Prognostic Signicance of Intra-tumoral Budding in Serous Ovarian Carcinomas

Intra-tumoral budding (ITB) has been well demonstrated to be an independent risk factor for adverse outcomes in colorectal carcinoma. This study investigated the prognostic signicance of ITB in serous ovarian carcinomas (SOCs). The medical records and slides of 84 SOCs, including 13 with neoadjuvant chemotherapy (NAC), were retrospectively reviewed. The following clinicopathological parameters were noted: age at the diagnosis, CA125 level, 2014 FIGO stage, architectural grade, nuclear grade, presence of ITB, lymphovascular space invasion (LVSI), mitotic index and lymph node metastasis. ITB was found in 65 (77.4%) of the 84 SOCs. The presence of ITB was signicantly correlated with a higher level of CA125, an advanced 2014 FIGO stage, a worse architectural grade, and the presence of LVSI. The median progression-free survival (PFS) was 20 months in women with SOC with ITB and 38 months in women with SOC without ITB (P<0.01), and their median overall survival (OS) was 52 months and 72 months (P=0.05). The multivariate analysis revealed that ITB was not an independent prognostic factor. ITB is a cost-effective prognostic indicator for women with SOC and ITB in ovarian tumor tissue is considered a useful histological biomarker of the progression of SOCs. serous ovarian carcinomas (SOCs) 3 , cases assigned to the low-grade category were characterized by the presence of mild to moderate nuclear atypia. As a secondary feature, they tended to show up to 12 mitoses/10 high-power elds (HPFs), whereas those in the high-grade category had marked nuclear atypia and as a secondary feature more than 12 mitoses/10 HPFs. Recent molecular genetic studies have shown that low-grade serous carcinomas demonstrate mutations in KRAS or BRAF and few chromosomal abnormalities. In contrast, high-grade serous carcinomas harbor mutations in TP53 and are, chromosomally, highly unstable 5 . Immunohistochemical algorithms and prediction models have been proposed for the ve major histologic types of epithelial ovarian carcinomas 6 . Based on a dualistic model of epithelial ovarian carcinogenesis, the importance of the molecular classication and origin of ovarian carcinomas was reported 5 . ovarian specimens were taken from 71 patients at primary surgery and 13 after neoadjuvant chemotherapy (NAC). All cases with available histopathologic slides were included in the present study, except for two women with complete regression of SOC after NAC. Hematoxylin and Eosin (H&E)-stained slides of the SOCs were cut from a median of 6 (range, 2–18) tissue blocks per case.


Introduction
Many authors have examined the prognostic signi cance of histopathologic features for invasive ovarian carcinomas. Although Shimizu and Silverberg 1 proposed a three-tier grading system, which was based on the dominant architectural pattern, degree of nuclear pleomorphism, and mitotic index, for all ovarian epithelial malignancies, the international collaboration on cancer reporting (ICCR) 2 recommends that different grading systems should be used for different morphological subtypes.
Ovarian high-and low-grade serous carcinoma, was de ned by Malpica et al. 3 in 2004 and subsequently adopted in the 2014 WHO Classi cation of Tumours of Female Reproductive Organs 4 . Using this two-tier grading system for serous ovarian carcinomas (SOCs) 3 , cases assigned to the low-grade category were characterized by the presence of mild to moderate nuclear atypia. As a secondary feature, they tended to show up to 12 mitoses/10 high-power elds (HPFs), whereas those in the high-grade category had marked nuclear atypia and as a secondary feature more than 12 mitoses/10 HPFs. Recent molecular genetic studies have shown that low-grade serous carcinomas demonstrate mutations in KRAS or BRAF and few chromosomal abnormalities. In contrast, high-grade serous carcinomas harbor mutations in TP53 and are, chromosomally, highly unstable 5 . Immunohistochemical algorithms and prediction models have been proposed for the ve major histologic types of epithelial ovarian carcinomas 6 . Based on a dualistic model of epithelial ovarian carcinogenesis, the importance of the molecular classi cation and origin of ovarian carcinomas was reported 5 .
Tumor budding (TB) and its association with disease progression in patients with various solid cancers was rst described by Imai in the 1950s 7 . TB is a morphological phenotype representing a destructive stromal invasion and was included as an additional prognostic factor for colorectal carcinoma in the Page 3/11 2019 WHO Classi cation of Digestive System Tumours 8 . The term intra-tumoral budding (ITB), which is TB found within the main tumor body, was introduced to distinguish this form of budding from the classic peritumoral budding 9 . The reported interobserver variability for assessing TB has ranged from moderate to very good, depending on the study 10 .
The prognostic signi cance of destructive invasive implants in extra-ovarian tissues has been well studied in serous borderline tumors (SBTs) of the ovary 11 . Such destructive invasion was suggested to be associated with a poor prognosis in patients with stage I endometrioid and mucinous ovarian carcinomas 12 . TB is an independent, unfavorable, prognostic factor for patients with early-stage cervical cancer following radical surgery 13 . However, ITB has not been well described in SOCs.
The present study evaluated the prognostic signi cance of ITB in SOCs, describing the correlation between ITB and other conventional histologic parameters.

Clinicopathologic features
The clinicopathologic data of 84 SOCs are shown in Table 1. ITB was found in 65 (77.4%) of 84 SOCs and presence of LVSI (P < 0.01) in women with SOC with ITB were signi cantly higher than those in women with SOC without ITB (Fig. 2). The differences in nuclear grade and lymph node metastasis between these two groups did not reach statistical signi cance (p = 0.06). The levels of CA125 before initial treatment and after 3cycles of cisplatin-based chemotherapy were measured in 84 and 75 women with SOC, respectively. The CA125 level after 3 cycles of chemotherapy was shown to be normal in all 16 women with SOC without ITB and 45 (76.2%) of 59 women with SOC with ITB. All 13 women with SOC who had undergone NAC showed ITB. Nine of these 13 SOCs showed a mitotic index ≤ 12 mitoses/10HPFs (SOCs with NAC vs. without NAC, P < 0.01). Poly (ADP-ribose) polymerase (PARP) inhibitor and bevacizumab were administered to three and three women with SOC without ITB, and ve and eight women with SOC with ITB, respectively.
SOCs with mild to moderate nuclear atypia are shown in Table 2. Three SOCs without ITB and an SOC with ITB (5.6%) showed histologic features of mild to moderate nuclear atypia and ≤ 12 mitoses/10 HPFs among 71 SOCs without NAC. The prognosis Figure 3 shows the differential Kaplan-Meier PFS and OS curves of women with SOC for the presence or absence of ITB. The median PFS was 20 months in 61 women with SOC with ITB (range, 2 to 156 months) and 38 months in 17 women with SOC without ITB (range, 12 to 148 months), and their median OS was 52 months (range, 6 to 189 months) and 72 months (range, 14 to 148 months).
Among the 13 patients treated with NAC, the prognostic statuses were dead of disease in 12 patients and alive with disease in 1 with median follow-up of 46 (range, 12 to 63) months.

Discussion
When we examined the presence of ITB as a maker of destructive stromal invasion of SOCs, it was found to be a signi cant poor prognostic indicator for the PFS of women with SOC, but did not reach statistical signi cance for the OS of women with SOC in the univariate analysis. In a multivariate analysis, ITB was not an independent prognostic indicator. The prognostic signi cance of ITB was considered to be strongly affected by the 2014 FIGO stage. The large size studies strati ed by the 2014 FIGO stage may be recommended.
It is well-known that TBs are part of the tumor microenvironment and are associated with epithelialmesenchymal transition (EMT) 7 . EMT is characterized by cytoskeletal rearrangements, cell motility and invasion, increased cell-associated proteolytic activity and reprogramming of the gene expression 14 .
Although the association between ITB and lymph node metastasis did not reach statistical signi cance, ITB was signi cantly associated with the incidence of LVSI in the present study. Increasing evidence has highlighted a close relationship between EMT and chemoresistance in epithelial ovarian carcinoma (EOC) 15 . In one study, the SKOV-3 EOC cells were shown to trigger both EMT and chemoresistance after treatment by carboplatin 16 . ITB was found in all SOCs after NAC, except for in two complete regression cases, and was associated with a low mitotic index of tumors and poor prognosis of patients.
The stromal-epithelial patterns of invasion in SBTs of the ovary have been subclassi ed as destructive and nondestructive. By de nition, well-differentiated serous tumors featuring destructive stromal invasion are classi ed as low-grade serous carcinomas whereas those with either no stromal invasion or stromal microinvasion are classi ed as SBTs of the ovary 17 . In a review, low-grade serous carcinoma was de ned as a serous neoplasm showing destructive invasion, mild to moderate cytologic atypia, and relatively low proliferative activity. Low-grade serous carcinoma accounts for 4.7% of SOCs and has an excellent prognosis, but long-term follow-up indicates that the prognosis for patients with stage III-IV disease is poor 18 . In the present study, 3 SOCs without ITB and 1 SOC with ITB (5.6%) showed both histologic features of mild to moderate nuclear atypia and ≤ 12 mitoses/10 HPFs in 71 SOCs without NAC.
Analyses of gene expression microarray data from The Cancer Genome Atlas (TCGA) project revealed that high-grade SOC could be classi ed as one of four gene expression subtypes: mesenchymal, immunoreactive, proliferative, or differentiated 19 . Tumors with the mesenchymal phenotype had poor prognoses, whereas those with the immunoreactive type had favorable prognoses. Murakami et al. 20 said that a feature of spindle and isolated cells with destructive stromal reaction was referred to as the mesenchymal phenotype. Tothill et al. 21 reported that a novel subtype of high-grade serous cancers re ected a mesenchymal cell type, characterized by the overexpression of N-cadherin and P-cadherin and low expression of differentiation markers, including CA125 and MUC1. A poor prognosis subtype was de ned by a reactive stroma gene expression signature, correlating with extensive desmoplasia in such samples.
In conclusion, recent studies concerning the origin and molecular pathogenesis of SOCs support the performance of prophylactic salpingo-oophorectomy in high-risk women and new molecular target therapy in women with high-grade SOC. ITB was shown to be a cost-effective prognostic indicator for women with SOC and a histopathologic biomarker of the progression of SOC. We may need to pay closer attention to the progression of SOCs

Case selection
After a review of the 318 malignant ovarian tumors found in the database of the Department of Pathology at the University of Occupational and Environmental Health Hospital between 2000 and 2017, 84 SOCs were selected. Twenty peritoneal carcinomas, de ned according to the Gynecological Oncology Group inclusionary criteria 22 , were not included in the present study. The ovarian specimens were taken from 71 patients at primary surgery and 13 after neoadjuvant chemotherapy (NAC). All cases with available histopathologic slides were included in the present study, except for two women with complete regression of SOC after NAC. Hematoxylin and Eosin (H&E)-stained slides of the SOCs were cut from a median of 6 (range, 2-18) tissue blocks per case.
Ethical approval for this study was granted by the Review Board of the University Hospital of Occupational and Environmental Health on Ethical Issues (H27-185). All methods were carried out in accordance with relevant guidelines and regulations. Informed consent was obtained from all participants.
The following clinicopathological parameters were evaluated: age at the diagnosis, CA125 level (before initial treatment and after three cycles of chemotherapy), 2014 FIGO stage, architectural grade, nuclear grade, ITB, lymphovascular space invasion (LVSI), mitotic index and lymph node metastasis while referring to previous studies 1, 3,23 . The postsurgical assessment of the interval debulking surgery was available for the 2014 FIGO stage of patients who received platinum-based NAC. Adjuvant platinumbased chemotherapy was not performed in one patient with stage IA tumor without ITB, and six patients with severe medical complication and/or advanced age, (including two with SOC without ITB and four with SOC with ITB,). These latter six patients were not included in the analysis of the patient prognosis.
According to the statements of the international tumor budding consensus conference (ITBCC) 10 , ITB was de ned as a single tumor cell or a cell cluster of up to four tumor cells at the invasive tumor front.
ITB was evaluated in the surgical specimens of the ovarian tumors to be clinically the site of the greatest tumor volume/size.
While we initially attempted to use a three-tier grading system of ITB (none, focal and diffuse) according to the recommendation of the ITBCC, there was no signi cant difference in the cumulative survival between women with SOC with focal and diffuse ITB. The SOCs were therefore ultimately divided into two groups (: those with and without ITB).

Statistical analyses
Statistical analyses were carried out using the IBM SPSS Statistics, version 27 (IBM SPSS Statistics for Windows, IBM Corporation, Armonk, NY, USA). Data for the clinicopathological factors were evaluated using the Chi-square test or Mann-Whitney U test. The progression-free survival (PFS) was de ned as the time from the date of initial treatment to the date of objective disease progression or last follow-up. The overall survival (OS) was de ned as the time from the date of initial treatment to the date of death or last follow-up. The PFS and OS curves were estimated using the Kaplan-Meier method and compared using the log rank test. Univariate and multivariate Cox proportional hazards models were used to determine the association between potential risk factors and progression as well as death from disease. Statistical signi cance was considered to exist at a value of P < 0.05.  Serous ovarian carcinoma in a 49-year-old woman. The stage IC1 tumor showed no intra-tumoral budding, architectural grade 1 (A), nuclear grade 3 and a high mitotic index (B).