Quanticision DB Diagnostics, Quantification of multiple biomarkers as absolute and continuous Inc P4-02-14 400 Park Offices Dr. RTP, NC 27709 www.quanticisiondiagnostics.com variables in 1049 FFPE breast cancer tissues using QDB method 1Fangrong Tang, 1Wenfeng Zhang, 1Jiahong Lv, 1Yunyun Zhang, 2Guohua Yu,3 Lei Ding & 1* Jiandi Zhang 1Yantai Quanticision Diagnostics, Inc, a division of Quanticision Diagnostics, Inc at RTP, NC, USA; 2 Yuhuangding Hospital, Affliated Hospital of Qingdao University, Yantai, China; 3 Xintai People’s Hospital, Xinta, China. * [email protected], 240-644-4229

Results Relationship between biomarkers as absolute and Abstract Introduction A: Developing QDB assay for Her2 C: Converting into dichotomous variables continuous variables (n=1049) using EP3 as example based on IHC scores. Immunohistochemistry (IHC) is plagued with the issues of subjectivity and inconsistency. The categorized re- 6 100 ER and Her2 ER vs PR ER vs Age 5 R² = 0.998 α EP3 sults of “+” vs “-” also provides limited information α EP3 3 3 3 4 about tissue biomarker levels in clinical diagnosis and n=1047 3 Area: 0.9998 ± 0.0002 prognosis. 50 95%CI: 0.9994~ 1 r=0.35, Pearson 2 ρ=0.22, Spearman Purpose: Immunohistochemistry (IHC) has inherent limitations in biomarker Defining of linear range p <0.0001 2 2 ρ=0.6, Spearman 2 p=6.7X10-13 Our company has developed Quantitative Dot Blot 1 of FFPE lysates ROC analysis p<0.0001 assessments. Method to measure biomarker at tissue level as absolute and Sensitivity % 0 (QDB) method to achieve absolute quantification of a 0 0.2 0.4 0.6 0 1 1 1

continuous variables with objectivity and consistency in daily practice represents nmole/g protein at tissue level objectively, consistently and in Breast cancer FFPE lysate (µg) 0 50 100 6 100% - Specificity% future in clinical diagnosis and prognosis. The feasibility of Quantiative Dot Blot high throughput format. 5 R² = 0.9976 100 4 α EP3 0 0 (QDB) method to achieve this goal was tested in this study using 1049 Formalin 0 10 20 30 40 0 10 20 30 40 The success of this method relies solely on the specific- 10 α EP3 0 20 40 60 80 100 Fixed Paraffin Embedded (FFPE) breast cancer tissue. 3 ity of the detection antibody. There are a rich resevior (Arbitary unit X1,000,000) Defining of linear range 1 nmole/g nmole/g Years Chemiluminesence reading Chemiluminesence reading 2 of extensively validated antibodies for IHC analysis of protein standard 1 0.1 (IHC antibodies) already. These antibodies may be ad- Proposed cutoff PR and Her2 Ki67 and ER Log scale 60 Ki67 and Her2 : The absolute contents of Her2, ER, PR, p53, Ki67, EGFR and PCNA 0 0.01 at 0.267 nmole/g 60 Methods opted directly in QDB analysis to measure tissue bio- 0 200 400 600 40 0.001 µ markers as absolute and continuous variables. 0 1 2 3 were measured using total tissue lysates prepared from the same 2X15 m FFPE Purified Her2 protein (pg) Her2 levels (nmole/g) 30 after deparaffination for QDB measurement using validated antibodies for Grouped by IHC result 40 40 slices The simplicity of the method allows it to be adopted in IHC analysis (IHC antibodies), including 4B5 and EP3 for Her2, MIB and regular lab to meet the daily clinical need of diagnosis A: Consistency of the results from two dif- D: Comparison with FISH 20 ferent antibodies for the same biomarker 20 and prognosis. FISH 20 UMAB107 for Ki67, SP1 for ER, 1E2 for PR, DO-7 for p53, PC10 for PCNA to Her2 EP3 Total QDB nmole/g 10 40 r=0.963 Negative Equivocal Positive Before ensure the specificity. Negative 89(90.8) 4 7(16.7) QDB method was used to measure multiple tissue bio- n=332 1 100 30 Her2 QDB 88.6% 0 0 0 markers (ER, PR, Ki67, Her2, p53, PCNA, Cyclin D1) Positive 9(9.2) 0 35(83.3) 44 0 10 20 30 40 0 10 20 30 40 0 1 2 3 20 using well accepted IHC antibodies (IVD or ASR grade) Total FISH 98 4 42 144 nmole/g Concordance : 88.6 %*, κ =0.732 by Kappa coefficient analysis. nmole/g nmole/g The method was indirectly validated by converting Her2 levels into dichotomous 10 2 in 1049 Formalin Fixed Paraffin Embedded (FFPE) 1 The cutoff value was set at 0.267 nmol/g based on ROC analysis of IHC results. 2 Equivocal cases were not includeded in the analysis. breast cancer specimens as absolute and continuous With EP3 (nmole/g) variables to compare with results from FISH and IHC analyses using ROC analysis 0 16 samples different from original FISH reports were variables simultanesouly. In this study, we reported our 0 10 20 30 40 50 and provided IHC results. The correlations among biomarkers and with other With 4B5 (nmole/g) submitted for third party FISH analysis with 50% results with ER, PR, Ki67 and Her2 in these samples. samples reclassified clinicopathological factors were also investigated statistically. QDB process 30 r=0.97 FISH Her2 EP3 Total QDB Conclusions 20 n=332 Negative Equivocal Positive After

Ki67 Negative 91(95.8) 5 4(9.3) Results: The intra- and inter-cv of all the QDB measurements were below 15% 1 100 10 QDB 94.2% from three independent experiments, each in triplicate. The consistency of Positive 4(4.2) 1 39(90.7) 44 ER, PR, Ki67, and Her2 were measured absolutely and quantiatively with QDB

With UMAB107 (nmole/g) Total FISH 95 6 43 144 Materials & Methods 0 2 methods using the same 2X15 mm FFPE slices and IHC antibodies (IVD or measurements from two independent antibodies for both Her2 and Ki67 were 0 5 10 15 20 25 Concordance : 94.2%*, κ =0.865 by Kappa coefficient analysis. 1 With MIB1 (nmole/g) The cutoff value was set at 0.267 nmol/g based on ROC analysis of IHC results. above 0.96 with Pearson correlation analysis. When converted Her2 level into Patients: The characteristiscs of the patients are available upon request. FFPE specimens were 2Equivocal cases were not includeded in the analysis. ASR). collected from local hospitals sequentially and non-selectively. All the specimens were provided dichotomous variables, QDB method achieved 94.2% concordance with FISH. as 2X15 µm FFPE slices in microtube anonymized, and the clinical data were approved by the We were also able to show the relationship among these biomarkers as absolute Ethical board of corresponding hospitals. Distributions (n=1049) QDB achieved more objective, comprehensive and accurate Her2 assessment 30 Preparation of FFPE samples: FFPE slices of 2X15 µm were de-paraffined directly in micro- 40 All experiments were and continous variables directly using scatterplots to illustrate the relationship 20 PR than FISH and IHC. tube, and total protein lysates were extracted using lysis buffer directly. The total protein amount Her2 repeated three times, among these biomarkers directly. was determined using BCA protein determination kit from Pierce. 30 15 each time in triplicate. QDB method allows absolute and quantiative assessment of multiple bio- Reagents and QDB plates: Antibodies were purchased from Roche, ZSBio and abcam. Pro- 20 10 Conclusions: For the first time, we were able to measure 6 biomarkers as tein standards were purchased from Sino Biologic, Beijing, China or manufactured in house. All All intra- and inter-cv markers with simplicity, consistency and objectivlty in daily clinical practice. other reagents were purchased from Fisher Scientific Inc. QDB plate was manufactured by 10 5 absolute and continuous variables in 1049 FFPE specimens to demonstrate the Yantai Zestern Biotechnique Co LTD, a division of Quanticision Diagnostics, Inc of USA at RTP, were below 15% NC (www.zestern.net) 0 feasility of QDB method in routine clinical diagnosis. The adoption of this method 3 50 Data of absolute and continuous nature allows development of a growing da- QDB measurement. The overall analytical process was described in detail in PMID 30199009 ER 30 Ki67 promises the transition in diagnostics of relative and discrere nature to absolute and 28938578 (keyword, QDB, Jiandi Zhang) with minor modifications. In short, total tissue ly- tabase for data mining to provide better diagnosis, prognosis and treatments. sates of 2µl/sample (0.2 µg to 0.5 µg total) were loaded to QDB plate, and incubated with IHC 2 and continuous variable. It also allows us to develop a growing database to full antibodies for Her2 (EP3 & 4B5), Ki67 (MIB1 & UMAB107), ER (SP1) and PR (1E2) overnight to 20 Median level explore the clinical usage of these biomarkers, and to better stratify patients for form immunocomplex on the QDB plate. The QDB plate was placed in the microplate reader to QDB method opens door to the transition from subjective, semi-quantitative, quantify the signal on the bottom of individual unit of QDB plate. 1 personalized treatments. 10 and relative to objective, quantitative, absolute in tissue biomarker assess-

Biomarker levels (nmole/g) 0 0 ment.