Patient characteristics
Patient characteristics are shown in Table 1. The median follow-up was 12.2 years and the median age was 70 years (range 53–85). Sixteen patients were female (38%). A majority of patients had locoregionally advanced disease with 32 (81%) having T3/T4 disease and 27 (64%) with positive lymph nodes. Six patients (14%) had positive margins and 22 (52%) had lymphovascular space invasion (LVI). Ten patients (24%) received NAC. None received radiation.
Table 1
Age
|
Number (n = 42)
|
Percent (%)
|
Median
|
70
|
|
Range
|
53–85
|
|
Sex
|
|
|
Male
|
26
|
62%
|
Female
|
16
|
38%
|
pT stage
|
|
|
pT1
|
1
|
2%
|
pT2
|
7
|
17%
|
pT3
|
22
|
52%
|
pT4
|
12
|
29%
|
pN stage
|
|
|
pN0
|
15
|
36%
|
pN1
|
10
|
24%
|
pN2
|
14
|
33%
|
pN3
|
3
|
7%
|
Margin Status
|
|
|
Positive
|
6
|
14%
|
Negative
|
35
|
83%
|
Unknown
|
1
|
2%
|
Presence of LVSI
|
|
|
Yes
|
22
|
52%
|
No
|
17
|
40%
|
Unknown
|
3
|
7%
|
Neoadjuvant chemo
|
|
|
Yes
|
10
|
24%
|
No
|
32
|
76%
|
Tumor vs normal tissue
There were notable differences between tumor and normal tissue with regard to biomarker expression (Table 2, Fig. 1). On IHC, tumor cells had an increased Ki-67 H score (33.3 vs 0, p < 0.001) and higher PD-L1 H score (6.93 vs 0, p = 0.034) and on IF, tumor had increased PD-L1 positive cells (237 vs 35, p = 0.019) and PD-L1 positive cell density (274 vs 53.4 cells/mm2, p = 0.034) compared to normal tissue. There were no differences in MRE11 expression between tumor and normal tissue.
Table 2
Tumor vs normal tissue values for Ki-67, MRE11, and PD-L1 biomarkers with comparative two-tailed t- tests. Significant p-values (p < 0.05) are in bold.
Molecular variable
|
Mean for normal
|
Mean for tumor
|
Tumor compared to normal
|
t-test p-value
|
PDL1 H score IHC
|
0
|
6.93
|
Increased
|
0.034
|
PDL1 CPS IHC (#<10, #>10)
|
(3,0)
|
(40,2)
|
No difference
|
1.00
|
Ki67 H score IHC
|
0
|
33.3
|
Increased
|
0.00016
|
Ki67 percentage IHC(< 1, > 1)
|
(3,0)
|
(33,9)
|
No difference
|
0.88
|
MRE11 Positive Cells per 20X field
|
912
|
2319
|
Increased
|
0.060
|
% MRE11 Positive Cells
|
66.2
|
52.8
|
Decreased
|
0.21
|
MRE11 Positive Cell density (cells/mm2)
|
1355
|
2749
|
Increased
|
0.17
|
Ki67 Positive Cells per 20X field
|
764
|
923
|
Increased
|
0.84
|
% Ki67 Positive Cells
|
39.0
|
22.2
|
Decreased
|
0.60
|
Ki67 Positive Cell density (cells/mm2)
|
1183
|
1115
|
Decreased
|
0.96
|
PD-L1 Positive Cells per 20X field
|
35
|
237
|
Increased
|
0.019
|
% PD-L1 Positive Cells
|
2.00
|
4.98
|
Increased
|
0.12
|
PD-L1 Positive Cell density (cells/mm2)
|
53.4
|
274
|
Increased
|
0.034
|
Associations with the pattern of recurrence and survival
On multivariate regression, positive surgical margin was the only variable associated with increased risk of recurrence (t = 2.14, p = 0.041). On student’s t-test (univariate analysis), three Ki-67 IF measurements were associated with a higher likelihood of LOR versus DR: Ki-67 positive cells (p = 0.036), percent Ki-67 positive cells (p = 0.026), and Ki-67 positive cell density (p = 0.034) (Table 3). Ki-67 cell density varied by recurrence type: LR (1354 cells/mm2), DR (557 cells/mm2) and NR (1111 cells/mm2) (p = 0.034). On multivariate Cox PH modeling the following variables were associated with worse overall survival: older age (HR 1.08, p = 0.015), local recurrence (HR 4.14, p = 0.022), distant recurrence (HR 50.7, p < 0.001), and positive LVI (HR 5.96, p = 0.015). The following were associated with lower recurrence free survival: age (HR 1.09, p = 0.016), pT3 (HR 8.16, p < 0.001), pT4 (HR 1.64, p = 0.004), pN2 (HR 4.60, p = 0.009), pN3 (HR 7.64, p = 0.010), and LVI (HR 41.5, p < 0.001). The only biomarker associated with improved OS was PD-L1 positive cells (HR 1.03, p = 0.036) although it was not associated with RFS.
Table 3
Two-tailed t-tests for recurrence patterns based on Ki-67, MRE11, and PD-L1 expression.
test on
|
Mean for NR
|
Mean for LOR
|
mean for DR
|
t-tests p-values
|
none vs. LOR
|
none vs. DR
|
LOR vs. DR
|
PDL1 H score IHC
|
1.22
|
12.5
|
8.63
|
0.14
|
0.39
|
0.72
|
PDL1 CPS IHC(#<10, #>10)
|
(17,1)
|
(13,3)
|
(7,1)
|
0.51
|
1.00
|
1.00
|
Ki67 H score IHC
|
30.7
|
43.9
|
17.9
|
0.57
|
0.30
|
0.19
|
Ki67 percentage IHC(#<1.#>1)
|
(14,4)
|
(12,4)
|
(7,1)
|
1.00
|
0.97
|
0.86
|
MRE11 Positive Cells per 20X field
|
2301
|
2394
|
2193
|
0.99
|
0.79
|
0.65
|
% MRE11 Positive Cells
|
51.3
|
55.1
|
51.2
|
0.80
|
0.99
|
0.70
|
MRE11 Positive Cell density (cells/mm2)
|
2676
|
2926
|
2530
|
0.77
|
0.77
|
0.44
|
Ki67 Positive Cells per 20X field
|
946
|
1084
|
489
|
0.74
|
0.069
|
0.036
|
% Ki67 Positive Cells
|
21.1
|
28.2
|
11.0
|
0.42
|
0.088
|
0.026
|
Ki67 Positive Cell density (cells/mm2)
|
1111
|
1354
|
577
|
0.59
|
0.082
|
0.034
|
PD-L1 Positive Cells per 20X field
|
156
|
345
|
202
|
0.34
|
0.82
|
0.58
|
% PD-L1 Positive Cells
|
3.10
|
7.11
|
4.92
|
0.28
|
0.72
|
0.71
|
PD-L1 Positive Cell density (cells/mm2)
|
180
|
397
|
231
|
0.34
|
0.83
|
0.58
|
NR = no recurrence, LOR = local only recurrence, DR = distant recurrence |
Developing a molecular signature
Of the 42 pathologic specimens included in this study, one sample was excluded due to unknown margin status bringing the total number of cases included in the PCAs to 41. We performed a series of PCAs to separate patients by recurrence pattern. The first analysis included all 15 biomarker variables (Fig. 2), the second included all 22 clinical and biomarker variables, and the third was limited to margin status and K-67 cell density which were significant on UVA. None of the PCAs were successful in distinguishing samples by recurrence pattern.