Study composition and patients’ characteristics
A total number of 371 patients with a diagnosis of ovarian cancer made between 2002 and 2017 were identified from the study sites. Following exclusions, two TMA’s were constructed representing 227 ovarian cancer cases. Further exclusions resulted in 210 ovarian cancer cases suitable for analysis, as shown in Figure 1. For all patients enrolled, the median age was 47.5 (Range 6 to 86) years. The patients presented with various symptoms and the most common symptoms were abdominal swelling, abdominal pain, abdominal distention, and abnormal uterine bleeding. The duration of the condition ranged from 2 weeks to 84 months, with a median duration of 6.5 months.
Menopausal status was available for 91 patients only, whereas 34 (37.4%) were post-menopausal. All patients underwent surgery, and the majority of the specimens received for investigation were ovarian masses 126 (60%), followed by hysterectomy and bilateral salpingo-oophorectomy. In a few cases, staging surgery was accompanied by omentectomy, bowel resection, and lymph node sampling. The stage of the disease was available in four patients only, and all were at an advanced stage. There was no data on disease recurrence or survival.
Ovarian tumor types in original and revised diagnoses
In the original diagnosis, the majority of ovarian cancers were epithelia (carcinomas), which constituted 147 (70%) of all the cases with a mean age of 50.5 (SE ±1.2) years. The second most common diagnosis was a sex cord stromal tumor in 24 (11.4%) cases with a mean age of 37 (SE± 3.0) years, followed by germ cell tumors 21 (10%), with a mean age of 22.8 (SE± 3.2) years. The diagnosis of ovarian lymphomas was rendered in 14 (6.7%) of the cases with a mean age of 15.9 (SE ±3.7) years. Other types included sarcoma in 2 (0.9%) cases and unspecified cancers in 2 (0.9%) cases.
Following morphological review and IHC integration, there was substantial agreement between original diagnosis and a revised diagnosis in the major categories of ovarian cancer (Kappa 0.702 with 95% CI of 0.619 to 0.791), as shown in table 1. In a revised diagnosis, the largest category was still epithelia in 127 (60.5%) of all the cases with a mean age of 52 (SE ±1.2) years. The second most common category was sex cord stromal tumors with 30 (14.3%) cases with a mean age of 45 (SE ±2.9) years, followed by germ cell tumors 27 (12.6%), and lymphomas 13 (6.2%). There was no significant mean age difference from the original to the revised diagnosis between ovarian tumor types.
Table 1. Original diagnosis and IHC integrated revised ovarian cancer types
|
Revised diagnosis
|
|
|
|
|
EPH
|
GCT
|
LYMH
|
SCST
|
SC
|
Others
|
NM
|
Total
|
Concordance (%)
|
Kappa
|
Original Diagnosis
|
|
|
|
|
|
|
|
|
|
|
EPH
|
123
|
9
|
0
|
9
|
0
|
0
|
6
|
147
|
83.7
|
|
GCT
|
3
|
17
|
0
|
1
|
0
|
0
|
0
|
21
|
81.0
|
|
LMPH
|
0
|
0
|
13
|
0
|
1
|
0
|
0
|
14
|
92.9
|
0.7019
|
SCST
|
1
|
1
|
0
|
20
|
1
|
0
|
1
|
24
|
83.3
|
|
SC
|
0
|
0
|
0
|
0
|
2
|
0
|
0
|
2
|
100.0
|
|
Others
|
0
|
0
|
0
|
0
|
1
|
1
|
0
|
2
|
50.0
|
|
Total
|
127
|
27
|
13
|
30
|
5
|
1
|
7
|
210
|
|
|
Concordance (%)
|
96.6
|
63.0
|
100
|
66.7
|
40.0
|
100.0
|
NA
|
|
|
|
EPH: Epithelia, GCT: Germ Cell Tumors, LMPH: Lymphomas, SCST: Sex Cord Stromal Tumors SC: Sarcomas, NM: Non Malignant
Histotype specific agreement between original diagnosis and revised diagnosis
In a revised diagnosis, the specific histotypes were assigned based on 2014 WHO Classification of Tumors of Female Reproductive Organs. There was a fair agreement between original diagnoses and revised diagnoses (Kappa=0.343, 95% CI: 0.277 to 0.409), as shown in Supplementary Table 2. In summary, a good concordance was seen in Lymphomas, followed by Germ Cell Tumors (92.9% and 81%, respectively). For epithelial tumors (carcinoma), a total number of 84 (57.9%) cases were not classified (carcinoma not otherwise specified (NOS)). Following review, a total number of 36 (42.9%) cases were reclassified to High Grade Serous Carcinoma (HGSC). Significant numbers of carcinoma NOS had poor H&E stains, in which the diagnosis was straightforward following recut and new H&E stain at Calgary laboratory. Two cases had a diagnosis of carcinoma NOS. Following H&E re-stain, one was adult granulosa cell tumor, and the other was HGSC with positive staining with WT-1 and p53 mutant type (see Figure 2). There were 13 cases of HGSC in the original diagnosis, and the concordance with the revised diagnosis was 76.9%. Among 17 cases originally diagnosed as Endometrioid Carcinoma (EC), the concordance with a revised diagnosis was 58.8%, and 5 (29.4%) were reclassified to HGSC. The concordance of 21.7% was seen in Mucinous Carcinoma (MC), and 6 (26.1%) were reclassified to EC, whereas 4 (17.4%) were metastasis from GI. There were three cases of Low Grade Serous Carcinoma (LGSC), with a concordance of 33.3%, one case was EC, and the other was a metastasis from GI. There was one case of neuroblastoma which was reclassified as small cell carcinoma hypercalcemic type (SCCOHT) with loss of BRG1 staining in tumor cells and positive staining of stromal cells (See Figure 3 A&B), and one case of carcinoma NOS was reclassified as mixed carcinoma (EC/LGSC).
IHC markers expression in ovarian cancers
The patterns of IHC markers expression across different ovarian cancer histotypes are shown in Table 2. PAX8 was expressed in all LGSC, in 87% of HGSC and in none of the mucinous carcinomas. The Fallopian tube marker WT-1 was positive in 89% of HGSC, all LGSC, and in 27% of EC, with no expression in clear cell carcinoma (CCC) and MC. Hormonal receptor expression (ER) was higher (75%) in HGSC compared to other histotypes. For endometrioid carcinoma, ER and PR receptors were observed in 34% and 21% of cases, respectively, and one case of EC with mucinous features had a loss of MSH6 and ARID1A in tumor cells with a positive stain in stromal tissue and lymphocytes (See Figure 3C). Abnormal staining of p53 was observed in 63% of HGSC, 18% of the EC, 20% of MC, and none of the LGSC. In sex cord stromal tumors, FOXL2 was expressed in 66% and 50% of adult granulosa cell tumor (AGST) and juvenile granulosa cell tumor (JGCT), respectively. SATB2 expression was predictive of ovarian metastatic mucinous tumor (p<0.0001). Additionally, two cases of immature teratoma and one case of AGCT were positive for SATB2.
For analysis of antibody performance, the known positive controls from APRL containing tonsil, Fallopian tube, placenta, and endometrium were included in each TMA. The performance of tested samples was assessed based on presence of internal controls for the tests, which require interpretation with internal controls (p53, ARID1A, MSH6, PMS2, and BRG1). All markers were not assessed equally across all the samples, as some samples were uninterpretable, or there was a loss of cores in the TMA. However, the majority of the samples were assessed for these markers, as shown in Table 3.
Table 2. Immunohistochemical marker expression across histotypes
Marker
|
Carcinoma (%)
|
Sex Cord Stromal Tumors (%)
|
HGSC
|
EC
|
CCC
|
MC
|
LGSC
|
AGCT
|
JGCT
|
PAX8: Positive
|
87.2
|
28.1
|
50
|
0
|
100
|
8.3
|
25
|
WT-1: Positive
|
89.5
|
27.6
|
0
|
0
|
100
|
47.8
|
0
|
p53: Mutant
|
63.2
|
18.8
|
0
|
20
|
0
|
4.2
|
0
|
p16 :Abnormal
|
75.4
|
53.1
|
50
|
60
|
50
|
54.2
|
75
|
Napsin-A: Positive
|
0
|
3.1
|
25
|
0
|
0
|
0
|
0
|
ER: Positive
|
75.4
|
34.4
|
25
|
10
|
50
|
37.5
|
50
|
PR: Positive
|
19.3
|
21.9
|
0
|
0
|
0
|
33.3
|
0
|
ARID1A: Negative
|
0
|
3.1
|
0
|
0
|
0
|
0
|
0
|
Vimentin: Diffuse
|
14
|
21.9
|
25
|
0
|
50
|
95.8
|
75
|
MSH6: Negative
|
0
|
3.1
|
0
|
0
|
0
|
0
|
0
|
PMS2: Negative
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
FOXL2: Positive
|
0
|
0
|
0
|
10
|
0
|
66.7
|
50
|
Table 3. IHC markers with no internal positive controls and level of the hospital
|
|
Hospital level
|
|
Marker
|
N (%)
|
Non-Tertiary (N=72)
|
Tertiary (N=147)
|
p-value
|
p53 (N=204)
|
18 (8.8)
|
3
|
15
|
0.178
|
ARID1A (N=208)
|
99 (47.6)
|
50
|
49
|
0.000**
|
MSH6 (N=208)
|
15( 7.2)
|
6
|
9
|
0.353
|
PMS2 (N=206)
|
26 (12.6)
|
9
|
17
|
0.913
|
BRG1 (N=201)
|
30(14.9)
|
12
|
18
|
0.716
|
**Majority of cases with positive internal control were from tertiary hospitals (87/109)
The most affected marker was ARID1A, whereby 47% of the cases had a loss of staining in tumor cells as well as stromal tissue and lymphocytes, which are the internal controls (See Figure 3D), followed by PMS2 (12%), and BRG1 (14%). When we analyzed for the primary site of sample fixation in relation to loss of internal control, there was no significant difference when the samples were fixed in tertiary or non-tertiary hospitals. However, for ARID1A, most of the samples with positive controls had primary fixation in a tertiary hospital. These results are summarized in Table 3.
Comparison of ovarian tumor types between East African and Alberta
The comparison of ovarian cancer tumor types and histotypes distribution was performed using the following two populations: i) accessible ovarian cancer cases from East Africa diagnosed over a period of 15 years, and ii) Alberta Cancer Registry for cases diagnosed over a period of 9 years (2007 to 2016). In Alberta, the women population at risk of ovarian cancer is estimated to be 1.4 million women compared to the catchment area of the selected centers in East Africa of 9 million women at risk. There were 8-times more ovarian cancer cases in Alberta, compared to the selected population in East Africa, which is 6-times larger than the population of Alberta. This rough estimate suggests that there are 48 times more ovarian cancer cases in the Alberta population compared to the selected East African population. The distribution of the major tumor types in both populations showed epithelial tumors being the commonest, 89.9% in Alberta, and 58% in East Africa.
For the carcinomas, when a comparison was made by specific histotypes, HGSC occurred with the same frequency in both populations, as shown in Table 4. Still, EC was more common in the East Africa population than the Alberta population (28.3% versus 11.3% respectively).
There was no striking difference for CCC, LGSC, and MC, and these cases occurred at a lower frequency in both populations. For Germ Cell Tumors, there was a predominance of Dysgerminoma and Immature Teratoma in the Alberta population (29.3% and 31%, respectively), compared to the East Africa population (19.2% and 15.4% respectively). However, the Yolk Sac Tumors were more common in East Africa compared to Alberta (53.8% and 17.2%, respectively). The Adult Granulosa Cell Tumor was the commonest sex cord stromal tumor in both East Africa and Alberta populations (80% and 70.6%, respectively), though in relatively low numbers in Alberta compared to the numbers of ovarian cancer cases. Juvenile Granulosa Cell tumors were exclusively seen in East Africa in 13.3% of the cases of SCST.
Table 4. Patterns of ovarian cancer tumor types in Tanzania compared with Alberta, Canada
|
|
Major categories
|
|
|
|
East Africa (n %)
|
Mean age (±SE)
|
Alberta (n %)
|
Mean age (±SE)
|
Epithelial
|
127 (60.5)
|
52.0 (1.2)
|
1645 (89.9)
|
61.8 (0.4)
|
Germ cell tumor
|
27 (12.6)
|
24.2 (2.7)
|
58 (3.2)
|
31.2 (1.5)
|
Lymphoma
|
13 (6.2)
|
15.3 ( 3.7)
|
NA
|
NA
|
Sex cord stromal tumor
|
30 (14.3)
|
45.0 (2.9)
|
17 (0.92)
|
57.1 (4.1)
|
Sarcoma
|
5 (2.4)
|
50.3 (6.7)
|
5 (0.3)
|
62.2 (6.1)
|
Others
|
1 (0.5)
|
30.0 (13.4)
|
104 (5.7)
|
79.0( 1.2)
|
Histotypes specific
|
|
|
|
|
|
Carcinoma (epithelia)
|
N= 113
|
|
N=1,646
|
|
HGSC
|
57 (50.4)
|
|
831 (50.5)
|
|
EC
|
32 (28.3)
|
|
186 (11.3)
|
|
CCC
|
4 (3.5)
|
|
132 (8.0)
|
|
LGSC
|
2 (1.8)
|
|
15 (0.9)
|
|
MC
|
10 (8.8)
|
|
120 (7.3)
|
|
Others
|
8 (7.1)
|
|
362 (22.0)
|
|
Germ cell tumors
|
N= 26
|
|
N=58
|
|
Dysgerminoma
|
5 (19.2)
|
|
17 (29.3)
|
|
Yolk Sac Tumor
|
14(53.8)
|
|
10 (17.2)
|
|
Immature Teratoma
|
4 (15.4)
|
|
18 (31.0)
|
|
Others
|
3 (11.6)
|
|
13 (22.4)
|
|
Sex cord stromal tumor
|
N= 30
|
|
N= 17
|
|
Adult granulosa Cell Tumor
|
24 (80.0)
|
|
12 (70.6)
|
|
Sertoli Leydig Cell Tumor
|
2 (6.7)
|
|
4 (23.5)
|
|
Juvenile Granulosa Cell Tumor
|
4(13.3)
|
|
0
|
|
Others
|
0
|
|
1(5.9)
|
|