient.

[0033] In an embodiment, at least 27 CD antigens are chosen,

[0034] Accordingly, taught herein is an assay to stratify a patient with CLL into a P form or an S/SP form of CLL, the method comprising contacting B-cells from the patient with an array of immunoglobulins immobilized to discrete regions on a solid support wherei the immunoglobulins in each discrete region are homogeneous with respect to their specificity to a CD antigen or a B-cell or a control wherein the array of immunoglobulins is specific for at least 27 CD antigens selected from the list comprising CDl la, CDl lb, CDl lc CD18, CD19, CD20 (2 epitopes), CD21, CD22, CD23, CD24, CD25, CD38, CD40, CD43, CD45, CD45RA, CD52, CD69, CD8i, CD84, CD9S, CD102, CD148, CD 180, CD 196 and CD270 wherein the differential abundanc of binding to the CD antigens distinguishes between a P form and an S/SP form of CLL wherein, a patient exhibits greater binding to the at least 27 CD antigens compared to an S/SP patient,

[00353 The signature of expression of CD antigens may be used to detect the P form or to select a non-P form or to select an S/S form or a non-S/S P form of CLL.

[0036] The numbe and type of CD antigens is selected based on the level of accuracy, sensitivity and specificity required. In an embodiment, accuracy, sensitivity and specificity is selected from at least 70%, 75% and 80%, respectively. This means at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 92, 93, 4, 95, 96, 7, 98, 9 or 100% accuracy, sensitivity and specificity. In an embodiment, if all 27 CD antigens are screened, the accuracy is at least 70%, sensitivity is at least 83% and specificity is at least 72%,.

[0037] The solid support is generally planar in nature and is selected from glass, cellulose, ceramic material, nitrocellulose, poly a.eryf amide, nylon, polystyrene, polystyrene derivatives, polyvinylidene difiuoride, methacrylate, methacrylate derivatives, pol vinylehloride and polypropylene. In an embodiment, the material formin the solid support is derivatized to receive or facilitate binding of the immunoglobulins.

[00383 Generally, the B-cells are CD19* B-cells.

[0039] In an embodiment, taught herein is an assay to stratify a patient with CLL into a P form or an S/SP form of CLL, the method comprising contacting CD_.19* B-cells from the patient with a array of immunoglobulins immobilized to discrete regions on a solid planar support comprising derivatized support selected from the group consisting of glass, cellulose, ceramic material, nitrocellulose, polyacrylamide, nylon, polystyrene, polystyrene derivatives, polyvinylidene difiuoride, methacrylate, methacrylate derivatives, polyvinyl chloride and polypropylene, wherein the immunoglobulins in each discrete region on the solid support are homogeneous with respect to their specificity to a CD antigen on a B-cell or a control wherein the array of immunoglobulins is specific for at least 18 CD antigens selected from those clinically show to distinguish between a P form and an S/SP form of CLL wherem a P patient exhibits greater binding to the at least 18 CD antigens compared to an S/SP patient.

[0040] Enabled herein is an assay t stratif a patient with CLL into a P form or an S/SP form of CLL, the method .comprising contacting CD1.9⁺ B-cells from the patient with an array of immunoglobulins immobilized to discrete region on a solid planar support comprising a derivatized support selected from the group consisting of glass, cellulose, ceramic material, nitrocellulose, polyacrylamide, nylon, polystyrene, polystyrene derivatives, polyvinylidene difiuoride, met haery late, methacrylate derivatives, polyvinylchloride and polypropylene, wherein the immunoglobulins in each discrete region on the solid support are homogeneous with respect to their specificity to a CD antigen on a B-cell or a control wherein the array of immunoglobulins is specific for at least 18 CD antigens selected from the list comprising CDl la, CDl lb, CD lie,. CD .1.8, CD19, CD20 (2 epitopes), CD21, CD22, CD23, CD24, CD25, CD38, CD4Q, CD43, CD45, CD45RA, CD52. CD69, CD81, CD84, CD98, CD102, CD148, CD180. CDl 6 and CD270 wherein the differential abundance of binding to the CD antigens distinguishes between a P and an S/SP fonn of CLL, wherein a P patient exhibits greater binding to the at least 1.8 CD of the antigens compared t an S/SP patient.

[00413 As indicated above, "at least 18" means 18, 1.9, 20, 21, 22, 23, 24, 25, 26 or 27. Any number of CD antigens may be substituted or added to the array of 18 to 27 discrete immunoglobulin regions t which a CD antigen binds, in an embodiment, the CD antigens comprise or are selected from the list of 27 CD antigens.

[0042] Another aspect, enabled herein is an assay device comprising a carrier stage adapted to receive a solid support having an array of immunoglobulins immobilized to discrete regions on the solid support wherein the immunoglobulins in each discrete region are homogeneous with respect to their specificity to a CD antigen on a B-cell or a control wherein the array of immunoglobulins is specific for at least 18 CD antigens selected from those which have an expression or non -expression- ignature which distinguish between a P form or S/SP form of CLL; a scanner comprisin a light source for illuminatin the carrier stage including the array of immunoglobulins on the solid support; drive mean for moving the carrier stage such that successive portions of the solid support are illuminated by the light source; and a digital optical camera system disposed such that, in use, it captures only the successive portions of light rays which emerge from, the solid support at an ffset angle relative to the light rays from the light source transmitted through and emerging from the solid support to generate a series of partial image arranged to be reconstructed into an image of the array of immobilized .immunoglobulins,

[00433 Generally, the device detects fluorescence after staining wit iluoieseent!y labeled antibodies. [0044] Another aspect enabled herein is an assay device comprising a carrier stage adapted to receive a solid support having an array of immunoglobulins immobilized, to discrete regions on the solid support wherein the immunoglobulins in each discrete region are homogeneous with respect to their specificity to a CD antigen on a B-cell or a control wherein the arra of immunoglobulins is specific for at least 18 CD antigens selected from the list comprising CDl la, GDI lb, CD l ie, CD 18, CD 1.9, CD20 (2 epitopes), CD21, CD22, CD23, CD24, CD25, CD38, CD4Q, CD43, CD45, CD45RA, CD52, CD69, CD81, CD84, CD98, CD 102, CD 148, CD 180, CD196 and CD270; a scanner comprising a light source for illuminating the carrier stage including the array of immunoglobulins on the solid support; drive means for moving the carrier stage such that successive portion of the solid support are illuminated by the light source; and a digital optical camera system disposed such that, in use, it captures only the successive portions of light rays which emerge from the solid support at an offset angle relative to the light rays from the light source transmitted through, and emerging from the solid support to generate a series of partial images arranged to be reconstructed into an image of the array of immobilized immunoglobulins.

[0045] In an embodiment, at least 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 CD antigens are selected. One or more other CD antigens may be substantially for any o the 27 or added to the 27 CD antigens.

[004ft] In an embodiment, the light source is a linear light source arranged to emit a substantially narrow beam, whereby successive portions of the solid support that are illuminated are band-like portions, and whereby the series of partial images are linear images. The digital optical camera system is disposed such that, in use, it receives substantially only ligh ray which are diffracted or otherwise deflected at the array of immunoglobulins on the solid support. In an embodiment, the digital optical camera system include discriminator means for preventing light rays which are not diffracted or otherwise deflected by the array from being captured by the camera system. This aspect includes at least one reflector positioned to direct diffracted or otherwise deflected light rays emerging from the solid support at the offset angle towards an imaging lens of the camera system. The digital optical camer system includes, in an embodiment, a line scan camera capable of sensing a linear image. In an embodiment, the digital optical camera system is disposed such that, in use, light rays emitted from fluorescent markers at the discrete immunoglobulin regions are captured. The digital optical camera system may also be arranged to operate in at least two modes, namely a diffraction or deflection mode, in which light rays diffracted or otherwise deflected at the array of immunoglobulins on the solid support are captured by the camera, and a fluorescent mode, i which light rays emitted from fluorescent markers on the array of immunoglobulins are captured. In the .deflection or diffraction mode, when the drive means moves the carrier stage in a first direction and is arranged to operate in the fluorescent mode when the drive means moves the carrier stage in. a second direction. Light rays in. both the visible and non-visible portions of the spectrum may be detected,

[0047] In use, B -cells from a C LL patient are contacted with the solid support resulting in a pattern of B-cells immobilized to the solid support at one or more of the discretely bound immunoglobulin regions .

[0048] A sampling compartment may also be provided in which, in use, the carrier stage is located, and an electrical component compartment, wherein the electrical component compartment, is fluid sealed from the sampling compartment, whereby, in use, fluid contamination of components inside the electrical component compartment from the solid support is inhibited, the carrier stage including a tray element disposed, in use, underneath the solid support for collecting fluid spilled from the solid support. The imaging device may also include an interface unit for interfacing to devices of a group including at least one of an external storage database, an external PC, and an external printer, The line scan capable camera is a line scan camer adapted to scan linear images having a width of one pixel.

[0049] Enabled herein is an assay to stratify a patient with CLL into a P form or an S/SP form of CLL, the method comprising contacting B -cells from the patient with an assay deviee comprising a carrier stage adapted to receive a solid planar support having an array of immunoglobulins immobilized to discrete regions on the solid support wherein the immunoglobulins in each discrete region are homogeneous with respect to their specificity to a CD antigen on a B-cell or a control wherein the array of immunoglobulins is specific for at least 18 CD antigens selected from the list comprising CDl la, CD l ib, CD 11c, CD1.8, CD19, CD20 (2 epitopes), CD2L C.D22, CD23, CD24, CD25, CD38, CD40, CD43, CD45, CD45RA, CD52, CD69, CD81, CD84, CD98, CD1.02, CD 148, CD180, CD 196 and CD270 a scanner comprising a light source for illuminating the carrier stage including the array of immunoglobulins on the solid support; drive means for moving the carrier stage such that successive portions of the solid support are illuminated by the light source; and a digital, optical camera system disposed such that in use, it captures only the successive portions of light rays which emerge from the solid support at an offset angle relative to the light rays from the light source transmitted through and emerging from the solid support to generate a series of partial images arranged to be reconstructed into an. image of the array of mimobilized immunoglobulins; wherein the differential abundance of binding to the CD antigens distinguish between P and an S/S.P form of CLL wherein a P patient exhibits greater binding to the at leas 18 CD antigen compared to an S/SP patient .

[0050] By reference to "at least I S" means 38, 19, 20, 21, 22, 23, 24, 25, 26 or 27. The solid support may be as listed above,

[0051] Reference to an "immunoglobulin'- includes a monoclonal or polyclonal antibody as well as an antigen-binding fragment thereof.

[0052] The immunoglobulin is capable of specifically or substantially specifically (that is with limited cross-reactivity) binding to an epitope on a selected CD antigen. Notwithstanding, immunoglobulins having multiple specificities for two or more epitopes on the one CD antigen are also contemplated herein. The immunoglobulin includes, without limitation: Fv fragments; single chain Fv (sePv) fragments; Fab' fragments; Ffab')2 fragments; humanized antibodies and antibody fragments; and multivalent versions of the foregoing. Multivalent binding reagents also may be used, as appropriate, including without limitation: monospecific or bispeeific antibodies which bind to two or more epitopes CD antigen; such as disulfide stabilized Fv fragments, scFv tandems [(scFv)? fragments], diabodies, tribodies or tetrabodies, which typically are eovalentfy linked or otherwise stabilized (ie. leucine zipper or helix stabilized) scFv fragments.

[00533 The antibodies may be generated specifically to a CD antigen or fragment thereof comprising an epitope or ma be engineered. In a embodiment, the antibody is of murine (i.e. mouse or rat origin), rabbit, goat, horse, avian, pig, camel or human origin. The terms "antibody" and "immunoglobulins" may be used interchangeably throughout the subject specification. Further taught herein is derivatization of the solid support and or the immunoglobulin to permit immobilization of the immunoglobulin to the solid phase. In an embodiment, an antibody is generated labeled with a reporter molecule such as a fluorophore which binds to a antigen common to all B -cells. Such an antibody is useful in detecting B-cell bound to the immunoglobulins in the array. Examples of fluorophores include Alexa dye, Cy dye, Quantum dot, I TIC and phyeoerythrin.

[0054] The immobilization is at discrete sites or regions in an array. In an embodiment, the array is a pattern having vertical and horizontal rows. In an embodiment,, the array is a pattern indicating a letter or grou of letters such as P, S/SP or CLL. The array may also be a position in the shape of a letter of the last name of a patient or other signifier of the patient. In this way, a multiplicity of plates can be screened and the resulting pattern correlated with a patient,

[0055] Another aspect .enabled herein is a kit for stratifying a patient as to a P or S/SP form of CLL, The kit, in an embodiment, comprises a device comprising a carrier stage adapted to receive a solid planar support having an array of immunoglobulins immobilized to discrete regions on the solid support wherein the immunoglobulins in each discrete region are homogeneous with respect to their specificity to a CD antigen on a B-cell or a control wherein the array of immunoglobulins is specific for at least 18 CD antigens selected from the list comprising CDl ia, CDl l b, CDl lc, CD18, CDI9, CD20 (2 epitopes), CD21, CD22, CD23, CD24, CD25, CD38, CD40, CD43, CD45- CD45RA. CD52, CD69, CD81 , CD84, CD98, CD102, CD 148, CD 180, CD 19.6 and CD270; a scanner comprising a light source for illuminating the carrier stage including the array of immunoglobulins on the solid support; drive means for moving the earner stage such that successive portions of the solid support are illuminated by the light source; and a digital optical camera system disposed such that, in use, it captures only the successive portions of light rays which emerge from the solid support at an offset angle .relative to the light rays from the ^'light source transmitted through and emerging from the solid support to generate a series of partial images arranged to be reconstructed into an image of the array of immobilized immunoglobulins. Generally, the device detects fluorescence after staining with iluorescently labeled antibodies. The kit may also contai compartments for receiving cells, blood samples, fluorescent! y labeled antibodies, excipients and the like.

[0056] Further enabled is a use of an assay device comprising a carrier stage adapted to receive a solid planar support having an array of immunogiobulins immobilized to discrete regions on the solid support wherein the immunogiobulins in eac discrete region are homogeneous with respect to their specificity to a CD antigen on a B-cell or a control wherein the array of immunoglobulins is specific for at least 1.8 CD antigens selected from the list comprising CDl la, CDl lb, CDLlc, CD 18, CD 19, CD20 (2 epitopes), CD2L CD22, CD23, CD24, CD25, CD38, CD40, CD43, CD45, CD45RA, CD52, CD69, CD81, CD84, CD98, CD1Q2, CD148. CD 180,_. C.D196 and CD270; a scanner comprising a. light source for illuminating the carrier stage including the array of immunoglobulins on the solid support drive means for moving the carrier stage such that successive portions of the solid support -are illuminated by the light source; and a digital optical camera ystem disposed such that, in. use, it. captures only the successive, portions of light rays which emerge from the solid support at an offset angle relative to the light rays from the light source transmitted through and emerging from the solid support to generate a series of partial images arranged to be .reconstructed into an image of the array of immobilized immunogiobulins, in the manufacture of a diagnostic assa to stratify a patient with CLL wit respect to having a P form or a S/SP form of CLL.

[0057] The subject specification further leaches a method of treating a patient wit CLL. the method comprising stratifying the patient with CLL into a P form or an S/SP form of CLL, the method comprising contacting B -cells from the patient with an array of immunoglobulins immobilized to discrete regions cm a solid support wherein the immunoglobulins in each discrete region are homogeneous with respect to thei specificity to a CD antigen on a B~ceU or a control wherein the array of immunoglobulins is specific for at least 1.8 CD antigens selected to enable a differential abundance of binding to the CD antigens which distinguishes betwee a P form and an S/SP form, of CLL wherein a P patient exhibits greater bindin to the at least 18 CD antigens compared to an S/SP patient. In an embodiment, the immunoglobulins bind to at least 18 of CD! la, CDl lb, CDl lc, CD18, CD19, CD20 (2 epitopes), CD21, CD22, CD23, CD24, CD25, CD38, CD40, C 43, CD45, CD45 A, CD52, CD69, CD81, CD84, CD98, CD 102, CD 148, CD 180, CD196 and CD270. However, one or more of these CD antigens may be substituted by other CD antigens where clinical data support a correlation between expression or non-expression and a P form or S/SP form of CLL, and the selecting a therapeutic protocol based on the P or S/SP stratification,

[0058] The subject specification further teaches a method of treating a patient with CLL, the method comprising stratifying the patient with CLL into a P form or an S/SP form of CLL, the method comprismg contacting B -cells from the patient with an array of immunoglobulins immobilized to discrete regions on a solid support wherein the immunoglobulins i each discrete region are homogeneous with respect to their specificity to a CD antigen on a B-cel! or a control wherein the array of immunoglobulins is specific for at least 18 CD antigens selected from the list comprising CDlla, CDl lb, CD Li e, GDI 8, CD19, CD20 (2 epitopes), CD21, CD22, CD23, CD24, CD25, C.D38, CD40, CD43, CD45, CD45RA, CD52, CD69, CD81. CDS4, CD98, CD102, CD148. CD 180, CD 196 and CD270 wherein the differential abundance of binding to the CD antigens distinguishes betwee a P form and an S/SP form of CLL wherein a P patient exhibits greater binding to the at least 18 CD antigens compared to a S/SP patient, and then selectin a therapeutic protocol based on the P or S/SP stratification, EXAMPI.ES

[0059] Aspects taught herein are further described by the following non-limiting examples.

Materials and Methods

Samples

[0060] A total of 100 samples was obtained from the Australasian Leukaemia and Lymphoma Group tissue bank (ALLG), Royal North Shore Hospital (St Leonards, Australia), and the University o Leicester (Leicester, UK), The diagnosis of CLL was made by standard clinical and surface marker criteria (CLL Guide-lines, Hallek et al (2008) Bloo 111 :5446-5456). The .disease status of each patient was assessed and defined as stable (S), slow-progressive (SP) or progressive (P) CLL using the cri teria in Table 1. isolation of B -lymphocytes from blood samples

[0061] Mononuclear cells were obtained from heparini.zed peripheral blood by FieoH- Hypaque gradient centrifugafi n. Cells were frozen at -80°C in fetal calf serum (PCS) with 10% w/v dimethyl sulfoxide (DMSO). Samples were thawed and B -lymphocytes, isolated using CD19 antibody-magnetic microbeads and MACS LS columns (Miltenyi Biotec Inc., Bergiseh Gladhach, Germany) according to the manufacturer's protocol, then profiled on DotScan (Trademark) mieroarrays. The enriched B-lymphoeyte samples were analysed by flow cytometry using CDS-fluoroscein isothiocyaiiate (FfTC) and CD20-phycoerythrin (PE) antibodies and the purity was >93¾. Only live cells bind to DotScan (Trademark) mieroarrays. The presence of microbeads on the purified cells did not interfere with their capture on mieroarrays (Kaufman et ui. (2010) Journal of Immunological Methods 358:23- 34).

Fluorescence in-situ hybridization { FISH)

[00623 Interphase FISH analysis was performed on some samples using a standard methodology and probe set. Briefly, commercially available probes (Vysis, Abbott Diagnostics, UK): 1 lq23(LSI ATM and CEPl l ), 13ql4(LSI D13S319 and LSI 13q34), trisomy 12 (CEP 12) and 17pl3(LSI P53 and CEP17) were hybridized according to the manuf cturers protocols in combination with a control probe, A minimum of 200 interphase nuclei was examined for each sample using a Zeiss Axioscop microscope with a lOOW mercury vapour UV lamp. Images were captured with a. Photometries CCD camera (Photometries, Tusco , AX. USA] and SmarfCapture (Trademark software (Digital Scienti ic, Cambridge, UK).

DotScan (Trademark) CD antibody mhraarrays

[0063] Microarrays were made by Medsaic Pty. Ltd. (Sydney, NSW, Australia), using FAST slides with a thin film o nitrocellulose (Grace Bio-Labs, Bend, OR, USA) as described by Belov et at (2003) Proteomics 3:2147-2154. The main section of the DotScan (Trademark.) CLL mieroarray (version 3) consisted of 182 antibodies in duplicate, selected from papers describing the prognostic significance of CD antigens on CLL, and B-cell CD antigens designated at the HLDA workshop (Barcelona, Spain, 2010). A second section of the mieroarray contained dilutions of isotype control antibodies. All antibody solutions contained 0.1 % w/v bovine serum albumin (BSA) and 0.1 % w/v sodium asride.

Surface profiling of CLL samples

[0064] The number of viable B -lymphocytes in each frozen sample was determined b trypan -blue exclusion. Control experiments have shown that frozen viable cells, when thawed, gave a similar immunophenotype to a fresh sample (Belov et at. (2003) supra). Viable cells (3 x 10⁶) in 300 L of PBS were applied to DotScan (Trademark) microarrays and incubated for 12 rain at room temperature. Unbound cells were gently washed off using PBS, and the bound cells were fixed with 3.7% w/v formaldehyde in PBS for 20-30 min. After removing residual fixative by washing in PBS, a digital image of the optical dot pattern of captured cells was obtained with a DotReader [Trademark] (Medsaic Ply. Ltd., Sydney, NSW, Australia).

Imaging of binding patterns and statistical an ysis

[0065] Dot patterns of captured cells on microarrays were scanned using an optical DotReader {Trademark] (Medsalc Ply. Ltd.) and densities of the dots were quantified on an 8-bit greyness scale (1-256), Any non-specific binding to the equivalent isotype control antibodies was subtracted from the binding values for CD antibodies. Several normalization options for data processed with the DotScan (Trademark) software (Medsaic Ply. Ltd.) were considered, including log-transformed normalisation, quantile. mean centred and cyclic loess. The quanti!e and cyclic loess normalization option are not well suited for antibod microarrays (Hamelinck et al. (2005) Mai Cell Proteomics 4:773-784; Sill et al. (2010) EMC Bioinformatics 1 : 56) therefore, mean-centred normalization was preferred, ensuring that data were normally distributed across all assays. Analysi of variance was run separately for each CD antibody. Antibodies with ANOVA / -valiie-s <0.05 after multiple testing correction, using the Benjamini and Hochberg false discovery rate method (Hamelinck et al. (2005) supra; Sill et al. (2010) supra), were retained as differentially expressed.

EXAMPLE I

CD signature

[0066] Figure 1C shows a typical DotScan (Trademark) dot pattern for a progressive CLL sample, and Figure IB shows a pattern for normal peripheral blood mononuclear cells. Erythrocytes were removed from bot samples by cefttrifugation on Fieoll-Hipaque, and the CLL sample was further fractionated using CD19 antibody-magnetic beads as described in the Materials and Methods. The CLL sample shows the characteristic expression of CD 19, CD20 and CD23 with CDS. CLL samples (100), obtained frozen in PCS with .1.0% w/v DMSO, were classified as stable (17), slow-progressive (23), progressive (56) and unknown (4) using the standard pathology criteria listed in Table 1. Surface profiles from the 100 CLL samples were obtained using a DotScan (Trademark) CLL microarray (Version 3) containing 182 CD antibodies in duplicate, and 12 isotype control antibodies.

EXAMPLE 2

Generation of heat map

[0067] After normalization, CD antibodies whose intensities were <2 (56) were eliminated along with the isotype control antibodies. Surface profiles were analyzed by unsupervised hierarchical clustering through a complete-linkage method, with Euclidean distances as measures of similar/dissimilar beha viou (Eisen et ai. (1998) Proceedings of the. National Academy of Sciences of the United Sums of America 95:14863-14868). This analysis yielded the heat map shown in Figure 2A; each colum of the map represents a patient sample; each horizontal row is a CD antigen of the microarray. Clusters 1 and 2 on the heat map are separated by a vertical red line and correlate with stable plus slow -progressive (S/SP, left side) CLL and progressive (P, right side) CLL, respectively. The tables below the heat map show the percentages of patients for each CLL subtype in the two clusters. The 45 patients in cluster 1 were classified as follows, one was of unknown CLL subtype, 8 were stable (8/17, 47.1%), 9 were slow-progressive (9/23, 39,1%), and 27 were progressive (27/56, 48.2%). The 55 patients in cluster 2 were classified as follows; 3 were of unknown CLL subtype, 9 were stable CLL (9/17, 52.9%), 14 were slo -progressive (14/23, 60.9%), and 29 were progressive (29/56, 51.8%). Each subtype of CLL totals 100% when clusters 1 and 2 are added together, for a total of 100 CLL samples, The red horizontal bar on the top right of Figure 2A indicates that cluster 2 was predominately progressive CLL.

EXAMPLE 3

Analysis of CD signature

[0068] To confirm the correlation -shown in Example 2, a 'partial least squares discriminant analysis' (PLS-DA) using the 'classification and regression training' (caret) R package, was undertaken. 'Leave -one-out cross-validation' was used to check the perfonnance of the discriminant analysis (Table 2A), This involves using a single observation from the original sample as the validation data, and the remaining observations as the training data, and is an analysis for estimating the performance of a predictive model. There were 56 CD antibodies that showed binding intensities of <2, leaving 126 antibodies with significant ceil capture. When these 126 antibodies were included (12 isotype control antibodies excluded), the accuracy of clustering samples according to clinical status was 71 >9% with a 95% confidence interval of 61.8-80.6%, The sensitivity (i.e., proportion of actual positives correctly identified) was 73.2%, and the specificity (i.e., proportion, of negatives correctly identified) was 70.0%.

[0069] A supervised analysis was performed that takes into account specific, pre-defined factors such as clinical status (Rigner and Peterson (2003) Biotechniques'.W). The 96 CLL cases with known CLL subtype were split into 3 groups, 56 progressive (P), 23 slow- progressive (SP) and 17 stable (S), A total of 37 CD antigens were identified as differentially abundant between the subtypes of CLL by supervised analysis. Of these 37 CD antigens, 1.0 had binding intensities <2 (after background subtraction and normalization), leaving 27 differentially abundant antigens between the 3 CLL subtypes. Supervised hierarchical clustering for the 96 classified CLL samples using the levels of these 27 CD antigens (Figure 2B) show that progressive (P CLL can be separated from S/SP with relatively high accuracy. Cluster 2 correlated with the progressive (P) CLL subtype (see the red horizontal bar on top right of Figure 2B). Cluster 2 contained 55 samples, only 6 stable (S, 6/17, 35.3%) and 4 slow -progressive (SP, 4/23, 17.4%;) were mixed with 45 progressive (P, 45/56, 80.4%), providing a high level of accuracy fo identification of the progressive CLL subtype, In cluster 1, there were 11 P (11/56, 19.6%), 11 S (11/17, 64.7%) and 19 SP (19/23, 82.6%). Mean levels for these CD antigens between the 3 CLL subtypes are shown in Table 3. These antigens were expressed at higher levels in progressive, compared with slow-progressive and stable CLL. Overall, the 2 clusters of Figure 2B discriminated well between SASP and P subtypes of CLL with 80.4% of progressive CLL found in cluster 2. When the surface profiling of CLL samples was restricted to just the 27 discriminatory antibodies, the accuracy of clustering samples according to CLL subtype was 79.2% with a 95% confidence interval of 69.7-86,8%. The sensitivity (i.e., proportion of actual positives correctly identified) was 83.9%;, and the specificit (i.e., proportion of negatives correctly identified) was 72.5%.

EXAMPLE 4

Significance of signature

[0070] There is a close correspondence between results for samples of B-CLL obtained as cell binding intensities from DolScan (Trademark) antibody microarrays, and mean fluorescence intensities MFI) from flow cytometry (see Table 1 of Belov et al. (2001) supra; Belov et al. (2006) supra; and Belov et al. (2003) supra). It is standard procedure in proteomics analyses to validate the differentially abundant proteins that have been identified and quantified, by an independent (orthogonal) procedure. The Dot-Scan (Trademark) microarrays provide high throughput, analyses giving patterns, profiles or signatures where 182 antibodies are screened concuiTently, rather than in individual assays. The surface profiles obtained are far more robust or stable than measurement of a single CD antigen.

EXAMPLE 5

Panel of CD antigens

[0071] Extensive surface profiles of CD antigens o cancer cells should reflect their genetic programs and correlate with clinical subtypes. CLL cells respond to micro- environmental signals through their surface molecules to confer growth and survival, advantages, providing a rationale for the prognostic value of CD antigens. There is a close correspondence between surface profiles of CD antigens obtained using a DotScan (Trademark) microarray, and collective flow cytometric data obtained from multiple analyses of the same samples (Belov ei al (2001) supra; Belov el til, (2006) supra and Belov el al (2003) supra). In accordance with these Examples, an expanded 1.82-antibody microarray (DotScan (Trademark) CLL microarray Version 3) has been used to obtain surface profiles of CD19-purified B -cells, eliminating contributions from other types of leukocytes. Twenty-seven CD antigens were identified that are differentially abundant between relatively stable CLL (S/SP) and disease that is progressive (P subtype). Using cell capture data for just these 27 discriminatory CD antigens, leave-one-out cross- validated discriminant analysis (implemented using partial least squares discriminant analysis (P^'LS-DA) from the caret R package) distinguishe S SP CLL from progressive CLL with an accuracy of 79.2%, a sensitivity of 83.9%, and specificity of 72.5%. This level of accurac using the DotScan (Trademark) CLL microarray (Version 3) should enable stratification of patients with stable or slow-progressive CLL (S/SP), from those with progressi ve disease (P). The horizontal color bar above the heat map of Figure 2B represents the subtypes of CLL as determined; the red bar to the right in cluster 2 indicates the predominance of progressive (P) CLL. Of all the progressive (P) samples, 80.4% are in cluster 2 (P),

[0072] This panel of 27 antibodies provides the basis for a simple prognostic test to triage patients with progressive CLL with higher accuracy than obtained with combinations of other markers such as un-mutated IGVH genes, high levels of ZAP-70 expression, and genomic abnormalities determined by FISH, This DotScan (Trademark) technology using an extensive surface phenotype may provide discriminative signal that is comparable to more complex and costly methods. In recent publication, Rossi et al. (2013) Blood 121:4902-4905, proposed a potential prognostic stratification into 4 groups using FISH and molecular data. High risk patients with TP53 and/or B1RC3 abnormalities had a 10-year survival of 29%, intermediate-risk with NOTCH! and/or SF3B I mutations and/or l lq- had a 10-year survival of 37%, low-risk with 12+ or normal -genetics .had a 10-year survival, of 57%, and very low -risk, with dell q.14 only had a 10-year survival of 69,3% that was not statistically different, from a matched general population. This stratification requires both FISH data and also sequencing for molecular genomic lesions by Next Generation Sequencing or similar technology and is therefore relatively costly compared to the phenofypic discrimination provided by DotScan (Trademark).

[0073] The proposed functions of some of these 27 antigens associated with progressive CLL are listed in Tabic 4 with possible roles in CLL pathogenesis. The identification of specific proteins and antigens on the cell surface and their known roles may allow for more direct targeting of proteins that are of direct biological significance in the pathophysiology, homing, and micro-environmental interaction important in CLL. In addition to confirming the value of known prognostic markers, a number of novel differentially expressed antigens were identified: CD8L CD98, CD148, CD 180, CD 196 and CD270 (Table 4). The color bars- beneath the heat map of Figure 2B are the results from FISH for the corresponding patient; only 27 of the 96 CLL samples were subjected to FISH. Several of these samples were .13q- singly or in combination with other chromosomal abnormalities as shown underneath Figure 2B: (blue, 13q- (3); purple, 13q-, 17p- (2); red, 12+, 13q- (1)).

[0074] CD49d and CD79b were not differentially abundant in this study, although they have been reported as prognostic markers for CLL correlating with un-mutated IGVH genes and elevated ZAP-70 (Buggins et al, (2011) British Journal of Haemamlogy 154 216-222; Gattei et al. (2008) Blood /7:865-873; Nuckel. et at, (2009) Clin Immunol /5i:472-480; Quijano et at. (2008) Cytometry B Clin Cytom 74: 139-149; Zucchetto et al. .(2-006) J Transl Med 4:11). The clusters of patients identified in these Examples are consistent with the combined (S plus SP) and (P) subtypes of CLL, displayed mixed IGVH and ZAP-70 status. [00753 A DotScan (Trademark) microarray containing discriminating CD antibodie identified herein is proposed to be used for triaging patients with progressive CLL who require and benefit from early treatment intervention.

Table 1

Criteria for classification of CLL

Course of CLL Criteria

Stable iS ) * Doubling of peripheral l mphocyte count >5 years

* Asymptomatic with nil or minimal clinical signs

Slow-progressive * Doubling of peripheral lymphocyte count >12 months and <5 (SP) years

* Usually Binet stage A o B: may have mild splenomegaly, hepatomegal , ly mphadenopamy

* Good response to treatment with time to second treatment of >3 years

Progressive (P) • Continuous progression, doubling of peripheral lymphocyte

count <1.2 months, absolute lymphocyte count >50 xlO^/L

• Commonly develop Binet stage C disease with marrow failure Massive splenomegaly or adenopathy (>10 cm in longest diameter)

• Refractory to treatment or time to second treatment of 3 year's [00763 Partial least squares discriminant analysis (PLS-DA) with leave-one-out cross- validation to cheek the performance of the classification of prognostic subsets of CLL. Data shown as the heat map in Figure 2A were analysed with omission of samples of unknown prognosis (n— 4), leaving classified CD 1.9+ CLL samples (n = 96). In leave-one- out cross validation each sample is predicted using a classifier trained on the data from all the remaining samples, then repeating the process; thus the data used to (rain the classifier does not include the sample used to test it Each sample in turn is predicted either correctly or incorrectly. A confidence interval (CI) is an interval estimation of a population parameter and is used to indicate the reliability of an estimate. That is. 95 CI indicates the range of values for which there is 95% confidence that the true value falls within the range. The results are shown in Table 2. (A) Leave-one-out cross-validation analysis using 126 CD antibodies. (B) Leave-one-out cross-validation analysis on the 27 differentially abundant (discriminatory) CD antibodies.

Table 2

A,

Stable/SP (actual) Progressive (actual)

Stable/SP (predicted) 28 15

Progressive (predicted) 12 41

Accurac (i.e., closeness of computations or estimates to the true values for the 96 samples that the statistics were intended to measure); (28 + 41 )x 100/96 - 71.9%.

95% confidence interval (CI): (0.618, 0.806).

Sensitivity (i.e., proportion of actual positives correctly identified): 41x1.00/(41 + 15) -

73.2%.

Specificity (i.e., proportion of actual negatives correctly identified): 28x 100/(28 + 12) -

70.0%.

B.

Stable/SP (actual) Progressive (actual)

Stable SP (predicted) 29 9

Progressive (predicted) 11 47

Accuracy: (29 + 47)xlOO/96 = 79.2%.

95% confidence interval (CI): (0.697, 0.868).

Sensitivity: 47x100/(47 + 9) = 83.9%.

Specificity; 29x100/(29 + 11) = 72.5%.

[0077] Differentially abundant CD antigens (27) between the three clinical subtypes of CLL; stable (S), slow-progressive (SP) and progressive (P). The results are shown in Table 3, The mean values represent levels of cells binding on DotSean (Trademark) antibody dots after background subtraction and normalisation. Mean P denotes the mean value of cell binding intensity on an antibody dot in the Progressive group. CD antigens are arranged in order of increasing mean intensity for the progressive sub-group The "IM" of CD20 IM distinguishes this Imnmiiotech antibody (clone B9E9(HRC20)) from the Beckman Coulter antibody 20 (clone H299(B 1)).

Table 3

CD antigen Mean S Mean SP Mean P p-¥alue

102 1.45 3.18 5.01 4.65E-02 lib 2.92 3.21 20.4 4.42E-02

52 1.23 0.95 22.3 6.47E-03

270 7,00 3.95 24.1 8.56E-04

81 10.0 7.05 32.9 4..65E-02

11a 5.70 7.40 34.6 3.63E-Q4

98 11.2 6.43 37.0 L.84E-02

19 4.04 11 .3 37.6 1.84E-Q2

180 21.4 7.96 38.2 3.86E-03

20 6.02 2.87 38.4 2.00E-06

38 8.40 13.8 41.5 3.78E-02

18 13.9 15.9 43.5 4.62E-02

24 2.09 3,01 47.6 1.95E-02

25 9.72 8.64 54.2 2.27E-04

11c 32.1 30.9 62.3 4..42E-02

69 19.2 1.8.5 62.5 1.12E-02

84 14.9 6.83 62.7 1.16E-03

22 28.5 17.7 66.3 3.63E-04

45 25.6 18.3 90.0 8.97E-05

19 32.8 33.4 91.5 L47E-03

20 IM 47.4 26.9 92.9 1.12E-02

21 39.6 39.2 101.3 3.86E-G3

40 49.3 37.8 103.5 3.1 1 E-03

43 2.9.6 26.9 112.6 1.45E-05

45RA 54.3 41 .6 114.0 1.37E-03

23 70.3 58.9 1 .4.2 4.42E-02

148 60.6 58.3 121.4 L95E-Q2 Table 4

Functions of CD antigens that are differentially abundant between 5, SP and P subtypes of CLL

[00783 Those skilled in the art will appreciate that the disclosure described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure contemplates all such vaiiations and modifications. The disclosure also enables all of the steps, features, compositions and compounds referred to or indicated i this specification, individually or collectively, and any and all combinations of any two or more of the steps or features or compositions or compounds.

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Claims

CLAIMS:

1. An assay to stratify a patient with B-cell chronic lymphocytic leukemia (CLL) into a progressive (P) form or a stahle/siow-progressive (S/SP) form of CLL, said method comprising contacting B-celis from the patient with an array of immunoglobulins immobilized to discrete regions on a solid support wherein the immunoglobulins in eac discrete region are homogeneous with respect to their specificity to a CD antigen on a B- cell or a. control wherein the array of immunoglobulins is specific for at least 18 CP antigens selected from the list consisting of CDl la, CD lib, CD.t le, CD18, CD 19, CD20 (2· epitopes), CD2L CD22, CD23, CD24, CD25, CD38, CD40, CD43, CD45, CD45RA, CD52, CD69, CD81, CD84, CD98, CD 102, CD 148, GDI 80, CD 196 and CD270 which enable a differential abundance of binding to the CD antigens which distinguishes between a P form and an S/SP form of CLL wherein a P patient exhibits greater binding to the at least 18 CD antigens compared, to an S/SP patient,

2. The assay of Claim 1 wherein the CD antigens screened are selected to give an accuracy, sensitivity and specificity of at least 70%, 75% and 80%, respectively,

3. The assay of Claim 1 wherein the B-celis are CD19⁺ B-celis,

4. The assay of Claim 1 wherein the solid support is selected from the listing consisting of glass, cellulose, ceramic material, nitrocellulose, polyacrylamide, nylon, polystyrene, polystyrene derivatives. polyv.inylMen.es diiluoride, methaerylate, methacrylate derivatives, polyvmyichloride and polypropylene or derivatized forms thereof.

5. The assay of Claim 1 wherein the solid support is selected from glass, cellulose and nitrocellulose and a cellulose or nitrocellulose film on glass or derivatized forms thereof,

6. The assay of Claim 1 wherein the discrete regions of immunoglobulins hind at least 19 out of the 27 CD antigens.

7. The assay of Claim 1 wherein the discrete regions of immunoglobulms bind at least

20 out of the 27 CD antigens.

8. The assay of Claim 1 wherein the discrete regions- of immunoglobulins bind at least

21 out of the 27 CD antigens.

9. The assay of Claim 1 wherein the discrete regions of immunoglobulins bind at least

22 out of the 27 CD antigens.

10. The assay of Claim 1 wherein the discrete regions of immunoglobulins bind at least

23 out of the 27 CD antigens.

11. The assay of Claim 1 wherein the discrete regions of immunoglobulins bind at least

24 out of the 27 CD antigens.

12. The assay of Claim 1 wherein the discrete regions of immunoglobulins bind at least

25 out of the 27 CD antigens.

13. The assay of Claim 1 wherein the discrete regions of immunoglobulins bind at least

26 out of the 27 CD antigens.

14. The assay of Claim 1 wherein the di screte region of immunoglobulins bind at least

27 out of the 27 CD antigens.

15. The assay of Claim 1 wherein the immunoglobulins are monoclonal antibodies or antigen-binding fragments thereof.

16. The assay of Claim 1 wherein the immunoglobulins are polyclonal antibodies or ant en-binding fragments thereof.

17. The assay of Claim 1 wherein the immunogiobulins are synthetic or chimeric antibodies or antigen-binding fragments thereof.

18. The assay of Claim 1 wherein th B -cells- are derived from a biological sample selected from whole blood, urine, lymph fluid, purulent discharge and a fraction of any of the above,

19. The assa of Claim i wherein the B-cells are derived from whole blood or a fraction thereof.

20. The assay of Claim 1 further comprising labeling bound cells with a detectable marker.

21. The assay of Claim 1 wherein the detectable marker is labeled immunoglobulin to a surface antigen common to all B -cells.

22. The assay of Cl im 20 or 21 wherein the label is a fluorophore.

23. The assay of Claim 21 wherein the fluoropbore is selected from an. Alexa dye, Cy dye. Quantum dot, FTTC and phycoerylhrin.

24. The assay -of Claim 1 wherein a portion of the solid support is illuminated and substantially only successive diffracted or otherwise defected portions of light rays which emerge from the solid support are captured generating a series of partial images arranged to the reconstructed into an image of th array of discrete sites.

25. The assay of Claim 24 whereby successive portions illuminated b a linear ligh source, and whereby the series of -partial images are captured as linear images.

26. The assay of Claim 25 comprising the step of capturin light rays emitted from the fluorescent markers on the solid support.

27. The assay of Claim 26 which includes capturing both light rays emitted in a diffraction or deflection mode and light rays emitted in a fluorescent mode from said fluorescent markers.

28. The assay of Claim 24 which includes processing the reconstructed image to arrive at a signature of relative abundance which is comparable with a library of signatures.

29. A kit for stratifying a patient to a P or S/SP form of CLL, said kit comprising a device a carrier stage for receiving solid planar support having an array of immunoglobulins immobilized to discrete region on the solid support wherein the immunoglobulins in each discrete region are homogeneous with respect to their specificity to a CD antigen on a B-cell or a control wherein the array of immunoglobulins is specific for at. least 18 CD antigens selected from the list comprisin CD 1.1a, CD lib, CD l ie, CD1.8, CD19, CD20 (2 epitopes), CD21, CD22, CD23, CD24, CD25, CD38, CD40, CD43, CD45, CD45RA, CD52, CD69. CD81, CD84, CD98, CD 102, CD148, CD 180, CD 19.6 and CD270; a scanner comprising a light source for illuminating the earner stage including the array of immunoglobulins on the solid support; drive means for moving the carrier stage such that successive portions of the solid support are illuminated by the light source; and a digital optical camera system disposed such that, in use, it captures only the successive portions of light rays which emerge from the solid support at an offset angle relative to the light rays from the light source hansmitted through and emerging from the solid support to generate a serie of partial images arranged to be .reconstructed into an image of the array of immobilized immunoglobulins ,

30. Use of an assay device comprising a carrier stage for receiving solid planar support having an array of immunoglobulin immobilized to discrete regions on the solid support wherein the immunoglobulins in each discrete region are homogeneous with respect to their specificity to a CD antigen on a B-cell or a. control wherei the array of immunoglobulins is specific for at least 18 CD antigens selected from., the list comprising CD1 la, CD lib, CDllc,. CD 18, CD 19, CD20 (2 epitopes), CD21 , CD22, CD23, CD24, C 25, CD38, CD40, CD43, CD45, CD45RA, CD52, CD69, CDS. I, CD84, CD98, CD102, CD1 8, CD18Q, CD1 6 and CD270; a scanner comprising a light source for illuminating the carrier stage including the array of immunoglobulins on the solid support; drive means for moving the carrier stage such that successive portions of the solid support are illuminated by the light source; and a digital optical camera system disposed suc that, in use, it captures .only the successive portions of light rays which emerge from the solid support at an offset angle relative to the fight rays from the light source transmitted throug and emerging from the solid support to generate a series of partial images arranged to be reconstructed into an image of the array of immobilized immunoglobulins in the manufacture of a diagnostic assay to stratify a patient with CLL with respect to having a P form or a S/SP form of CLL,

31 , A method of treating a patient with CLL, said method comprising stratifying the patient with CLL into a P form or an S/SP form of CLL, the method comprising contacting B-ceiis from the patient with an array of immunoglobulins immobilized to discrete region on a solid support wherein the immunoglobulins in each discrete region are homogeneous with respect to their specificity to a CD antigen on a B-cell or a control, wherein the array of immunoglobulins is specific for at least 18 CD antigens selected from the list comprising CD 1 1 a, CD l lb, CD lie, CD18, CD19, CD20 (2 epitopes), CD21 , CD22, CD23, CD24, CD25, CD38, CD40, CD43, CD45, CD45RA, CD52, CD69, CDS!, CD84, CD98, CD102, CD148, CD180, CD196 and CD270 wherein the differential abundance of binding to the CD antigens distinguishes between a P form and an S/SP form of CLL wherein the CLL cells of a P patient exhibit greater binding to the at least 18 CD antigens compared to an S/SP patient, and then selecting a therapeutic protocol based on the P or S/SP stratification.