Clinicopathological features and outcomes in gastric-type of HPV-independent endocervical adenocarcinomas

Background We aimed to analyze the clinicopathological features and outcomes of patients with gastric-type of HPV-independent endocervical adenocarcinoma (GAS HPVI ECA), and compare them with non-GAS HPVI ECA cases. Methods Thirty-eight GASs [including 17 minimal deviation adenocarcinoma (MDA), 21 non-MDA GAS] and 17 non-GAS HPVI ECAs were studied. Data of clinical features, pathological characteristics, treatment, and outcomes were evaluated. Results The median age of patients with GAS and non-GAS HPVI ECA was 46 and 48 years, respectively (p = 0.93). Compared with non-GAS HPVI ECAs, GAS had more common complains of vaginal watery discharge (p = 0.04). GAS cases were also associated with higher clinical stage (p = 0.036), more common in deeper cervical stromal invasion (p = 0.002) and lymphoavascular invasion (p = 0.044). GAS was associated with worse median progression-free survival (PFS) (p = 0.02) and median overall survival (OS) (p = 0.03) over patients with non-GAS HPVI ECAs. MDA had similar clinical and pathological features and prognosis compared with non-MDA GAS. Of note, serum CA19–9 levels were significantly higher in GAS than that in non-GAS HPVI ECA cases. Conclusions GAS cases were more likely to have high risk pathological factors and poorer PFS and OS compared with non-GAS HPVI ECAs. Serum CA19–9 may be helpful for diagnosis and screening in patients with GAS. Supplementary Information The online version contains supplementary material available at 10.1186/s12885-021-08792-7.

groups [10][11][12], GAS, including MDA, is the second most common subtype of ECA and the most common subtype of HPVI ECAs [13]. Although considered rare in Western countries [14,15], GAS accounts for up to 25% of all ECAs in Asian population [11,16]. Other types of HPVI ECAs are rare, and data about their clinical behavior is limited.
GASs are frequently present with an advanced stage, poor prognosis, and diverse clinical features with different subtypes [11,15]. However, these cases are rare and large clinicopathologic studies in this field are limited. Here, we conduct a relatively large retrospective analysis focused on the clinicopathological features and outcome of GASs to provide a useful reference for the diagnosis and treatment of such tumors.

Case selection
Patients with a final diagnosis of GAS and non-GAS HPVI ECA from 2014 to 2020 in our hospital were identified. Hematoxylin and eosin (H&E) and immunohistochemical (IHC) staining slides were reviewed by 2 gynecologic pathologists (F.Z. and X.Z.) in a blinded fashion and the pathologic diagnosis was confirmed. All related clinical data including age, symptoms, imaging materials, level of serum CA19-9, treatment, clinical outcome were collected from the electronic clinical information system database. All tumors were classified according to the 2020 WHO Classification of Female Genital Tumors. Patients were clinically staged using the 2018 International Federation of Gynecology and Obstetrics (FIGO) system. The results of Thinprep cytologic test (TCT), high-risk HPV (hrHPV) (tested by Aptima, Cervista or Hybrid capture 2 assay), and p16 performed as part of clinical care were recorded. Informed consent was obtained from all subjects involved in the study. IRB approval was obtained by the Ethics Committee of our hospital.

Serum CA19-9 examination
Serum CA19-9 was estimated by using an automated chemiluminescence analyzer (Shanghai Roche Diagnostic products Co., LTD., China). All assay procedures were performed based on manufacturer instructions. The normal upper limitation is 39 U/ml.

Statistical analysis
Non-normal distributed parametric variables and categorical data were separately compared by the Mann-Whitney U-test and Chi-square test in the IBM SPSS software environment (version 26.0). The Kaplan-Meier method was used to generate survival curves and the log-rank method was used for statistical testing. Survival curves and the log-rank test were both performed using the R software (version 4.1.0; www.r-project.org). Progression-free survival (PFS) was calculated from the date of diagnosis to the date of tumor recurrence, progression, or death. The overall survival (OS) was defined as the time between the date of surgery and the last date of follow-up or death from any cause. Two-sided p values were reported. p values less than 0.05 were considered statistically significant.
Clinical features of GAS and compared to non-GAS HPVI ECA The median age of patients with GAS was 46 years old (IQR: 41.8, 59.3), with no significant difference compared with that of patients with non-GAS HPVI ECA (48 years, IQR: 40.5, 59.0, p = 0.93). 27 patients with GAS had clinical symptoms, with 18/27 (66.7%) complained of irregular bleeding or contact bleeding and 9/ 27 (33.3%) experienced vaginal watery discharge. Meanwhile, 14 patients with non-GAS HPVI ECAs had clinical symptoms, in which 100% patients had irregular vaginal bleeding or contact bleeding. Vaginal watery discharge was more frequently noted in GAS cases than in non-GAS HPVI ECAs (p = 0.04). Combined pelvic examination with imaging data, although there was no significant difference in lesion size of the greatest dimension between the two groups, 17 patients (44.7%) in the GAS group had a tumor size larger than 4 cm, while only 4 patients (23.5%) in the non-GAS HPVI ECA group had a tumor size larger than 4 cm (p = 0.24). This information is illustrated inTable1.
GAS was associated with worse pathological risk factors, higher stage, and poorer prognosis compared to non-GAS HPVI ECA In total, 47 hysterectomies (34 GAS and 13 non-GAS HPVI ECAs) were available for analyzing ancillary factors. The incidence of deep stromal invasion and lymphovascular space invasion (LVSI) in GAS were significantly higher than that of non-GAS HPVI ECAs (88.2% vs 38.5%, p = 0.002; 44.1% vs 7.7%, p = 0.044, respectively). The incidence of lymph nodes metastasis in GAS was also higher than that in non-GAS HPVI ECAs, although no statistical difference was found (29.4% vs 7.7%, p = 0.23). Seen Table 1.
Among the 50 HPVI ECAs (37 GAS and 13 non-GAS HPVI ECAs) with clinical pathological staging information, 17/37 (46.0%) were staged as I-IIA and 20/37 (54.0%) were staged as IIB-IV for GAS. Meanwhile, for non-GAS HPVI ECAs, 11/13 (84.6%) were staged I-IIA and 2/13 (15.4%) was staged IIB-IV. Here we used IIA as the boundary to state which type of HPVI ECA is more likely to infiltrate into the parametrial, pelvic cavity or have distant metastasis. Patients with GAS were more likely to have an advanced clinical stage by infiltration into parametrial and remote organs compared with those of non-GAS HPVI ECAs (p = 0.036).
Complete follow-up information was available and included in a survival analysis for 55 patients with HPVI ECA. In the GAS group, 16 patients relapsed, with lesions in the pelvic cavity, vaginal stump, great omentum metastasis, intestinal metastasis, pulmonary metastasis, etc. A total of 12 patients died. In the non-GAS HPVI ECA group, 2 cases recurred and 1 case died as of the last follow-up. GAS patients showed significant reduction in the median PFS over patients in the non-GAS HPVI ECA group with hazard ratio (HR) 0.16 (95% CI: 0.03, 0.73), p = 0.02. And GAS patients   also showed significant reduction in the median OS over patients in the non-GAS HPVI ECA group with HR 0.10 (95% CI: 0.01, 0.83), p = 0.03 (Fig. 2).
Comparison of clinicopathological features and outcomes between MDA and non-MDA GAS In GAS cases, 17/38 (44.7%) were diagnosed as MDA and 21/38 (55.3%) were diagnosed as non-MDA GAS. There were no statistical differences between the two groups in age (p = 0.86), symptoms (p = 0.34), and tumor sizes (p = 0.56). Although the cytological results of TCT with NILM were more common in MDA group than that in non-MDA GAS group, no significantly difference was found (63.0% vs 20.0%, p = 0.06). Additional histologic risk factors were also evaluated, with lymph node metastasis (p = 0.95), deep stromal invasion (p = 0.43), and LVSI (p = 0.44) were all similar between MDA and non-MDA GAS (  (Fig. 3).

GAS was associated with higher level of serum CA19-9 compared to non-GAS HPVI ECA
We compared the level of serum CA19-9 among MDA, non-MDA GAS, and non-GAS HPVI ECA types. The median level of CA199 was 161.0 U/ml (IQR: 17.2, 712.14) in MDA type, 22.8 U/ml (IQR: 12.5, 176.0) in non-MDA GAS type, and 10.1 U/ml (IQR: 6.3, 40.4) in non-GAS HPVI ECA type. The level of CA199 in MDA type was not significantly higher compared with the non-MDA GAS group (p = 0.121). Compared with the non-GAS HPVI ECA group, the CA199 levels in MDA and non-MDA GAS types were all significantly higher (p = 0.001 and p = 0.024). The number of patients with abnormal CA19-9 levels (> 39 U/ml) in different groups was also analyzed. The cases with elevated CA19-9 were more in MDA group than that in the non-MDA GAS group (p = 0.022) and non-GAS HPVI ECA group (p = 0.006), while no significant difference was found between non-MDA GAS and non-GAS HPVI ECA types. Seen Table 3.

Discussion
This is a relatively large retrospective study of GASs, which were confirmed by clinicopathological characteristics including p16 and hrHPV results. In our study, GAS cases had a different constellation of clinical presentation and laboratory results compared with non-GAS HPVI ECA cases, including vaginal watery discharge and elevated serum CA19-9. In addition, GAS cases were more likely to have deep cervical stromal invasion, LVSI and an advanced stage when compared against those of non-GAS HPVI ECAs. Finally, GAS cases were more commonly found to have prognosis with poorer PFS and OS. In this series of HPVI ECA cases, GAS accounted for 69.1% of all studied cases, followed by CCC (21.8%). The prevalence of different histologic types was similar to Stolnicu et al.'s report [13], in which they studied 40 cases of HPVI ECAs. Of those 40 cases, GAS and CCC accounted for 67.3 and 20%, respectively. Stolnicu et al. [13] reported patients with non-GAS HPVI ECA tended to be older. However, the ages of different subtypes of HPVI ECA were similar in other reports [17,18]. These discrepant results may be related to the limited number of cases of these studies. In our cohort, the ages of patients with GAS or non-GAS HPVI ECAs were similar, with the median age being 46 (IQR: 43.5, 62) in MDA, 43 (IQR: 38.5, 58) in non-MDA GAS, and 48 (IQR: 40.5, 59) in non-GAS HPVI ECAs.
Unlike usual type HPVA ECAs, GAS is frequently located in the upper endocervix and present with a bulky cervix [7,8]. Because the number of such cases is relatively limited, reports about the comparison of clinical characteristics, pathological features and outcomes between GAS and non-GAS HPVI ECA are rare [11,15]. According to our findings, the clinical manifestations of GAS included vaginal watery discharge and/or bleeding, while patients with non-GAS HPVI ECA mostly complained of vaginal bleeding. Consensus guidelines for management of cervical dysplasia in the screening setting have not yet been reached to accommodate the three most widely available screening strategies: primary HPV testing, co-testing with HPV testing and cervical cytology, and cervical cytology alone [19]. This is a critical need for these guidelines because HPVI ECAs are negative for hrHPV, and cytology results become more important especially in those without abnormal appearance of cervix. According to previous reports, the positivity rate of TCT screening in ECAs is 40-50%, which is much lower than that in SCCs (above 90%) [20]. Nakamura et al. [21] reported 78% NILM of TCT were found in the GAS group. Our study indicated a similar result that TCT had a low positivity rate for HPVI ECAs (61.5% for GAS and 55.6% for non-GAS HPVI ECA).  Table 3 Comparing of levels and cases of serum CA199 among MDA, non-MDA and non-GAS Median levels and cases of MDA compared with non-MDA, ‡ Median levels and cases of MDA compared with non-GAS. ※ Median levels and cases of non-MDA compared with non-GAS Thus, some of these cases may be missed during conventional screening because of negative results from both hrHPV and cytology. As a result, the patient with HPVI ECA is frequently diagnosed at a relatively later stage. Although no significant difference was detected, the TCT results showed higher rate of NILM in MDA than the rate of NILM in non-MDA GAS (63.6% vs 20.0%, p = 0.06). This reminds us that MDA might be more prone to be missed and misdiagnosed clinically. CA19-9 is of great clinical importance in the diagnosis, treatment and prognosis of gastrointestinal malignancies, and it is closely related to disease progression [22][23][24]. However, elevated serum CA19-9 in ECAs has rarely been reported. Until now, only Nakamural et al. [21] reported that serum CA19-9 in GAS was higher than that in non-GAS HPVI ECAs. They compared the rate of cases with elevated CA19-9 levels, and found it was higher than the rate of levels among non-GAS HPVI ECAs. However, in their report, some cases with hrHPV infection were also included in the non-GAS HPVI ECAs. In our study, we found that the serum CA19-9 level of patients with MDA GAS was significantly higher than that of patients with non-GAS HPVI ECA (161.0 U/ml vs 10.1 U/ml, p = 0.001). Although no significant difference was found between MDA and non-MDA GAS (161.0 U/ml vs 22.8 U/ml, p = 0.121), we believed that there may be certain testing deviations for this is a retrospective study. While the number of cases with elevated serum CA19-9 was more in MDA than in both non-MDA GAS and non-GAS HPVI ECA (p = 0.022 and p = 0.006 respectively). It demonstrated that the elevated serum CA19-9 mainly occurred in MDA cases. Thus, it might be an effective tumor marker for the differential diagnosis of MDA, and non-MDA GAS or non-GAS HPVI ECA. As mentioned before, MDA is more prone to be missed by cytology. Taken together, CA19-9 might be an effective tumor marker for clinical diagnosis of GAS, especially for MDA.
Kojima et al. [17] demonstrated higher frequencies of destructive invasive patterns, LVSI, and advanced stage in HPVI ECAs. We further analyzed the stage and pathological features of GAS and non-GAS HPVI ECAs. In our study, GAS cases were more likely to be in the stage above IIB than those of non-GAS HPVI ECAs (54.0% vs 15.4%, p = 0.036). Karamurzin et al. [15] reported that 59% of GAS were staged over II, which was a significant difference when compared with HPVA ECA cases. A previous report showed that GAS had always been diagnosed at a more advanced stage than usual type HPVA ECAs [11]. According to our results, GAS cases were more likely to infiltrate into the parametrial and pelvic organs than non-GAS HPVI ECA cases. In addition, there were significant differences in presence of deep stromal invasion and LVSI in GAS compared with non-GAS HPVI ECAs (p = 0.002 and p = 0.044, respectively). Although no significant difference was found in lymph nodes metastasis between GAS and non-GAS HPVI ECAs, the incidence seems to be higher in GAS (29.4% vs 7.7%).
It has been reported that HPVI ECAs portend worse prognosis than HPVA ECAs, including OS, DFS, and PFS. Kojima et al. [11] demonstrated that patients with HPVI ECA (including 12 GAS and 4 MDA) had significantly decreased 5-year DFS compared with usual type HPVA ECA. In addition, GAS was associated with an increased risk of recurrence compared with non-GAS HPVI ECA. Karamurzin et al. [15] reported that disease specific survival (DSS) at 5 years was 42% for GAS compared with 91% for usual type HPVA ECAs. Few studies have focused on outcomes of GAS and non-GAS HPVI ECAs. We found that the prognosis of GAS was worse than that of non-GAS HPVI ECAs. Compared with GAS patients, patients with non-GAS had an 84% reduction in the risk of relapse (p = 0.02), and 90% reduction in the risk of mortality (p = 0.03). In our study, the most common postoperative adjuvant therapy was RT combined with CT for GAS, regardless of eligibility according to the SEDLIS criteria by NCCN. The value of adjuvant therapy after surgery needs further investigation.
There has been considerable debate about clinical outcomes of MDA since compared with HPVA ECA. Several studies had indicated its relatively aggressive nature [7]. Nishio et al. [25] reported that more than half the patients died of disease, and only three patients were alive without recurrence after 2 years of follow-up. Karamurzin et al. [15] reported forty cases of GAS including 13 of MDA and 27 of non-MDA GAS subtype and found no clinical and survival difference between MDA and non-MDA GAS. Similar to the results of Karamurzin et al., we found no differences between these two groups, including clinical complaints, tumor size, stage, lymph node metastasis, LVSI, deep stromal invasion, and survival outcomes. This reminded us although MDA was a kind of well differentiated adenocarcinoma, the prognosis was almost as poor as non-MDA GAS.

Conclusions
Screening for successful diagnosis is difficult for patients with GAS. GAS had different clinical presentation with genital watery discharge compared with non-GAS HPVI ECA cases. Comparison with those of non-GAS HPVI ECAs, GAS cases were more likely to have high risk pathological factors such as deep stromal invasion, LVSI, and advanced stage, with poorer PFS and OS. Serum CA19-9 may be helpful for diagnosis and screening in patients with GAS, especially those with MDA.