br Two researchers independently in a blinded manner
Two researchers independently, in a blinded manner, evaluated the intensity and distribution of positive staining of each slide. Slides and scores were reviewed together using a dual-headed scope to reach a final consensus score for any specimens that were discordant. A stan-dard 4-point scale scoring system as (absent), 1 (weak), 2 (moderate), and 3 (strong) was used to score intensity to study origin (stroma vs. cancer) and gene expression alteration (primary vs. metastatic sites). In expression alteration analysis, the average of 2 scores of metastatic sites was compared with the score of primary site.
2.5. Statistical analysis
The analysis of protein expression was performed using GraphPad Prism 7 software and statistical differences were evaluated using t-test.
3.1. Patient demographics
Patients' characteristics are shown in Table 1. All patients had MES subtype of HGSOC, and underwent cytoreductive surgery. The vast ma-jority of patients had either high or intermediate complexity surgery. All patients had b0.5 cm of gross residual disease at the conclusion of sur-gery with 40% having complete gross resection. Most patients were ei-ther platinum sensitive (47%) or resistant (47%) showing disease progression within 6 months of completion of chemotherapy.
3.2. Gene signatures
Based on existing literature, an initial candidate list selected proteins that are recognized to be highly expressed in MES vs. non-MES sub-types. We then narrowed this candidate list to focus on those with established GSK3 that further performed well in testing in FFPE samples to arrive at the 8 proteins selected. Published reports of micro-array analyses show correlation with overexpression and prognosis for many of these genes (Table 2). These proteins include alpha-smooth muscle (ACTA2), collagen type V alpha 1 (COL5A1), collagen type XI
(40%) metastatic sites, compared with absent expression in only 2/15
this likely reflects small sample size.
In contrast, COL11A1 demonstrated increased expression in the
tionship was reversed when considering expression from cancer cells
instead of stroma: COL11A1 expression was reduced in metastatic vs.
Sensitivity to platinum
Collectively these results suggest that collagen proteins, as the major
component of stroma play important but different roles in cancer
alpha 1 (COL11A1), fibroblast activation protein (FAP), periostin metastasis.
(POSTN), phosphorylated-SMAD2 (p-SMAD2), versican (VCAN), and
zinc finger E-box binding homeobox 1 (ZEB1). The representative im- P-SMAD2 was expressed in the nucleus of both cancer cells and
ages of IHC staining of these proteins are shown in Fig. 1A.
stroma (Fig. 1). We observed that nearly all cancer nuclei expressed p-
3.3. Protein localization
SMAD2 (Fig. 3B & Supplementary Table 1). In contrast there was wider
variation in nuclear expression of p-SMAD2 in stromal cells. Specifically,
The staining intensity was classified as: absent, weak, moderate, or when evaluating the primary cancer, 40% of tumors demonstrated only
rare (0–30%) nuclear expression – this decreased in the metastatic sites
strong. We used the broader 4-score grading system to help discern
(Fig. 3B & Supplementary Table 1). We did not characterize the specific
even low levels of protein expression which may be important in cancer
stromal cell type to be able to determine if this decrease in stromal
expression was confined to a specific cell type. These results support
VCAN) were expressed exclusively in stroma and they were expressed
that TGF-β signaling is important in stromal activation associated with
COL11A1, POSTN, p-SMAD2 and ZEB1), stromal expression was the
most commonly observed pattern (Fig. 1B). These data confirm that 4. Discussion
the molecular description of the MES subtype of HGSOC reflects a signif-
icant stromal contribution.
The MES subtype of HGSOC is associated with several high risk clin-
ical features including worse overall survival (OS), higher intraperito-
neal disease dissemination patterns, and low rates of resection .
Molecularly, MES subtype is characterized by the overexpression of
We next investigated the differences in expression between primary many genes which have been associated with activated stroma [3,4],
and metastatic disease sites. We used the 2-score grading system of though the stromal component to the MES classification has not been