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The presence of 18q loss of heterozygosity (LOH) predicts decreased disease-free and overall survival in stage II colon cancer: A study of CALGB Protocol 9581
CALGB 9581: Phase III study of monoclonal antibody 17-1A versus observation following surgery for stage II colon cancer
Thomas Colacchio, Dartmouth-Hitchcock, Treatment Study PIRobert Warren, Correlative Science Study PIDonna Niedzwiecki, Donna Hollis, Study Statisticians
Accrual began August 1997, ended May 20021738 enrolled; median follow-up 7.4 yearsTreatment trial result:
no difference in 5-year DFS or OS between the two study armsColacchio, et al, manuscript in prep
ran
do
miz
e
Surgical resection of pT3N0 or pT4bN0 colon cancer
MoAb 17-1A500 mg IV initial treatment then 100 mg IV q28 days x 4
Observation
M.M. Bertagnolli1,4, D. Niedzwiecki2, M. Hall2, S.D. Jewell3, R.J. Mayer4, R.M. Goldberg5, T.A. Colacchio6, R. S. Warren7, M. Redston1
1Brigham and Women’s Hospital, Boston, MA; 2CALGB Statistical Center, Duke University Durham, NC; 3CALGB Pathology Coordinating Office, Ohio State University, Columbus, OH; 4Dana Farber Cancer Institute, Boston, MA, 5University of North Carolina-Chapel Hill, Chapel Hill, NC; 6Dartmouth-Hitchcock Medical Center, Dartmouth, NH; 7University of California-San Francisco, San Francisco, CA
Overall treatment trial result: no difference in 5-year DFS or OS
between thetwo study arms
Colacchio, et al, manuscript in prep
0 2 4 6 8 10
Years from Study Entry
0.0
0.2
0.4
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1.0
Dis
ease
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e S
urvi
val P
roba
bilit
y
0 2 4 6 8 10
0.0
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0.4
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1.0
Adjuvant MoAb 17-1A Observation
N= 865N= 873
Events= 210Events= 215
Median= NAMedian= NA
Chi-square=p-value=
00.96
DFS by treatment for the entire 9581 cohort (n=1,738).
OS by treatment for the entire 9581 cohort (n=1,738).
0 2 4 6 8 10
Years from Study Entry
0.0
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1.0
Ove
rall
Sur
viva
l Pro
babi
lity
0 2 4 6 8
0.0
0.2
0.4
0.6
0.8
1.0
Adjuvant MoAb 17-1A Observation
N= 865N= 873
Events= 157Events= 173
Median= NAMedian= NA
Chi-square=p-value=
0.60.44
35.1/
/66.0
2#1#
2#1# PeakAreaPeakArea
PeakAreaPeakArea
NN
TT
Scoring AlgorithmFor any marker: If the ratio was ≥0.66 and ≤1.35, then the marker indicated that heterozygosity was present (18q intact) If the ratio is outside this range, then the marker indicated LOH (LOH present)
Case calls: If LOH was present at any of the 5 markers, then the patient’s tumor was called LOH+ (LOH present) The case was called s non-informative for 18q if either the non-tumor sample was not heterozygous for the marker or if DNA amplification failure occurred
CALGB 9581 Tissue Bank
Tumor and normal tissue obtained from CALGB Pathology Coordinating Office
Pathology review, dissection to achieve optimal tumor and normal samples, then DNA extraction by standard methods
Tumors without either MSI-H or loss of expression of MLH1 or MSH2 genotyped to detect 18qLOH using theD18S55 marker
Multiplex PCR using fluorescent dye labeled primers; analysis using 310 Genetic Analyzer with
GeneScan/Genotyper software
Results
Chromosomal location 18q is a common site of loss of heterozygosity in colon cancer
18q contains several genes implicated in tumor progression including: SMAD4 – encodes a nuclear transcription factor mediating TGF- signaling SMAD22 – encodes a gene associated with endodermal differentiation DCC – a site encoding a netrin-1 receptor; loss of expression of DCC protein by immunohistochemistry was associated with poor prognosis in stage II and III colon cancers (Shibata, et al)
A retrospective analysis of tissues from randomized phase III cooperative group trials showed that 18qLOH was associated with significantly worse survival in high risk stage II and III colon cancer patients treated with 5- fluorouracil-based chemotherapy (Watanabe et al). No studies to date have prospectively validated the utility of 18qLOH as a prognostic marker for early stage colon cancer.
Background
Saltz LB, Niedzwiecki D, Hollis D, Goldberg RM, Hantel A, Thomas JP, Fields AL, Mayer RJ. Irinotecan fluorouracil plus leucovorin is not superior to fluorouracil plus leucovorin alone as adjuvant treatment for stage II colon cancer: results of CALGB 89803. J Clin Oncol 2007; 25:3456. Shibata D, Reale MA, Lavin P, Silverman M. Fearon ER, Steele G, Jessup JM, Loda M,Summerhayes IC. The DCC protein and prognosis in colorectal cancer. N Engl J Med 1996; 335:1727.Watanabe T, Wu TT, Catalano PJ, Ueki T, Satriano R, Haller DG, Benson AB 3 rd, Hamilton SR. Molecular patterns of survival after adjuvant chemotherapy for colon cancer. N Engl J Med 2001; 344:1196.
References
Study Cohort
Treatment Trial Result
Primary Objective:To evaluate a panel of prognostic markers
in order to determine the relationship between pre-treatment tumor marker status, overall clinical outcome, and response to MoAb 17-1A
Hypothesis: Patients whose tumors demonstrate 18qLOH will have
poorer study outcomes (DFS and OS) than those whose tumors do not demonstrate 18qLOH
Other markers tested: Microsatellite instability by genotyping and MLH1, MSH2 immunohistochemistry, Crohn’s-like peritumoral
inflammatory cell infiltrate
The original study design called for analysis of tumor DCC expression, which was ultimately not completed due to lack of DCC antibody availability
Correlative Science Protocol
Conclusions
D18S55 Marker N= 537 stage II colon cancers:
18q IntactNon-informative or insufficientDNA for
testing
18q LOH
MSI-H,MMR-D
DFS:
OS:
Characteristic All patients
Patients with 18q intact
tumors
Patients with 18qLOH tumors
p=
Number of patients in category 1738 49 101
Treatment – no. (%) 0.388
MoAb17-1A 865 (49.8) 26 (53.0) 46 (45.5)
Observation 873 (50.2) 23 (47.0) 55 (54.5)
Age – yr median (range) 66 (24-90) 64 (37-89) 68 (33-86) 0.163
Gender – no. (%) 0.149
Male 901 (51.8) 22 (44.9) 58 (57.4)
Female 837 (48.2) 27 (55.1) 43 (42.6)
Site of tumor – no. (%)1 0.054
Proximal 1048 (60.3) 31 (63.2) 47 (46.5)
Distal 678 (39.0) 18 (36.7) 54 (53.5)
Unknown 12 (0.7) 0 0
Tumor differentiation – no. (%) 0.134
Well 146 (8.4) 7 (14.2) 8 (7.9)
Moderate 1305 (75.1) 35 (71.6) 86 (85.2)
Poor/undifferentiated 268 (15.4) 7 (14.2) 7 (6.9)
Unknown 19 ( 1.1) 0 (0) 0 (0)
Analysis of tumor microsatellite instability status by either genotyping using Bethesda markers to detect high levels of
microsatellite instability (termed MSI-H) or immunohistochemistry to detect loss of expression of MLH1 or MSH2 (termed mismatch
repair protein deficient, or MMR-D)
Cases analyzed for mismatch repair status
N=537
MMR-I casesN=413
5-yr DFS=0.795-yr OS=0.87
5-yr DFS=0.78 5-yr OS=0.85
5-yr DFS=0.735-yr OS=0.82
5-yr DFS=0.925-yr OS=0.98
5-yr DFS=0.775-yr OS=0.89
5-yr DFS=0.855-yr OS=0.91
5-yr DFS=0.805-yr OS=0.86
p=0.01, 0.04
p=0.03, 0.01
MMR-D casesN=124
Cases analyzed for 18qLOHN=239
Non-informativeN=89
18qLOH+N=101
18q intactN=49
Cases not further tested
N=174
0 2 4 6 8 10
Years from Study Entry
0.0
0.2
0.4
0.6
0.8
1.0
Dise
ase-
free
Surv
ival P
roba
bility
0 2 4 6 8 10
0.0
0.2
0.4
0.6
0.8
1.0
LOH18q-Neg LOH18q-Pos
N= 49N= 101
Events= 5Events= 28
Median= NAMedian= NA
Chi-square=p-value=
4.780.029
0 2 4 6 8 10
Years from Study Entry
0.0
0.2
0.4
0.6
0.8
1.0
Ove
rall S
urviv
al P
roba
bility
0 2 4 6 8
0.0
0.2
0.4
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0.8
1.0
LOH18q-NegLOh18q-Pos
N= 49N= 101
Events= 2Events= 23
Median= NAMedian= NA
Chi-square=p-value=
7.170.007
D18S55 Marker N= 537 stage II colon cancers:
18q IntactNon-informative or insufficientDNA for
testing
18q LOH
MSI-H,MMR-D
DFS:
OS:
Characteristic All patients
Patients with 18q intact
tumors
Patients with 18qLOH tumors
p=
Number of patients in category 1738 49 101
Treatment – no. (%) 0.388
MoAb17-1A 865 (49.8) 26 (53.0) 46 (45.5)
Observation 873 (50.2) 23 (47.0) 55 (54.5)
Age – yr median (range) 66 (24-90) 64 (37-89) 68 (33-86) 0.163
Gender – no. (%) 0.149
Male 901 (51.8) 22 (44.9) 58 (57.4)
Female 837 (48.2) 27 (55.1) 43 (42.6)
Site of tumor – no. (%)1 0.054
Proximal 1048 (60.3) 31 (63.2) 47 (46.5)
Distal 678 (39.0) 18 (36.7) 54 (53.5)
Unknown 12 (0.7) 0 0
Tumor differentiation – no. (%) 0.134
Well 146 (8.4) 7 (14.2) 8 (7.9)
Moderate 1305 (75.1) 35 (71.6) 86 (85.2)
Poor/undifferentiated 268 (15.4) 7 (14.2) 7 (6.9)
Unknown 19 ( 1.1) 0 (0) 0 (0)
Cases analyzed for mismatch repair status
N=537
MMR-I casesN=413
5-yr DFS=0.795-yr OS=0.87
5-yr DFS=0.78 5-yr OS=0.85
5-yr DFS=0.735-yr OS=0.82
5-yr DFS=0.925-yr OS=0.98
5-yr DFS=0.775-yr OS=0.89
5-yr DFS=0.855-yr OS=0.91
5-yr DFS=0.805-yr OS=0.86
p=0.01, 0.04
p=0.03, 0.01
MMR-D casesN=124
Cases analyzed for 18qLOHN=239
Non-informativeN=89
18qLOH+N=101
18q intactN=49
Cases not further tested
N=174
0 2 4 6 8 10
Years from Study Entry
0.0
0.2
0.4
0.6
0.8
1.0
Dise
ase-
free
Surv
ival P
roba
bility
0 2 4 6 8 10
0.0
0.2
0.4
0.6
0.8
1.0
LOH18q-Neg LOH18q-Pos
N= 49N= 101
Events= 5Events= 28
Median= NAMedian= NA
Chi-square=p-value=
4.780.029
0 2 4 6 8 10
Years from Study Entry
0.0
0.2
0.4
0.6
0.8
1.0
Ove
rall S
urviv
al P
roba
bility
0 2 4 6 8
0.0
0.2
0.4
0.6
0.8
1.0
LOH18q-NegLOh18q-Pos
N= 49N= 101
Events= 2Events= 23
Median= NAMedian= NA
Chi-square=p-value=
7.170.007
Preliminary results using D18S55 as a single marker support a role for 18qLOH as a prognostic marker in stage II colon cancer
The high rate of non-informative cases in this series likely results from use of a single 18q marker
Interrogation of 4 additional 18q markers, including D18S58, D18S61, D18S64, and D18S69, is underway.
Tumor Analysis Method
