# Presentation: 069282

## Validation of Biomarkers for Hepatocellular Carcinoma

- Jorge A. Marrero, M.D., M.S.

## Overview

- Epidemiology of Hepatocellular Carcinoma (HCC)
- Current Markers for HCC
- Validation of Biomarkers for HCC
- Des-gamma carboxyprothrombin (DCP)
- Others

## Trends in Incidence and Death Rates1994-2003

- www.seer.cancer.gov

## HCC Progression

**Chronic ****	****                  Cirrhosis****	****	****      HCC**
**Liver Disease****	**

- Nat Genet. 2002;31:339-46.

**Notes:**
It is postulated that the progression to HCC starts with chronic hepatitis or chronic liver disease. This progresses to cirrhosis and then to high grade dysplasia and finally to HCC. The exact molecular mechanisms have not been elucidated and is an area of research.

## Incidence of Hepatocellular Carcinoma in HCV and HBV

- Fattovich G, et al. Gastroenterology 2004;127:S35

**Hepatitis C**

**Hepatitis B**

## Overview

- Epidemiology of Hepatocellular Carcinoma (HCC)
- Current Markers for HCC
- Validation of Biomarkers for HCC
- Des-gamma carboxyprothrombin (DCP)
- Others

## Screening for HCC:AASLD Recommendations

- Surveillance should be performed with ultrasonography (level II)
- AFP alone should not be used for screening unless it is with ultrasound (level II)
- Screening should occur every 6-12 months (level II)
- Need for better screening tests

- Bruix J, et al. Hepatology 2005;42:1208

## Alpha-fetoprotein Cross-Sectional Studies

- Marrero JA. Clin Liver Dis 2005;9:235.

| Author | Cutoff | No. of HCC | Sensitivity  Specificity
%                   % |
| --- | --- | --- | --- |
| Peng | 20 | 205 | 65                   88 |
| Trevisani | 16# | 170 | 60                   90 |
| Cedrone | 100# | 74 | 25                   95 |
| Soresi | 30# | 197 | 65                   89 |
| Lee | 200 | 54 | 53                   79 |
| Nguyen | 20 | 163 | 63                   79 |

- # determined by ROC curves

## Alpha-fetoprotein Prospective Cohort Studies

| Author | No. of      No. of
cirrhotics  HCC | PPV         NPV
   %              % | Sensitivity     Specificity
        %                    % |
| --- | --- | --- | --- |
| Pateron | 118             14 |    33              - | 50                  86 |
| Oka | 260              55 | 32             82 | 39                  76 |
| Bolondi | 313             61 | 46             85 | 41                  82 |
| Tong | 602             31  | 12             99 | 41                  95 |
| Chalasani | 285             27 |    30              - | 63                  87 |

- Marrero JA. Clin Liver Dis 2005;9:235.

## Ultrasound in HCC in Cohort Studies

- Author       Year       Sens%       Spec%        Pos          	Neg
- Okazaki       84	86	       99		66		0.14
- Maringhni    84	92	       86		6.5       	0.09
- Kobayashi   85 	75	       98		32.6		0.26
- Tanaka        86	47	       100	589		0.41
- Dodd	        92	43	       98		21.5		0.58
- Saada	        97	33	       100	333		0.67
- Chalasani    99	59	       92		8.4		0.45
- Rode	        01	46	       95		9.2		0.57
- Bennett       01		30	       97		7.4		0.72
- Teefey	        03	89	       73		3.3		0.15
- Libbrecht     03	40	       100	400		0.6

- Likelihood Ratio

- Pooled Estimates	60.5	        96.9         17.7                0.5
- (95%CI)	           (44-76)            (95-98)       (8.5-36.9)            (0.4-0.6)

## Overview

- Epidemiology of Hepatocellular Carcinoma (HCC)
- Current Markers for HCC
- Validation of Biomarkers for HCC
- Des-gamma carboxyprothrombin (DCP)
- Others

## Phases of Biomarker Development for Early Detection of Cancer

- Pepe MS, et al. J NCI 2001;93:1054

- Phase 1:	Preclinical exploratory studies
- Phase 2:	Clinical Assay Development 				for Clinical Disease
- Phase 3:	Retrospective Longitudinal Study
- Phase 4:	Prospective Screening Studies
- Phase 5:	Cancer Control Studies

## Increased prothrombin precursor

The activity of the -glutamyl-carboxylase has been shown to be decreased in HCC 

The increase production of precursor is named des-gamma carboxy prothrombin

- The activity of the -glutamyl-carboxylase has been shown to be decreased in HCC
- The increase production of precursor is named des-gamma carboxy prothrombin

- Des-gamma carboxyprothrombin (DCP) in HCC

- Tsai et al Hepatology 1990;11:481

## Lectin-bound Alpha-fetoproteinAFP-L3

- The sugar chain structures of AFP obtained from patients with LC and HCC have different affinities for lectins
- One subspecies, *Lens culinaris* agglutinin (LCA)-reactive AFP (AFP-L3) is more specific to HCC

- Yamagata et al. Clin Chim Acta. 2003;327:59

- AFP
- AFP-L3%

## Phase I validation study of DCP in HCC

- 4 Groups
*Group 1*: normal volunteers: 60
*Group 2*: non-cirrhotic chronic hepatitis: 61
*Group 3*: cirrhosis: 63
*Group 4*: HCC: 65
- Blood was obtained and centrifuged before treatment at the time of diagnosis (after sitting in 32F for 12 hrs), aliquoted and stored at -80C
- Each sample barcoded to link to clinical data
- DCP performed by sandwich ELISA in duplicates in an external lab and measured blindly
- AFP-L3 was performed by Wako diagnostics in a blind fashion
- Assay intra- and interassay variability ~ 10%

- Marrero, Hepatology 2003

## AFP, AFP-L3 and DCP

- AFP			AFP-L3%		      DCP

- Control  Case

- Control  Case

- Control  Case

**Control =  cirrhosis without HCC (n=159)**
**Case = HCC with underlying cirrhosis (n=84)**

## DCP Differentiates Cirrhosis from HCC

**AUROC **
**** DCP = 0.928
- ----AFP = 0.821
- p = 0.0022

- *

*****

- _* Cutoff Values_
- DCP = 150 mAU/ml
-      Sens: 89%
-      Spec: 96%
-      PPV: 91%
-      NPV: 88%
- AFP = 13 ng/ml
-      Sens: 62%
-      Spec: 76%
-      PPV: 78%
-      NPV: 71%

- Marrero, Hepatology 2003

- Marrero et al; Hepatology 2003;37:490

## Performance Characteristics of Markers-Early Stage (n=52)

| Marker | AUROC | Sens | Spec | +LR | -LR |
| --- | --- | --- | --- | --- | --- |
| AFP
14 ng/mL | 0.81 | 64 | 88 | 8.5 | 0.38 |
| AFP-L3
3% | 0.71 | 50 | 88 | 4.5 | 0.56 |
| DCP
150 mAU/ml | 0.93 | 92 | 93 | 13.9 | 0.08 |
| Comb | 0.94 | 90 | 91 | 10.7 | 0.11 |

## DCP Validation Study DesignPhase 2

- Aims
- To determine the sensitivity and specificity of des-gamma carboxyprothrombin (DCP) for the diagnosis of early hepatocellular carcinoma (HCC).
- compare performance characteristics of DCP and Alpha-fetoprotein (AFP) in the diagnosis of early HCC.
- To determine whether demographic or etiology of underlying liver disease alter the expression of DCP or AFP.
- Case-control study
- cases: modified TNM stage I and II HCC (eligible for liver transplant), prior to any cancer therapy
- controls: cirrhosis without tumor

## Participating Centers

- University of Michigan
- Mount Sinai University Hospital
- University of Pennsylvania
- Mayo Clinic, Rochester
- Mayo Clinic, Jacksonville
- Saint Louis University
- Stanford University

## Sample size

- AFP
- 	True Positive Fraction = 0.57  (0.65 for power analysis)
- 	False Positive Fraction = 0.22 (0.20 for power 			analysis)
- DCP
- 	True Positive Fraction = 0.85
- 	False Positive Fraction = 0.10
***  *****Power 90% and 92% for TPF and FPF**
**  ****We will need to recruit 190 early HCC cases and **
**    ****410 cirrhosis controls.**

## Recruitment

- Patients are identified at the time of the clinic visit, consented prior to the physician seeing the patient
- VSIMS has a tool to determine if a potential control matches to a case (gender, age ± 10 y, etiology of liver disease –viral vs nonviral liver disease)
- Cases
- Histology or 2 imaging tests showing characteristics of HCC (> 2 cm)
- Most sites have a centralized area for the care of patients with primary liver tumors
- Multidisciplinary liver tumor clinic
- Controls
- Histology or evidence of portal hypertension (splenomegaly, low platelets, presence of esophageal varices)
- Liver clinics because controls have chronic liver disease
- Patients are confirmed if they meet the eligibility criteria and if serum was obtained
- Data is collected regarding demographics, family history, social history, medical history, detail data about their liver disease, obesity

## Samples

- Serum collection
- Phlebotomy, blood sits for 30 minutes and then 12 hrs in 32C, followed by centrifuge and aliquoted in 500 μL, then stored at -80C. Sites ship to central facility monthly located at the University of Michigan.
- Each aliquot is barcoded for identification purpose and link to clinical data
- Data systems allow for sample tracking (VSIMS)
- An aliquot (500 L) from each patient will be shipped to UCLA for assay, aliquot identified by DMCC
- UCLA then send raw results to DMCC for analysis
- Assays
- DCP will be performed as sandwich ELISAs’ in a blind fashion at UCLA in duplicates
- AFP and AFP-L3 will be performed singly UCLA (already FDA approved)
- 10% of all samples will undergo QC
- DCP at UMich
- AFP and AFP-L3% at Wako
- Additional samples will be collected and stored at NCI Frederick once the study concludes

## Data Management

- DMCC (FHCRC) will perform data coordination and management
- Database utilized is the EDRN Validation Study Information Management System (VSIMS)
- Data Quality Monitoring Board in place

## Next Step

- Phase 2 study will be the largest involving early stage tumors. Important to determine:
- Appropriate cutoffs for the biomarkers
- Determine performance characteristics in early stage HCC
- To better select the population for future studies
- A phase 3 study: a cohort of patients with cirrhosis to determine efficacy of biomarker to detect preclinical HCC
- 2 ongoing studies

## AFP-L3

- FDA approved for the determination of risk of HCC among patients with cirrhosis
- Approval based on a multicenter US study (as well as data from Japan)
- Prospective study of patients with cirrhosis 332 of which 34 had HCC
- Each site had its own definition of HCC
- Could not go back to when HCC first diagnosed
- Serum obtained locally and AFP-L3 done centrally at Wako
- Unclear if they studied other important risk factors for the development of HCC such as liver function, alcohol, tobacco exposures, obesity

## Conclusion

- For the validation of biomarkers we have developed a reference set for discovery, followed by a large phase 2 study powered for novel biomarker(s) to “beat” AFP (standard), and then a phase 3 study for the ability of the biomarker to detect preclinical disease
- It is important to test the quality of the assay as well as the design evaluating the clinical indication
- A DMCC is critical in the validation process