Clinical pathological and molecular features of 100 patients with gastric-type cervical adenocarcinoma

Clinical pathological and molecular features of 100 patients with gastric-type cervical adenocarcinoma | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Clinical pathological and molecular features of 100 patients with gastric-type cervical adenocarcinoma Shangshu Gao, Yan Song This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6295926/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 Jun, 2025 Read the published version in Diagnostic Pathology → Version 1 posted 9 You are reading this latest preprint version Abstract Objective: To investigate the clinicopathological and molecular features, diagnosis, and differential diagnosis of gastric-type cervical adenocarcinoma (GAS). Methods: A retrospective analysis was conducted on 100 patients diagnosed with GAS at the National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences, from January 2017 to December 2024. Clinicopathological data were collected, histological characteristics and immunohistochemical expression patterns were analyzed, and the relevant literature was reviewed. Results: The ages of the 100 patients ranged from 25-73 years, with a median age of 50 years. The most common clinical manifestations included abnormal uterine bleeding and vaginal discharge, and the majority of patients were in FIGO stages II-IV. The GAS tumor glands were irregularly branched or cystically dilated, and the cells lining the glands had eosinophilic cytoplasm, with apical mucin secretion observed and the formation of mucus lakes in the stroma. Highly differentiated GAS may be misdiagnosed as heterogeneous hyperplasia of the cervical glands because of its relatively regular glandular structure. Irregularity of glandular architecture is observed in poorly differentiated areas, with fusion and a back-to-back arrangement occurring alongside an interstitial profibrotic reaction. Immunohistochemical markers, including MUC6, P16, P53, PAX8, ER, and PR, can provide additional diagnostic clues for GAS. Intestinal markers, such as CK7, CK20, CDX2, SATB2, Villin, CA19-9, CEA, and CA125, are not substantially helpful in distinguishing GAS from pancreaticobiliary adenocarcinoma. PAX2 aids in differentiating between GAS and precancerous lesions of benign gastric-type adenopathy. Positive staining for gastric markers, such as MUC6, HIK1083, and CAIX, helps indicate gastric-type differentiation. HNF1β in combination with NapsinA aids in differentiating GAS from clear-cell carcinoma. Patients with HER2 overexpression or PD-L1 expression (CPS ≥1) may benefit from targeted therapy or immunotherapy. Next-generation sequencing (NGS) analysis of 11 cases revealed recurrent somatic mutations in critical oncogenic pathways, including TP53 (72.7%, 8/11), KRAS (45.5%, 5/11), SMAD4 (45.5%, 5/11), CDKN2A (36.4%, 4/11), PIK3CA (27.3%, 3/11), and STK11 (18.2%, 2/11). No statistically significant associations were identified between p53 mutation status and FIGO stage (p=0.32), depth of myometrial invasion (p=0.45), lymphovascular invasion (LVSI) (p=0.67), or lymph node metastasis (LNM) (p=0.89), as determined by Fisher’s exact test. Conclusions: GAS is a highly malignant, HPV-independent cervical adenocarcinoma characterized by atypical clinical symptoms and complex, varied histology. It can be accurately diagnosed based on histologic features and immunohistochemical staining. cervical cancer gastric-type cervical adenocarcinoma clinical pathological features molecular features Figures Figure 1 Figure 2 Figure 3 Background Despite advancements in screening and therapeutic strategies, endocervical adenocarcinomas (ECAs), accounting for 20–32% of all cervical malignancies, continue to pose a substantial public health challenge.( 1 , 2 ) The human papillomavirus (HPV) is well established as a primary etiological factor in ECAs. The 2020 World Health Organization (WHO) classification of female genital tumors introduced a revised framework highlighting the role of HPV in pathogenesis, classifying ECAs into two distinct categories: HPV-associated adenocarcinomas (HPVA) and HPV-independent adenocarcinomas (HPVIA).( 3 ) Among HPVIA subtypes, gastric-type endocervical adenocarcinoma (GAS) represents the most prevalent and clinically aggressive variant, exhibiting resistance to conventional therapies and a poorer prognosis than its HPV-associated counterparts.( 4 )Notably, the expanding global implementation of HPV vaccination may paradoxically increase the relative incidence of HPVIA, including GAS, highlighting the pressing need to address the diagnostic and therapeutic challenges associated with this emerging subgroup. The diagnosis of GAS presents significant challenges because of its deceptively bland histological appearance, closely resembling benign conditions such as lobular endocervical glandular hyperplasia. Furthermore, the mucin-rich cytoplasm of GASs frequently requires differentiation from metastatic gastrointestinal adenocarcinomas. Adding to these challenges, GAS demonstrates unique therapeutic resistance, necessitating customized chemotherapy regimens distinct from those used for usual-type ECAs.( 5 – 7 )The current diagnostic framework predominantly depends on histomorphological evaluation and a limited set of immunohistochemical markers (e.g., HIK1083 and MUC6); however, systematic studies integrating comprehensive immunoprofiles, molecular signatures, and clinical characteristics remain scarce. This gap in knowledge impedes the establishment of standardized diagnostic algorithms and targeted therapeutic strategies, thereby contributing to the low 3-year progression-free survival (PFS) rate of patients with GAS (44.4%).( 8 ) To overcome these limitations, a large-scale cohort study was conducted to systematically examine the clinicopathological and molecular characteristics of gastric-type adenocarcinoma (GAS). The study encompasses the following aspects: immunohistochemical profiling, including hormone receptors (ER/PR), proliferation markers (Ki-67), HPV surrogate markers (p16), lineage-specific markers (PAX2/PAX8), therapeutic targets (HER2, PD-L1), gastric-type markers (MUC6, HIK1083, CAIX), intestinal differentiation markers (CK7/CK20, CDX2, SATB2, Villin, CA19-9), and clear cell carcinoma discriminators (HNF1β, Napsin A); molecular profiling aimed at identifying recurrent genomic alterations; and clinicopathological correlations to define diagnostic criteria and prognostic predictors. By comprehensively characterizing the multidimensional features of GAS, this study aims to increase diagnostic accuracy, distinguish GAS from benign mimics and metastatic carcinomas, and identify potential therapeutic targets, thereby bridging critical gaps between translational research and clinical practice. MATERIALS AND METHODS Patients and tissue samples A retrospective cohort of 100 patients with newly diagnosed and recurrent GAS was assembled from pathological specimens obtained from the Cancer Hospital Chinese Academy of Medical Sciences between January 2017 and January 2025. The samples included colposcopic biopsies, cervical conization samples, simple hysterectomy samples, radical hysterectomy samples, and cytoreductive surgery samples. All enrolled patients had undergone ThinPrep cytologic test and high-risk HPV testing at our institution or referring hospitals before enrollment. The study cohort consisted of patients aged 25–73 years, with a mean age of 50 years. Histopathological confirmation was achieved through a blinded independent review conducted by two gynecological pathologists (S.G. and Y.G.), who examined hematoxylin‒eosin (HE) staining slides without prior knowledge of the clinical data. Tumor classification adhered to the diagnostic criteria outlined in the 2020 WHO Classification of Female Genital Tumors. Clinicopathological parameters, including patient age and clinical stage, were systematically retrieved from the electronic clinical information system database maintained by the Cancer Hospital, Chinese Academy of Medical Sciences. HE staining and immunohistochemistry All the samples were fixed in 10% neutral buffered formalin, subjected to standard dehydration protocols, and embedded in paraffin. Tissue sections, each 4 µm thick, were prepared for HE staining and immunohistochemical analysis via the Roche BenchMark ULTRA automated staining platform. The antibody panel comprised the following: ER (clone: SP1), HER2 (clone: 4B5), p16 (clone: E6H4), PR (clone: 1E2), and PD-L1 (clone: SP263) from Roche Diagnostics; CA125 (clone: OC125), CA19-9 (clone: C241:5:1:4), CK7 (clone: RN7), CDX2 (clone: EP25), CK20 (clone: EP23), CAIX (clone: Poly), Villin (clone: EP163), HNF1β (clone: OTIR2E9), PAX2 (clone: GR007), MUC6 (clone: MRD220), and HIK1083 (clone: M-GGMC-1) from Zhongshan Golden Bridge Biotechnology; Ki-67 (clone: MIB-1), PAX8 (clone: EP298), P53 (clone: DO-7), NapsinA (clone: MX015), and SATB2 (clone: OTI5H7) from Maixin Biotech; and CEA (clone: II-7), MLH1 (clone: ES05), MSH2 (clone: FE11), PMS2(clone:EP51)and MSH6 (clone: EP49) from Dako. Positivity for ER, PR, P53, Ki-67, PAX2, and PAX8 was defined by nuclear localization. P53 expression patterns were categorized as either mutant type (≥ 80% diffuse nuclear positivity, complete negativity, or cytoplasmic-only staining) or wild type (< 80% heterogeneous nuclear staining). Membrane and cytoplasmic markers, including CA125, CA19-9, Villin, and CEA, require combined localization, whereas CK7, HIK1083, MUC6, and CK20 exhibit cytoplasmic specificity, with HIK1083 positivity defined as > 5%. HER2 scoring was conducted in accordance with the College of American Pathologists (CAP) gastric carcinoma guidelines. Mismatch repair (MMR) status was determined by the nuclear loss of ≥ 1 protein (MLH1, PMS2, MSH2, MSH6) for dMMR classification, whereas intact expression was defined as pMMR, with internal positive controls mandatory. A comprehensive analysis of clinicopathological parameters, including FIGO stage, histopathological features, tumor differentiation, depth of cervical stromal invasion, LVSI, nodal and metastatic status, and postoperative management, was performed. All HE staining and immunohistochemical slides were subjected to blinded review by two pathologists to ensure diagnostic consensus. Hybrid capture-based targeted NGS Formalin-fixed paraffin-embedded (FFPE) tissue blocks were selected on the basis of corresponding HE staining slides with pathological confirmation performed by certified pathologists according to the aforementioned evaluation criteria. Genomic DNA was isolated from selected FFPE tissue blocks via the QIAamp DNA FFPE Tissue Kit (Qiagen, Germany). DNA concentration and integrity were evaluated via a Qubit 3.0 fluorometer (Thermo Fisher Scientific, USA) and verified via 1% agarose gel electrophoresis. The DNA NGS workflow involved genomic DNA fragmentation, library preparation, and sequencing on the Illumina NextSeq 550 platform (Illumina, USA). Driver mutations were detected via an internally developed molecular diagnostic management system. Variant calling was performed with a minimum variant allele frequency threshold of ≥ 2% and a sequencing depth of ≥ 1000× to ensure analytical validity. Statistical analysis Statistical analyses were conducted via SPSS 27.0 software. Categorical variables are expressed as frequency counts and percentages, and between-group comparisons were performed via the chi-square (χ²) test. A two-tailed p value < 0.05 was regarded as statistically significant. Results Clinicopathological features and histological analysis of GAS The cohort consisted of 100 GAS patients with a median age of 50 years (range: 25–73 years). The initial clinical manifestations included abnormal vaginal bleeding (85%, 85/100), vaginal discharge (60%, 60/100), and pelvic pain (20%, 20/100). Additionally, 15% (15/100) of the patients were diagnosed incidentally during routine gynecological examinations because of the detection of cervical masses. Histopathological confirmation was obtained through biopsy in 11 patients, with FIGO staging available for 89 patients: Stage I (22.6%, 15/66), Stage II (28.1%, 25/89), Stage III (28.1%, 25/89), and Stage IV (29.2%, 26/89). Notably, all the patients tested negative for high-risk HPV. (Table 1 ) Table 1 Descriptive statistics of the study cohort (n = 100) Clinicopathologic parameter N/Range Percentage(%)/Median (age) Age 25–73 50 HPV status Negative 100 100 Positive 0 0 FIGO stage I 15 22.6 II 25 28.1 III 25 28.1 IV 26 29.2 NA 11 - Deep stromal invasion Yes 57 79.2 No 15 20.8 NA 28 - Lymphovascular invasion Yes 38 51.4 No 36 48.6 NA 26 - Distant metastasis Yes 24 24 No 76 76 GAS exhibited a heterogeneous morphology characterized by irregularly sized and shaped, branching, or cystically dilated glands. Well-differentiated areas retain a relatively intact glandular architecture, frequently displaying "sawtooth" or "antler-like" luminal contours, which resemble the morphology of the gastric pyloric gland (Fig. 1A). In contrast, poorly differentiated regions exhibited glandular fusion and back-to-back arrangements (Fig. 1B), occasionally progressing into nested growth patterns (Fig. 1C) with a desmoplastic stromal response. The glandular lumina appeared distended by abundant eosinophilic or pale-staining mucin, with extracellular mucin extravasation forming stromal "mucin lakes" in select cases (Fig. 1D), necessitating differentiation from metastatic mucinous adenocarcinomas. Neoplastic cells displayed abundant eosinophilic or clear cytoplasm resembling gastric-type mucinous cells, frequently exhibiting apical snouting, which is characterized by a distinctive protrusion of apical cytoplasm with mucin secretion (Fig. 1E). While partial cellular polarity persisted in well-differentiated zones, a progressive loss of architectural organization was observed, which was correlated with decreased differentiation. Poorly differentiated foci exhibited marked nuclear atypia, including nuclear enlargement, hyperchromasia, increased nuclear‒cytoplasmic ratios, prominent nucleoli, and frequent mitotic activity (> 5 mitoses per 10 high-power fields [HPF]) (Fig. 1F). Immunohistochemical analysis of GAS Among the 100 GAS cases analyzed, key immunohistochemical findings were categorized as follows: p16 protein expression was not detected in 94.7% of the cases (71/75), whereas only 5.3% (4/75) exhibited diffuse strong positivity (Fig. 2 A). Mutant P53 expression (diffuse strong or null staining) was detected in 50% of the patients (37/74, Fig. 2 B). ER expression (8/50, 16.0%) and PR expression (9/50, 18.0%) were predominantly negative or weakly positive in focal areas. Nuclear Pax8 expression was present in 74% of the samples (37/50), whereas PAX2 expression remained consistently negative in all the samples. Intestinal differentiation markers: CK7 was strongly diffusely positive in all patients (53/53, 100%), whereas CK20 expression was low (9/48, 18.8%). CDX2 was detected in 64.4% (29/45) of the patients, whereas Villin exhibited focal positivity in 36.4% (4/11) of the patients. CEA was strongly positive in 77.8% of the patients (42/54), whereas SATB2 was completely absent (0/23). CA19-9 displayed focal staining in a single sample (1/1), and CA125 positivity was observed in 60% (3/5) of the tested samples. Gastric-type differentiation markers: MUC6 was strongly expressed in 98.4% of the patients (62/63, Fig. 2 C). HIK1083 positivity was detected in 66.7% (4/6) of the patients, with CAIX demonstrating universal expression (6/6, 100%). Clear Cell Carcinoma Discriminators: HNF1β positivity was detected in 92.9% of the patients (13/14), whereas NapsinA was consistently negative (0/18). Therapeutic biomarkers: HER2 overexpression (3+) was detected in 6.9% of the patients (2/29). PD-L1 positivity (combined positive score [CPS] ≥ 1) in immune cells was detected in 67.9% (19/28) of the patients, including one patient with a CPS = 80 (Fig. 2 D), who demonstrated only a mild-to-moderate response to immunotherapy. Tumor proliferation activity exhibited significant heterogeneity, with Ki-67 indices ranging from 10–90% (median: 50.0%). Immunohistochemical analysis of mismatch repair proteins (MLH1, MSH2, MSH6, and PMS2) was conducted in 22 cases, with intact nuclear expression noted in all the tested samples, confirming a pMMR status. Molecular characteristics NGS analysis of 11 cases revealed recurrent somatic mutations in critical oncogenic pathways, including TP53 (72.7%, 8/11), KRAS (45.5%, 5/11), SMAD4 (45.5%, 5/11), CDKN2A (36.4%, 4/11), PIK3CA (27.3%, 3/11), and STK11 (18.2%, 2/11).Notably, no HER2 amplification or microsatellite instability-high (MSI-H) status was observed within the cohort (Fig. 3 ). Survival Outcomes During a follow-up period of 3 months to 8 years, disease progression resulted in mortality in 36% (36/100) of patients. Correlation between the P53 mutation profile and clinicopathological indicators Among the 74 patients assessed for P53 mutational status, the following clinicopathological data were available: FIGO stage (69/74, 93.2%), depth of myometrial invasion (56/74, 75.7%), lymphovascular space invasion (LVSI) (59/74, 79.7%), and lymph node metastasis (LNM) (50/74, 67.6%). No statistically significant associations were identified between P53 mutation status and FIGO stage (p = 0.32), depth of myometrial invasion (p = 0.45), LVSI (p = 0.67), or LNM (p = 0.89), as determined by Fisher’s exact test (Table 2 ). Table 2 Correlations between the P53 mutation profile and clinicopathological indicators P53 P value Mutant-type Wild-type High FIGO stage(III, IV) + 21 19 0.949 - 15 14 Deep stromal invasion + 24 23 0.716 - 4 5 lymphovascular space invasion (LVSI) + 17 12 0.240 - 13 17 lymph node metastasis(LNM) + 11 9 0.729 - 15 15 Discussion In the 2020 WHO classification of female genital tract tumors, a novel classification of endocervical adenocarcinoma (ECA) was introduced. This system classifies cervical adenocarcinoma into HPV-associated and HPV-independent subtypes, which are further categorized histologically into gastric, mesonephric, clear cell, and endometrioid variants. ( 3 )This classification emphasizes the role of HPV in the pathogenesis of cervical adenocarcinoma. Among HPV-independent ECAs, gastric-type adenocarcinoma of the cervix (GAS) represents the most prevalent subtype. The atypical pathological characteristics and clinical manifestations of GAS pose significant challenges for early diagnosis. In this study, the median age of patients with GAS was 50 years, with ages ranging from 25–73 years. Although abnormal vaginal bleeding (85%) and discharge (60%) were identified as the predominant symptoms, their nonspecific nature frequently results in misdiagnosis. Therefore, a comprehensive evaluation that integrates imaging studies (such as MRI) with histopathological characteristics is crucial for an accurate diagnosis. In this case series, 57.2% of patients were diagnosed with stage III-IV disease, which is consistent with the documented high invasiveness of GAS.( 4 , 9 ) The histological features of GAS observed via HE staining are highly indicative. The tumor glands exhibit irregular branching or cystic dilation, and the lining cells display abundant eosinophilic cytoplasm along with evidence of apical mucus secretion. The presence of mucin lakes in the stroma and significant nuclear atypia, characterized by prominent hyperchromasia and nucleoli, serve as key distinguishing features from benign lesions.( 5 ) However, well-differentiated GAS may be misinterpreted as cervical adenosis because of its relatively regular glandular structures. In such cases, attention should be given to the pattern of stromal invasion, such as desmoplastic reactions or the “pushing” growth of glands. Poorly differentiated areas manifest as solid nests with frequent mitotic activity (> 10/10 HPF). Previous studies have demonstrated that the grading of GAS lacks prognostic value.( 6 , 7 ) In differential diagnosis, conventional cervical adenocarcinoma is characterized by a more regular glandular arrangement, a lack of gastric-type mucin secretion, and diffuse positivity for p16. In contrast, metastatic gastric adenocarcinoma can be differentiated via the use of immunohistochemical markers such as PAX8 and CDX2. Although HE morphology remains the cornerstone of diagnosis, the integration of molecular features (such as TP53 mutations) and gastric-type markers (such as MUC6) can significantly increase diagnostic accuracy, thereby preventing underdiagnosis or overdiagnosis. The majority of GAS cases do not express p16, with only 5.3% (4/75) showing positive expression (all four with diffuse positivity). p16 is a cyclin-dependent kinase-4 inhibitor that is overexpressed in the presence of oncogenic HPV. ( 8 )GAS is unrelated to HPV infection. Therefore, the fact that 94.7% of GAS cases lack p16 expression suggests that p16 is highly sensitive for HPVA. p16 staining can differentiate between gastric adenocarcinoma (negative or focally positive) and HPV-associated adenocarcinoma (diffusely positive). In this study, four patients with gastric adenocarcinoma exhibiting diffuse p16 positivity, a characteristic typically associated with HPV-associated tumors, underwent HPV testing and were all found to be HPV-negative. Therefore, diffuse p16 expression in cervical adenocarcinoma does not necessarily indicate the presence of oncogenic HPV; it may result from an unknown mechanism causing abnormalities in the Rb pathway.( 10 ) Moreover, p16 staining can serve as a valuable tool for distinguishing between HPVIA and HPVA. Additionally, abnormal P53 staining was observed in 50% (37/74) of the GAS patients, suggesting a mutated state of the TP53 gene. This observation is relatively rare in other cancer types and occurs at a markedly higher frequency than in related malignancies. In the majority of cases, ER and progesterone receptor PR expression was weakly positive or focal. In contrast, a significant proportion of endometrial cancers with mucinous differentiation exhibit positive ER and PR staining. Consequently, the assessment of ER and PR staining may serve as a valuable diagnostic tool for distinguishing between these two malignancies. In various types of tumors, mutations in the TP53 gene are typically linked to more aggressive biological behavior and a poorer prognosis.( 11 ) Significant prognostic factors, including FIGO stage, LNM and LVSI, have been identified. ( 12 )In our study, TP53 mutations were detected in approximately 50% of the patients; however, no statistically significant associations were identified between TP53 mutation status and either FIGO stage or other pathological risk factors. These findings suggest that, in GAS, the TP53 mutation status may not be significantly correlated with prognostic stratification. In GAS, Pax8 was positive in 74% of cases (37/50). The expression of Pax8 is observed in normal thyroid, renal, and Müllerian epithelial cells, as well as in the majority of cancers originating from these organs.( 13 ) Within the female genital system, Pax8 is detected in most cancers arising from the ovary, fallopian tube, endometrium, and cervical adenocarcinoma. Notably, Pax8 expression is higher in endometrioid adenocarcinoma and nonmucinous ovarian adenocarcinoma than in cervical adenocarcinoma. However, it is typically negative in primary ovarian mucinous adenocarcinoma, showing at most weak focal immunoreactivity.( 14 ) In the vast majority of gastrointestinal adenocarcinomas, including those of the pancreas and biliary tract, Pax8 is negative.( 13 ) Therefore, Pax8 can serve as a valuable marker for distinguishing between adenocarcinomas of female genital origin and those of gastrointestinal origin. Nonetheless, the absence of Pax8 staining does not provide information regarding the primary tumor site. Research has shown that PAX2 expression is common in nonneoplastic epithelial cells of the female genital tract but is often absent in various gynecologic adenocarcinomas and their precursor lesions, including cervical adenocarcinoma in situ and typical HPV-associated cervical adenocarcinomas.( 15 – 17 ) Our immunohistochemical analysis indicated that PAX2 was not expressed in any of the GAS cases examined. These results suggest that PAX2 could serve as a valuable marker for distinguishing between GAS, its precursor lesions, and benign gastric-type adenomatous growth. In our study, all the GAS samples were positive for CK7 and CA19-9, with CK7 typically showing diffuse and strong positivity. The positivity rates for CK20 and CDX2 were 18.8% (9/48) and 64.4% (29/45), respectively, with immunoreactivity ranging from focal to diffuse in some cases. The positivity rate for CEA was 77.8% (42/54), with the majority of patients exhibiting moderate positivity. For CA125, the positivity rate was 60%. In our series, SATB2 was negative in all patients (23/23), whereas Villin was positive in 36.4% (4/11) of the patients. CK7 staining is nearly universally positive in adenocarcinomas. In the majority of common HPV-associated cervical adenocarcinomas, CK7 is positive, whereas CK20 and the intestinal transcription factor CDX2 are negative. However, CK20 and CDX2 may be positive in tumors that exhibit intestinal differentiation. SATB2 is more specific for tumors of intestinal origin; however, its sensitivity for gastrointestinal tumors is suboptimal. In contrast, CDX2 and Villin exhibit increased sensitivity for tumors of gastrointestinal origin but lack specificity. The immunohistochemical profile of primary pancreatic or biliary tract cancers typically includes positivity for CK7, CEA, CA19-9, and CA125, whereas CK20 and CDX2 may be either positive or negative. Consequently, these intestinal markers may not offer significant utility in distinguishing GASs from adenocarcinomas of pancreaticobiliary origin. Positive staining for gastric markers, such as MUC6 and HIK1083, plays a crucial role in the classification of these tumors as the gastric type.( 5 , 18 , 19 ) Like MUC6, HIK1083 is expressed in the pancreaticobiliary tract. Furthermore, the gastric marker CAIX has been found to be positive in conventional HPV-associated cervical adenocarcinoma in situ, adenocarcinoma, GAS, and lobular endocervical glandular hyperplasia. ( 20 , 21 )In our study, MUC6 positivity was detected in 98.4% of the samples, which exhibited staining patterns ranging from focal to diffuse, with only one sample testing negative. HIK1083 staining was positive in 66.7% (4/6) of the patients. CAIX staining was positive in all six cases evaluated. When used as a marker for GAS, HIK1083 has moderate overall diagnostic accuracy and is characterized by high specificity but low sensitivity. In contrast, MUC6 exhibits good sensitivity but limited specificity.( 22 ) A comprehensive understanding of the strengths and limitations of each marker, along with their combined utility, is essential for improving diagnostic accuracy in clinical practice. The characteristic eosinophilic and clear cytoplasm of GAS requires differentiation from cervical clear cell carcinoma. HNF1β is recognized as a sensitive marker for ovarian and endometrial clear cell carcinoma; however, it lacks specificity.( 20 , 23 ) A prior study reported positive nuclear staining for HNF1β in 42 of 56 cases (75%) of cervical adenocarcinoma; however, the morphological subtypes were not specified. ( 24 )Another study demonstrated that 13 of 14 GAS cases (93%) exhibited positive staining for HNF1β. ( 23 )These findings are consistent with our results, suggesting that HNF1β lacks utility in differentiating gastric-type adenocarcinoma from clear cell carcinoma. NapsinA has recently been identified as a valuable marker for gynecological clear cell carcinoma, demonstrating greater specificity than HNF1β. In our study, none of the 18 samples stained with NapsinA were positive, indicating its potential utility in distinguishing these two entities. In our study, HER2 overexpression (3+) was identified in 2 of 29 patients (6.9%), a prevalence notably lower than that reported in gastric adenocarcinoma (approximately 20–30%).( 25 ) Notably, TP53 mutations were absent in HER2-positive patients, implying that HER2 may contribute to tumor progression independently of P53 pathway dysregulation, although the underlying mechanisms warrant further investigation. The heterogeneous expression of HER2, such as focal membranous staining, may impede the efficacy of targeted therapies, mirroring the challenges associated with HER2 heterogeneity in gastric cancer. ( 26 )Future investigations should assess the clonality of HER2 via multiregional sequencing and examine the therapeutic potential of combining HER2-targeted treatments with PI3K/mTOR inhibitors. The PD-L1 positivity rate in immune cells (CPS ≥ 1) was 67.9% (19/28). PD-L1-positive patients may respond to immunotherapy, particularly when immunotherapy is combined with radiotherapy or antiangiogenic agents such as bevacizumab. ( 27 )However, gastric-type adenocarcinoma (GAS) is characterized predominantly by an immune-cold phenotype. Consistent with the sequencing results and immunohistochemical findings, all patients were microsatellite stable (MSS) with a low tumor mutational burden (TMB; mean 2/Mb). A patient with PD-L1 expression (CPS = 80) exhibited only a mild-to-moderate response to immunotherapy. Thus, further validation of the predictive value of biomarkers and treatment efficacy is needed. In this study, genomic analysis revealed a distinct mutational landscape in GAS. The most frequently mutated genes were TP53 (8/11, 72.7%), KRAS (5/11, 45.5%), SMAD4 (5/11, 45.5%), CDKN2A (4/11, 36.4%), PIK3CA (3/11, 27.3%), and STK11 (2/11, 18.2%). These genes are predominantly involved in key biological processes, including signal transduction pathways, cell cycle regulation networks, and epithelial‒mesenchymal transition. Notably, compared with HPV-associated cervical adenocarcinoma (UEA), endometrial cancer, and cervical squamous cell carcinoma patients, the GAS patients in this study presented mutations in TP53, KRAS, CDKN2A, and PIK3CA. The frequencies of TP53 and KRAS mutations were significantly greater than those reported in the aforementioned three malignancies. Importantly, inactivation of the clinically relevant PTEN gene and mutations in the POLE gene, which are frequently identified in endometrial cancer, were not observed in this cohort. These molecular pathological differences may offer a potential explanation for the distinct aggressive phenotype of GAS. Furthermore, the high-frequency mutational profile of GAS genes identified in the present study, including KRAS, TP53, SMAD4, and CDKN2A, demonstrated significant molecular homology with the core driver genes of pancreatic cancer. These genes are among the four genes most frequently mutated in pancreatic cancer. Additional analysis revealed partial overlap in the mutation frequencies of TP53 (81.8%) and BRCA2 (9.1%) with the molecular characteristics of cholangiocarcinoma, in which these genes ranked among the ten most frequently mutated genes. Notably, the presence of STK11-inactivating mutations (18.2%) in GAS further supports a molecular pathological association between GAS and tumors associated with Peutz–Jeghers syndrome.( 28 ) Therefore, the molecular landscape of gastric-type adenocarcinoma (GAS) is markedly distinct from that of other tumors arising in the female genital tract. Nevertheless, GAS shares certain molecular similarities with gastric and pancreaticobiliary adenocarcinomas. This molecular overlap may provide insights into the aggressive nature of GAS and reinforce its distinct pathogenesis. Notably, none of the patients analyzed in this study exhibited HER2 amplification or a microsatellite instability-high (MSI-H) status. These findings suggest that GAS may exhibit limited sensitivity to trastuzumab or immune checkpoint inhibitors. Thus, further investigations into personalized treatment strategies based on molecular subtyping are essential for optimizing therapeutic approaches for GAS. This study constitutes the most extensive clinical and pathological investigation of GAS to date, encompassing 100 cases, a scale that markedly surpasses those reported in previous literature.( 29 )The novelty of this study is primarily reflected in two key aspects. First, it offers the first comprehensive analysis of the immunophenotypic characteristics of GAS. This analysis encompasses markers of gastric differentiation (MUC6, HIK1083, CAIX), intestinal differentiation (CK20, CDX2, SATB2), differentiation from clear cell carcinoma (HNF1β, Napsin A), mismatch repair protein expression, and treatment-related markers (HER2, PD-L1). This approach addresses the limitations of prior studies, which have focused predominantly on a limited number of markers.( 22 , 30 ) This study independently investigated the immunohistochemical characteristics and molecular landscape of GAS rather than analyzing it in conjunction with other cervical adenocarcinoma subtypes. Through a systematic comparison of GAS with other related malignancies, distinct molecular differences were identified (e.g., TP53 mutations and PIK3CA-driven pathways), along with the establishment of disease-specific diagnostic markers. These findings offer compelling evidence to enhance the classification framework for precision pathology. This study has several limitations. To ensure the broad inclusion of real-world data, this study incorporated a diverse array of clinical samples, including initial biopsy samples, hysterectomy samples without lymph node dissection, and postoperative consultation cases from external institutions. Although this approach improved the comprehensiveness of the study, it resulted in limitations concerning the completeness of the data on FIGO staging and pathological risk factors. Conclusion The presence of abnormal uterine bleeding and vaginal discharge, in conjunction with a negative HPV test, should prompt suspicion for GAS. Histologically, GAS is defined by a gastric-type mucinous epithelium, where varying degrees of differentiation contribute to structural and cytological atypia. A comprehensive panel of immunohistochemical markers, including ER, PR, P53, p16, Ki-67, PAX2, HIK1083, MUC6, PAX8, and CK7, serves as a valuable tool for accurate diagnosis. At the molecular level, GAS is similar to gastric and pancreaticobiliary adenocarcinomas, particularly concerning genetic alterations such as TP53 mutations and PIK3CA-driven pathways. These shared molecular characteristics may provide insight into the aggressive nature of GAS and contribute to an understanding of its distinct pathogenesis. Abbreviations CA125 Cancer antigen 125 CA19-9 Carbohydrate antigen 19-9 CAIX Carbonic anhydrase IX CDX2 Caudal type homeobox 2 CEA Carcinoembryonic antigen CK7 Cytokeratin 7 CK20 Cytokeratin 20 CPS Combined Positive Score DNA Deoxyribonucleic acid ER Estrogen receptor ECA endocervical adenocarcinoma FFPE Formalin-fixed paraffin-embedded GAS Gastric-type cervical adenocarcinoma HE hematoxylin‒eosin HNF1β Hepatocyte nuclear factor 1 beta HPF high power field HPV human papillomavirus HPVA HPV-associated adenocarcinoma HPVIA HPV-independent adenocarcinoma LNM lymph node metastasis LVSI Lymphovascular invasion MMR Mismatch repair Muc6 Mucin 6 NGS Next generation sequencing p16 Cyclin-dependent kinase inhibitor 2A P53 Tumor protein P53 PAX2 Paired box 2 Pax8 Paired box 8 PD-L1 Programmed death-ligand 1 PFS progression-free survival PR Progesterone receptor SATB2 Special AT-rich sequence-binding protein 2 WHO World Health Organization Declarations Authors’ contributions S.G. made substantial contributions to conception and design of study, acquisition of data, drafting of the manuscript and critical revision of the manuscript. Y.S. supervised the study involved in conception and critical revision of the manuscript. All authors read, revise and gave approval of the manuscript. Funding There was no funding available for this manuscript. Data availability No datasets were generated or analysed during the current study. Ethics approval and consent to participate The National Cancer Center/Cancer Hospital Ethics Committee, Chinese Academy of Medical Sciences, and Peking Union Medical College approved this study. Consent to publish Not applicable. Competing interests The authors declare no competing interests. Author details 1 Department of Pathology, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, China References Adegoke O, Kulasingam S, Virnig B. Cervical cancer trends in the United States: a 35-year population-based analysis. Journal of women's health (2002). 2012;21(10):1031-7. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2020. CA: a cancer journal for clinicians. 2020;70(1):7-30. Höhn AK, Brambs CE, Hiller GGR, May D, Schmoeckel E, Horn L-CJGuF. 2020 WHO classification of female genital tumors. 2021;81(10):1145-53. Stolnicu S, Hoang L, Chiu D, Hanko-Bauer O, Terinte C, Pesci A, et al. Clinical Outcomes of HPV-associated and Unassociated Endocervical Adenocarcinomas Categorized by the International Endocervical Adenocarcinoma Criteria and Classification (IECC). Am J Surg Pathol. 2019;43(4):466-74. Mikami Y, McCluggage WG. Endocervical glandular lesions exhibiting gastric differentiation: an emerging spectrum of benign, premalignant, and malignant lesions. Advances in anatomic pathology. 2013;20(4):227-37. Ehmann S, Sassine D, Straubhar AM, Praiss AM, Aghajanian C, Alektiar KM, et al. Gastric-type adenocarcinoma of the cervix: Clinical outcomes and genomic drivers. Gynecologic oncology. 2022;167(3):458-66. Karamurzin YS, Kiyokawa T, Parkash V, Jotwani AR, Patel P, Pike MC, et al. Gastric-type Endocervical Adenocarcinoma: An Aggressive Tumor With Unusual Metastatic Patterns and Poor Prognosis. Am J Surg Pathol. 2015;39(11):1449-57. O'Neill CJ, McCluggage WG. p16 expression in the female genital tract and its value in diagnosis. Advances in anatomic pathology. 2006;13(1):8-15. Nishio S, Mikami Y, Tokunaga H, Yaegashi N, Satoh T, Saito M, et al. Analysis of gastric-type mucinous carcinoma of the uterine cervix - An aggressive tumor with a poor prognosis: A multi-institutional study. Gynecologic oncology. 2019;153(1):13-9. Houghton O, Jamison J, Wilson R, Carson J, McCluggage WG. p16 Immunoreactivity in unusual types of cervical adenocarcinoma does not reflect human papillomavirus infection. Histopathology. 2010;57(3):342-50. Li VD, Li KH, Li JT. TP53 mutations as potential prognostic markers for specific cancers: analysis of data from The Cancer Genome Atlas and the International Agency for Research on Cancer TP53 Database. Journal of cancer research and clinical oncology. 2019;145(3):625-36. Kamijo K, Miyamoto T, Oshima S, Asaka S, Shinagawa M, Sato Y, et al. Extensive Pathologic Invasion and Prognostic Implication of Gastric-Type Cervical Adenocarcinoma: A Comparative Analysis With Human Papillomavirus-Associated Adenocarcinoma. Am J Surg Pathol. 2025. Laury AR, Perets R, Piao H, Krane JF, Barletta JA, French C, et al. A comprehensive analysis of PAX8 expression in human epithelial tumors. Am J Surg Pathol. 2011;35(6):816-26. Yemelyanova A, Gown AM, Wu LS, Holmes BJ, Ronnett BM, Vang R. PAX8 expression in uterine adenocarcinomas and mesonephric proliferations. International journal of gynecological pathology : official journal of the International Society of Gynecological Pathologists. 2014;33(5):492-9. Ordóñez NG. Value of PAX2 immunostaining in tumor diagnosis: a review and update. Advances in anatomic pathology. 2012;19(6):401-9. Shukla A, Thomas D, Roh MH. PAX8 and PAX2 expression in endocervical adenocarcinoma in situ and high-grade squamous dysplasia. International journal of gynecological pathology : official journal of the International Society of Gynecological Pathologists. 2013;32(1):116-21. Rabban JT, McAlhany S, Lerwill MF, Grenert JP, Zaloudek CJ. PAX2 distinguishes benign mesonephric and mullerian glandular lesions of the cervix from endocervical adenocarcinoma, including minimal deviation adenocarcinoma. Am J Surg Pathol. 2010;34(2):137-46. Kojima A, Mikami Y, Sudo T, Yamaguchi S, Kusanagi Y, Ito M, et al. Gastric morphology and immunophenotype predict poor outcome in mucinous adenocarcinoma of the uterine cervix. Am J Surg Pathol. 2007;31(5):664-72. McCluggage WG. Recent Developments in Non-HPV-related Adenocarcinomas of the Lower Female Genital Tract and Their Precursors. Advances in anatomic pathology. 2016;23(1):58-69. Mikami Y, Minamiguchi S, Teramoto N, Nagura M, Haga H, Konishi I. Carbonic anhydrase type IX expression in lobular endocervical glandular hyperplasia and gastric-type adenocarcinoma of the uterine cervix. Pathology, research and practice. 2013;209(3):173-8. Liao SY, Rodgers WH, Kauderer J, Darcy KM, Carter R, Susumu N, et al. Endocervical glandular neoplasia associated with lobular endocervical glandular hyperplasia is HPV-independent and correlates with carbonic anhydrase-IX expression: a Gynaecological Oncology Group Study. British journal of cancer. 2013;108(3):613-20. Fulgione C, Raffone A, Travaglino A, Arciuolo D, Santoro A, Cianfrini F, et al. Diagnostic accuracy of HIK1083 and MUC6 as immunohistochemical markers of endocervical gastric-type adenocarcinoma: A systematic review and meta-analysis. Pathology, research and practice. 2023;241:154261. Carleton C, Hoang L, Sah S, Kiyokawa T, Karamurzin YS, Talia KL, et al. A Detailed Immunohistochemical Analysis of a Large Series of Cervical and Vaginal Gastric-type Adenocarcinomas. Am J Surg Pathol. 2016;40(5):636-44. Němejcová K, Cibula D, Dundr P. Expression of HNF-1β in cervical carcinomas: an immunohistochemical study of 155 cases. Diagnostic pathology. 2015;10:8. Vakiani E. HER2 testing in gastric and gastroesophageal adenocarcinomas. Advances in anatomic pathology. 2015;22(3):194-201. Ieni A, Cardia R, Lentini M, Tuccari G. Intratumoral HER2 heterogeneity in early gastric carcinomas: potential bias in therapeutic management. Virchows Archiv : an international journal of pathology. 2019;474(3):401-2. Colombo N, Dubot C, Lorusso D, Caceres MV, Hasegawa K, Shapira-Frommer R, et al. Pembrolizumab for Persistent, Recurrent, or Metastatic Cervical Cancer. 2021;385(20):1856-67. Beggs AD, Latchford AR, Vasen HF, Moslein G, Alonso A, Aretz S, et al. Peutz-Jeghers syndrome: a systematic review and recommendations for management. Gut. 2010;59(7):975-86. Yang J, Peng Y, Ding Y, Liu Y, Wang Y, Liu Y, et al. The Clinicopathological and Molecular Characteristics of Endocervical Gastric-Type Adenocarcinoma and the Use of Claudin18.2 as a Potential Therapeutic Target. Mod Pathol. 2024;37(10):100569. Lu S, Shen D, Zhao Y, Kang N, Wang X. Primary endocervical gastric-type adenocarcinoma: a clinicopathologic and immunohistochemical analysis of 23 cases. Diagnostic pathology. 2019;14(1):72. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 10 Jun, 2025 Read the published version in Diagnostic Pathology → Version 1 posted Editorial decision: Revision requested 30 Apr, 2025 Reviews received at journal 26 Apr, 2025 Reviews received at journal 13 Apr, 2025 Reviewers agreed at journal 05 Apr, 2025 Reviewers agreed at journal 02 Apr, 2025 Reviewers invited by journal 02 Apr, 2025 Editor assigned by journal 25 Mar, 2025 Submission checks completed at journal 25 Mar, 2025 First submitted to journal 24 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6295926","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":442394034,"identity":"d8acf922-8b44-49ed-ab90-cd46e4885de3","order_by":0,"name":"Shangshu Gao","email":"","orcid":"","institution":"National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College","correspondingAuthor":false,"prefix":"","firstName":"Shangshu","middleName":"","lastName":"Gao","suffix":""},{"id":442394036,"identity":"7ae9ff81-ef2b-4bdf-9075-41a8cb752a62","order_by":1,"name":"Yan Song","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9UlEQVRIie3PMUvDQBjG8TsCl+Wyv5iS+woXXqhCv0yyZAqCuHQoeiXQLNX5BO3H6Bw46HQ4d3CJARcd6iKZpNTN5ZJR8H7bwfOH9wjxvD8tXC67fg6JGJ9wY5DYC0zV6ASKIqareU6agaGo78w7X7wk56qU8dUGMqqC9nXvSKR9LmZ894aTxkp82MJlSBhi6UqgnCJnJtd0LbNoC9dUcRa7EqFPybe51QGXTfQIuWoGErIvsYtWJgPG8ipSIxJp7ZQ+3ZtU88BQvQNMq4G/iHqNh48vI0C0dX9Y3CQirNrOeRgh7Iz/egfu+c/ksx8eeZ7n/WdHUFhMRbsEteMAAAAASUVORK5CYII=","orcid":"","institution":"National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College","correspondingAuthor":true,"prefix":"","firstName":"Yan","middleName":"","lastName":"Song","suffix":""}],"badges":[],"createdAt":"2025-03-24 13:38:37","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6295926/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6295926/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13000-025-01666-7","type":"published","date":"2025-06-10T15:57:01+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82055649,"identity":"b3982543-d102-4c7c-8b9e-e31379204068","added_by":"auto","created_at":"2025-05-06 10:25:15","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":118424,"visible":true,"origin":"","legend":"\u003cp\u003eHistopathological features of gastric adenocarcinoma of the cervix (GAS)\u003c/p\u003e\n\u003cp\u003e(1A) Well-differentiated areas exhibit relatively intact glandular structures, with lumens displaying characteristic \"serrated\" or \"antler-like\" branching, resembling pyloric glands of the stomach (×200 magnification).\u003c/p\u003e\n\u003cp\u003e(1B) Poorly differentiated regions demonstrate glandular fusion and a back-to-back arrangement of glands (×200 magnification).\u003c/p\u003e\n\u003cp\u003e(1C) Nested architectural patterns may be observed, often accompanied by a desmoplastic stromal reaction (×200 magnification).\u003c/p\u003e\n\u003cp\u003e(1D) Glandular lumens contain abundant eosinophilic or lightly stained mucin. In certain cases, mucin extravasation into the stroma results in \"mucin lakes\" (×100 magnification).\u003c/p\u003e\n\u003cp\u003e(1E) Tumor cells display abundant eosinophilic or clear cytoplasm, frequently exhibiting an \"apical mucin secretion\" pattern (×200 magnification).\u003c/p\u003e\n\u003cp\u003e(1F) Poorly differentiated areas reveal marked nuclear atypia, characterized by enlarged, hyperchromatic nuclei, an increased nuclear‒cytoplasmic ratio, prominent nucleoli, and frequent mitotic figures (×200 magnification).\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6295926/v1/ea29fbbc6d817f094a68568b.jpg"},{"id":82055648,"identity":"3df8030f-d67d-4374-a821-1b7eb3189708","added_by":"auto","created_at":"2025-05-06 10:25:15","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":87849,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemical features of gastric adenocarcinoma of the cervix (GAS)\u003c/p\u003e\n\u003cp\u003e(2A) Negative immunohistochemical staining for p16, indicative of HPV-independent tumorigenesis (×40 magnification).\u003c/p\u003e\n\u003cp\u003e(2B) Aberrant P53 expression was consistent with a missense mutation pattern (×100 magnification).\u003c/p\u003e\n\u003cp\u003e(2C) Strong diffuse positivity for MUC6, supporting gastric differentiation (×100 magnification).\u003c/p\u003e\n\u003cp\u003e(2D) PD-L1 expression with a high combined positive score (CPS) of 80, suggesting potential sensitivity to immunotherapy (×100 magnification).\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6295926/v1/618fa6cb8bbeca59fdaa17e6.jpg"},{"id":82055646,"identity":"a359a3ae-ac89-4205-8636-838c1ff5675f","added_by":"auto","created_at":"2025-05-06 10:25:15","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":40871,"visible":true,"origin":"","legend":"\u003cp\u003eSummary of the molecular features of gastric adenocarcinoma of the cervix (GAS).\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6295926/v1/4af82915139880cf74e14e7f.jpg"},{"id":84726490,"identity":"6812500a-b33f-4bca-943d-60289aef8879","added_by":"auto","created_at":"2025-06-16 16:05:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1038371,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6295926/v1/4101dd10-9dd4-4fb3-bedf-015dc183e0ff.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Clinical pathological and molecular features of 100 patients with gastric-type cervical adenocarcinoma","fulltext":[{"header":"Background","content":"\u003cp\u003eDespite advancements in screening and therapeutic strategies, endocervical adenocarcinomas (ECAs), accounting for 20\u0026ndash;32% of all cervical malignancies, continue to pose a substantial public health challenge.(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) The human papillomavirus (HPV) is well established as a primary etiological factor in ECAs. The 2020 World Health Organization (WHO) classification of female genital tumors introduced a revised framework highlighting the role of HPV in pathogenesis, classifying ECAs into two distinct categories: HPV-associated adenocarcinomas (HPVA) and HPV-independent adenocarcinomas (HPVIA).(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) Among HPVIA subtypes, gastric-type endocervical adenocarcinoma (GAS) represents the most prevalent and clinically aggressive variant, exhibiting resistance to conventional therapies and a poorer prognosis than its HPV-associated counterparts.(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e)Notably, the expanding global implementation of HPV vaccination may paradoxically increase the relative incidence of HPVIA, including GAS, highlighting the pressing need to address the diagnostic and therapeutic challenges associated with this emerging subgroup.\u003c/p\u003e \u003cp\u003eThe diagnosis of GAS presents significant challenges because of its deceptively bland histological appearance, closely resembling benign conditions such as lobular endocervical glandular hyperplasia. Furthermore, the mucin-rich cytoplasm of GASs frequently requires differentiation from metastatic gastrointestinal adenocarcinomas. Adding to these challenges, GAS demonstrates unique therapeutic resistance, necessitating customized chemotherapy regimens distinct from those used for usual-type ECAs.(\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e)The current diagnostic framework predominantly depends on histomorphological evaluation and a limited set of immunohistochemical markers (e.g., HIK1083 and MUC6); however, systematic studies integrating comprehensive immunoprofiles, molecular signatures, and clinical characteristics remain scarce. This gap in knowledge impedes the establishment of standardized diagnostic algorithms and targeted therapeutic strategies, thereby contributing to the low 3-year progression-free survival (PFS) rate of patients with GAS (44.4%).(\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eTo overcome these limitations, a large-scale cohort study was conducted to systematically examine the clinicopathological and molecular characteristics of gastric-type adenocarcinoma (GAS). The study encompasses the following aspects: immunohistochemical profiling, including hormone receptors (ER/PR), proliferation markers (Ki-67), HPV surrogate markers (p16), lineage-specific markers (PAX2/PAX8), therapeutic targets (HER2, PD-L1), gastric-type markers (MUC6, HIK1083, CAIX), intestinal differentiation markers (CK7/CK20, CDX2, SATB2, Villin, CA19-9), and clear cell carcinoma discriminators (HNF1β, Napsin A); molecular profiling aimed at identifying recurrent genomic alterations; and clinicopathological correlations to define diagnostic criteria and prognostic predictors. By comprehensively characterizing the multidimensional features of GAS, this study aims to increase diagnostic accuracy, distinguish GAS from benign mimics and metastatic carcinomas, and identify potential therapeutic targets, thereby bridging critical gaps between translational research and clinical practice.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatients and tissue samples\u003c/h2\u003e \u003cp\u003eA retrospective cohort of 100 patients with newly diagnosed and recurrent GAS was assembled from pathological specimens obtained from the Cancer Hospital Chinese Academy of Medical Sciences between January 2017 and January 2025. The samples included colposcopic biopsies, cervical conization samples, simple hysterectomy samples, radical hysterectomy samples, and cytoreductive surgery samples. All enrolled patients had undergone ThinPrep cytologic test and high-risk HPV testing at our institution or referring hospitals before enrollment. The study cohort consisted of patients aged 25\u0026ndash;73 years, with a mean age of 50 years. Histopathological confirmation was achieved through a blinded independent review conducted by two gynecological pathologists (S.G. and Y.G.), who examined hematoxylin‒eosin (HE) staining slides without prior knowledge of the clinical data. Tumor classification adhered to the diagnostic criteria outlined in the 2020 WHO Classification of Female Genital Tumors. Clinicopathological parameters, including patient age and clinical stage, were systematically retrieved from the electronic clinical information system database maintained by the Cancer Hospital, Chinese Academy of Medical Sciences.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eHE staining and immunohistochemistry\u003c/h3\u003e\n\u003cp\u003eAll the samples were fixed in 10% neutral buffered formalin, subjected to standard dehydration protocols, and embedded in paraffin. Tissue sections, each 4 \u0026micro;m thick, were prepared for HE staining and immunohistochemical analysis via the Roche BenchMark ULTRA automated staining platform. The antibody panel comprised the following: ER (clone: SP1), HER2 (clone: 4B5), p16 (clone: E6H4), PR (clone: 1E2), and PD-L1 (clone: SP263) from Roche Diagnostics; CA125 (clone: OC125), CA19-9 (clone: C241:5:1:4), CK7 (clone: RN7), CDX2 (clone: EP25), CK20 (clone: EP23), CAIX (clone: Poly), Villin (clone: EP163), HNF1β (clone: OTIR2E9), PAX2 (clone: GR007), MUC6 (clone: MRD220), and HIK1083 (clone: M-GGMC-1) from Zhongshan Golden Bridge Biotechnology; Ki-67 (clone: MIB-1), PAX8 (clone: EP298), P53 (clone: DO-7), NapsinA (clone: MX015), and SATB2 (clone: OTI5H7) from Maixin Biotech; and CEA (clone: II-7), MLH1 (clone: ES05), MSH2 (clone: FE11), PMS2(clone:EP51)and MSH6 (clone: EP49) from Dako.\u003c/p\u003e \u003cp\u003e Positivity for ER, PR, P53, Ki-67, PAX2, and PAX8 was defined by nuclear localization. P53 expression patterns were categorized as either mutant type (\u0026ge;\u0026thinsp;80% diffuse nuclear positivity, complete negativity, or cytoplasmic-only staining) or wild type (\u0026lt;\u0026thinsp;80% heterogeneous nuclear staining). Membrane and cytoplasmic markers, including CA125, CA19-9, Villin, and CEA, require combined localization, whereas CK7, HIK1083, MUC6, and CK20 exhibit cytoplasmic specificity, with HIK1083 positivity defined as \u0026gt;\u0026thinsp;5%. HER2 scoring was conducted in accordance with the College of American Pathologists (CAP) gastric carcinoma guidelines. Mismatch repair (MMR) status was determined by the nuclear loss of \u0026ge;\u0026thinsp;1 protein (MLH1, PMS2, MSH2, MSH6) for dMMR classification, whereas intact expression was defined as pMMR, with internal positive controls mandatory.\u003c/p\u003e \u003cp\u003eA comprehensive analysis of clinicopathological parameters, including FIGO stage, histopathological features, tumor differentiation, depth of cervical stromal invasion, LVSI, nodal and metastatic status, and postoperative management, was performed. All HE staining and immunohistochemical slides were subjected to blinded review by two pathologists to ensure diagnostic consensus.\u003c/p\u003e\n\u003ch3\u003eHybrid capture-based targeted NGS\u003c/h3\u003e\n\u003cp\u003eFormalin-fixed paraffin-embedded (FFPE) tissue blocks were selected on the basis of corresponding HE staining slides with pathological confirmation performed by certified pathologists according to the aforementioned evaluation criteria. Genomic DNA was isolated from selected FFPE tissue blocks via the QIAamp DNA FFPE Tissue Kit (Qiagen, Germany). DNA concentration and integrity were evaluated via a Qubit 3.0 fluorometer (Thermo Fisher Scientific, USA) and verified via 1% agarose gel electrophoresis. The DNA NGS workflow involved genomic DNA fragmentation, library preparation, and sequencing on the Illumina NextSeq 550 platform (Illumina, USA). Driver mutations were detected via an internally developed molecular diagnostic management system. Variant calling was performed with a minimum variant allele frequency threshold of \u0026ge;\u0026thinsp;2% and a sequencing depth of \u0026ge;\u0026thinsp;1000\u0026times; to ensure analytical validity.\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were conducted via SPSS 27.0 software. Categorical variables are expressed as frequency counts and percentages, and between-group comparisons were performed via the chi-square (χ\u0026sup2;) test. A two-tailed p value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was regarded as statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eClinicopathological features and histological analysis of GAS\u003c/h2\u003e \u003cp\u003eThe cohort consisted of 100 GAS patients with a median age of 50 years (range: 25\u0026ndash;73 years). The initial clinical manifestations included abnormal vaginal bleeding (85%, 85/100), vaginal discharge (60%, 60/100), and pelvic pain (20%, 20/100). Additionally, 15% (15/100) of the patients were diagnosed incidentally during routine gynecological examinations because of the detection of cervical masses. Histopathological confirmation was obtained through biopsy in 11 patients, with FIGO staging available for 89 patients: Stage I (22.6%, 15/66), Stage II (28.1%, 25/89), Stage III (28.1%, 25/89), and Stage IV (29.2%, 26/89). Notably, all the patients tested negative for high-risk HPV. (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDescriptive statistics of the study cohort (n\u0026thinsp;=\u0026thinsp;100)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eClinicopathologic parameter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN/Range\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePercentage(%)/Median (age)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25\u0026ndash;73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHPV status\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePositive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFIGO stage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e28.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e28.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e29.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDeep stromal invasion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e79.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLymphovascular invasion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e51.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e48.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDistant metastasis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e76\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eGAS exhibited a heterogeneous morphology characterized by irregularly sized and shaped, branching, or cystically dilated glands. Well-differentiated areas retain a relatively intact glandular architecture, frequently displaying \"sawtooth\" or \"antler-like\" luminal contours, which resemble the morphology of the gastric pyloric gland (Fig.\u0026nbsp;1A). In contrast, poorly differentiated regions exhibited glandular fusion and back-to-back arrangements (Fig.\u0026nbsp;1B), occasionally progressing into nested growth patterns (Fig.\u0026nbsp;1C) with a desmoplastic stromal response. The glandular lumina appeared distended by abundant eosinophilic or pale-staining mucin, with extracellular mucin extravasation forming stromal \"mucin lakes\" in select cases (Fig.\u0026nbsp;1D), necessitating differentiation from metastatic mucinous adenocarcinomas. Neoplastic cells displayed abundant eosinophilic or clear cytoplasm resembling gastric-type mucinous cells, frequently exhibiting apical snouting, which is characterized by a distinctive protrusion of apical cytoplasm with mucin secretion (Fig.\u0026nbsp;1E). While partial cellular polarity persisted in well-differentiated zones, a progressive loss of architectural organization was observed, which was correlated with decreased differentiation. Poorly differentiated foci exhibited marked nuclear atypia, including nuclear enlargement, hyperchromasia, increased nuclear‒cytoplasmic ratios, prominent nucleoli, and frequent mitotic activity (\u0026gt;\u0026thinsp;5 mitoses per 10 high-power fields [HPF]) (Fig.\u0026nbsp;1F).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eImmunohistochemical analysis of GAS\u003c/h3\u003e\n\u003cp\u003eAmong the 100 GAS cases analyzed, key immunohistochemical findings were categorized as follows: p16 protein expression was not detected in 94.7% of the cases (71/75), whereas only 5.3% (4/75) exhibited diffuse strong positivity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Mutant P53 expression (diffuse strong or null staining) was detected in 50% of the patients (37/74, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). ER expression (8/50, 16.0%) and PR expression (9/50, 18.0%) were predominantly negative or weakly positive in focal areas. Nuclear Pax8 expression was present in 74% of the samples (37/50), whereas PAX2 expression remained consistently negative in all the samples.\u003c/p\u003e\n\u003ch3\u003eIntestinal differentiation markers:\u003c/h3\u003e\n\u003cp\u003eCK7 was strongly diffusely positive in all patients (53/53, 100%), whereas CK20 expression was low (9/48, 18.8%). CDX2 was detected in 64.4% (29/45) of the patients, whereas Villin exhibited focal positivity in 36.4% (4/11) of the patients. CEA was strongly positive in 77.8% of the patients (42/54), whereas SATB2 was completely absent (0/23). CA19-9 displayed focal staining in a single sample (1/1), and CA125 positivity was observed in 60% (3/5) of the tested samples.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eGastric-type differentiation markers:\u003c/h2\u003e \u003cp\u003eMUC6 was strongly expressed in 98.4% of the patients (62/63, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). HIK1083 positivity was detected in 66.7% (4/6) of the patients, with CAIX demonstrating universal expression (6/6, 100%).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eClear Cell Carcinoma Discriminators:\u003c/h2\u003e \u003cp\u003eHNF1β positivity was detected in 92.9% of the patients (13/14), whereas NapsinA was consistently negative (0/18).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eTherapeutic biomarkers:\u003c/h2\u003e \u003cp\u003eHER2 overexpression (3+) was detected in 6.9% of the patients (2/29). PD-L1 positivity (combined positive score [CPS]\u0026thinsp;\u0026ge;\u0026thinsp;1) in immune cells was detected in 67.9% (19/28) of the patients, including one patient with a CPS\u0026thinsp;=\u0026thinsp;80 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), who demonstrated only a mild-to-moderate response to immunotherapy. Tumor proliferation activity exhibited significant heterogeneity, with Ki-67 indices ranging from 10\u0026ndash;90% (median: 50.0%). Immunohistochemical analysis of mismatch repair proteins (MLH1, MSH2, MSH6, and PMS2) was conducted in 22 cases, with intact nuclear expression noted in all the tested samples, confirming a pMMR status.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMolecular characteristics\u003c/h2\u003e \u003cp\u003eNGS analysis of 11 cases revealed recurrent somatic mutations in critical oncogenic pathways, including TP53 (72.7%, 8/11), KRAS (45.5%, 5/11), SMAD4 (45.5%, 5/11), CDKN2A (36.4%, 4/11), PIK3CA (27.3%, 3/11), and STK11 (18.2%, 2/11).Notably, no HER2 amplification or microsatellite instability-high (MSI-H) status was observed within the cohort (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eSurvival Outcomes\u003c/h2\u003e \u003cp\u003eDuring a follow-up period of 3 months to 8 years, disease progression resulted in mortality in 36% (36/100) of patients.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eCorrelation between the P53 mutation profile and clinicopathological indicators\u003c/h2\u003e \u003cp\u003eAmong the 74 patients assessed for P53 mutational status, the following clinicopathological data were available: FIGO stage (69/74, 93.2%), depth of myometrial invasion (56/74, 75.7%), lymphovascular space invasion (LVSI) (59/74, 79.7%), and lymph node metastasis (LNM) (50/74, 67.6%). No statistically significant associations were identified between P53 mutation status and FIGO stage (p\u0026thinsp;=\u0026thinsp;0.32), depth of myometrial invasion (p\u0026thinsp;=\u0026thinsp;0.45), LVSI (p\u0026thinsp;=\u0026thinsp;0.67), or LNM (p\u0026thinsp;=\u0026thinsp;0.89), as determined by Fisher\u0026rsquo;s exact test (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCorrelations between the P53 mutation profile and clinicopathological indicators\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP53\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eP value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMutant-type\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWild-type\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHigh FIGO stage(III, IV)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.949\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDeep stromal invasion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.716\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003elymphovascular space invasion (LVSI)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.240\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003elymph node metastasis(LNM)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.729\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the 2020 WHO classification of female genital tract tumors, a novel classification of endocervical adenocarcinoma (ECA) was introduced. This system classifies cervical adenocarcinoma into HPV-associated and HPV-independent subtypes, which are further categorized histologically into gastric, mesonephric, clear cell, and endometrioid variants. (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e)This classification emphasizes the role of HPV in the pathogenesis of cervical adenocarcinoma. Among HPV-independent ECAs, gastric-type adenocarcinoma of the cervix (GAS) represents the most prevalent subtype. The atypical pathological characteristics and clinical manifestations of GAS pose significant challenges for early diagnosis.\u003c/p\u003e \u003cp\u003eIn this study, the median age of patients with GAS was 50 years, with ages ranging from 25\u0026ndash;73 years. Although abnormal vaginal bleeding (85%) and discharge (60%) were identified as the predominant symptoms, their nonspecific nature frequently results in misdiagnosis. Therefore, a comprehensive evaluation that integrates imaging studies (such as MRI) with histopathological characteristics is crucial for an accurate diagnosis. In this case series, 57.2% of patients were diagnosed with stage III-IV disease, which is consistent with the documented high invasiveness of GAS.(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eThe histological features of GAS observed via HE staining are highly indicative. The tumor glands exhibit irregular branching or cystic dilation, and the lining cells display abundant eosinophilic cytoplasm along with evidence of apical mucus secretion. The presence of mucin lakes in the stroma and significant nuclear atypia, characterized by prominent hyperchromasia and nucleoli, serve as key distinguishing features from benign lesions.(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e) However, well-differentiated GAS may be misinterpreted as cervical adenosis because of its relatively regular glandular structures. In such cases, attention should be given to the pattern of stromal invasion, such as desmoplastic reactions or the \u0026ldquo;pushing\u0026rdquo; growth of glands. Poorly differentiated areas manifest as solid nests with frequent mitotic activity (\u0026gt;\u0026thinsp;10/10 HPF). Previous studies have demonstrated that the grading of GAS lacks prognostic value.(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e) In differential diagnosis, conventional cervical adenocarcinoma is characterized by a more regular glandular arrangement, a lack of gastric-type mucin secretion, and diffuse positivity for p16. In contrast, metastatic gastric adenocarcinoma can be differentiated via the use of immunohistochemical markers such as PAX8 and CDX2. Although HE morphology remains the cornerstone of diagnosis, the integration of molecular features (such as TP53 mutations) and gastric-type markers (such as MUC6) can significantly increase diagnostic accuracy, thereby preventing underdiagnosis or overdiagnosis.\u003c/p\u003e \u003cp\u003eThe majority of GAS cases do not express p16, with only 5.3% (4/75) showing positive expression (all four with diffuse positivity). p16 is a cyclin-dependent kinase-4 inhibitor that is overexpressed in the presence of oncogenic HPV. (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e)GAS is unrelated to HPV infection. Therefore, the fact that 94.7% of GAS cases lack p16 expression suggests that p16 is highly sensitive for HPVA. p16 staining can differentiate between gastric adenocarcinoma (negative or focally positive) and HPV-associated adenocarcinoma (diffusely positive). In this study, four patients with gastric adenocarcinoma exhibiting diffuse p16 positivity, a characteristic typically associated with HPV-associated tumors, underwent HPV testing and were all found to be HPV-negative. Therefore, diffuse p16 expression in cervical adenocarcinoma does not necessarily indicate the presence of oncogenic HPV; it may result from an unknown mechanism causing abnormalities in the Rb pathway.(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e) Moreover, p16 staining can serve as a valuable tool for distinguishing between HPVIA and HPVA.\u003c/p\u003e \u003cp\u003eAdditionally, abnormal P53 staining was observed in 50% (37/74) of the GAS patients, suggesting a mutated state of the TP53 gene. This observation is relatively rare in other cancer types and occurs at a markedly higher frequency than in related malignancies. In the majority of cases, ER and progesterone receptor PR expression was weakly positive or focal. In contrast, a significant proportion of endometrial cancers with mucinous differentiation exhibit positive ER and PR staining. Consequently, the assessment of ER and PR staining may serve as a valuable diagnostic tool for distinguishing between these two malignancies.\u003c/p\u003e \u003cp\u003eIn various types of tumors, mutations in the TP53 gene are typically linked to more aggressive biological behavior and a poorer prognosis.(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e) Significant prognostic factors, including FIGO stage, LNM and LVSI, have been identified. (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e)In our study, TP53 mutations were detected in approximately 50% of the patients; however, no statistically significant associations were identified between TP53 mutation status and either FIGO stage or other pathological risk factors. These findings suggest that, in GAS, the TP53 mutation status may not be significantly correlated with prognostic stratification.\u003c/p\u003e \u003cp\u003eIn GAS, Pax8 was positive in 74% of cases (37/50). The expression of Pax8 is observed in normal thyroid, renal, and M\u0026uuml;llerian epithelial cells, as well as in the majority of cancers originating from these organs.(\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e) Within the female genital system, Pax8 is detected in most cancers arising from the ovary, fallopian tube, endometrium, and cervical adenocarcinoma. Notably, Pax8 expression is higher in endometrioid adenocarcinoma and nonmucinous ovarian adenocarcinoma than in cervical adenocarcinoma. However, it is typically negative in primary ovarian mucinous adenocarcinoma, showing at most weak focal immunoreactivity.(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e) In the vast majority of gastrointestinal adenocarcinomas, including those of the pancreas and biliary tract, Pax8 is negative.(\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e) Therefore, Pax8 can serve as a valuable marker for distinguishing between adenocarcinomas of female genital origin and those of gastrointestinal origin. Nonetheless, the absence of Pax8 staining does not provide information regarding the primary tumor site.\u003c/p\u003e \u003cp\u003eResearch has shown that PAX2 expression is common in nonneoplastic epithelial cells of the female genital tract but is often absent in various gynecologic adenocarcinomas and their precursor lesions, including cervical adenocarcinoma in situ and typical HPV-associated cervical adenocarcinomas.(\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e) Our immunohistochemical analysis indicated that PAX2 was not expressed in any of the GAS cases examined. These results suggest that PAX2 could serve as a valuable marker for distinguishing between GAS, its precursor lesions, and benign gastric-type adenomatous growth.\u003c/p\u003e \u003cp\u003eIn our study, all the GAS samples were positive for CK7 and CA19-9, with CK7 typically showing diffuse and strong positivity. The positivity rates for CK20 and CDX2 were 18.8% (9/48) and 64.4% (29/45), respectively, with immunoreactivity ranging from focal to diffuse in some cases. The positivity rate for CEA was 77.8% (42/54), with the majority of patients exhibiting moderate positivity. For CA125, the positivity rate was 60%. In our series, SATB2 was negative in all patients (23/23), whereas Villin was positive in 36.4% (4/11) of the patients. CK7 staining is nearly universally positive in adenocarcinomas. In the majority of common HPV-associated cervical adenocarcinomas, CK7 is positive, whereas CK20 and the intestinal transcription factor CDX2 are negative. However, CK20 and CDX2 may be positive in tumors that exhibit intestinal differentiation. SATB2 is more specific for tumors of intestinal origin; however, its sensitivity for gastrointestinal tumors is suboptimal. In contrast, CDX2 and Villin exhibit increased sensitivity for tumors of gastrointestinal origin but lack specificity. The immunohistochemical profile of primary pancreatic or biliary tract cancers typically includes positivity for CK7, CEA, CA19-9, and CA125, whereas CK20 and CDX2 may be either positive or negative. Consequently, these intestinal markers may not offer significant utility in distinguishing GASs from adenocarcinomas of pancreaticobiliary origin.\u003c/p\u003e \u003cp\u003ePositive staining for gastric markers, such as MUC6 and HIK1083, plays a crucial role in the classification of these tumors as the gastric type.(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e) Like MUC6, HIK1083 is expressed in the pancreaticobiliary tract. Furthermore, the gastric marker CAIX has been found to be positive in conventional HPV-associated cervical adenocarcinoma in situ, adenocarcinoma, GAS, and lobular endocervical glandular hyperplasia. (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e)In our study, MUC6 positivity was detected in 98.4% of the samples, which exhibited staining patterns ranging from focal to diffuse, with only one sample testing negative. HIK1083 staining was positive in 66.7% (4/6) of the patients. CAIX staining was positive in all six cases evaluated. When used as a marker for GAS, HIK1083 has moderate overall diagnostic accuracy and is characterized by high specificity but low sensitivity. In contrast, MUC6 exhibits good sensitivity but limited specificity.(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e) A comprehensive understanding of the strengths and limitations of each marker, along with their combined utility, is essential for improving diagnostic accuracy in clinical practice.\u003c/p\u003e \u003cp\u003eThe characteristic eosinophilic and clear cytoplasm of GAS requires differentiation from cervical clear cell carcinoma. HNF1β is recognized as a sensitive marker for ovarian and endometrial clear cell carcinoma; however, it lacks specificity.(\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e) A prior study reported positive nuclear staining for HNF1β in 42 of 56 cases (75%) of cervical adenocarcinoma; however, the morphological subtypes were not specified. (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e)Another study demonstrated that 13 of 14 GAS cases (93%) exhibited positive staining for HNF1β. (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e)These findings are consistent with our results, suggesting that HNF1β lacks utility in differentiating gastric-type adenocarcinoma from clear cell carcinoma. NapsinA has recently been identified as a valuable marker for gynecological clear cell carcinoma, demonstrating greater specificity than HNF1β. In our study, none of the 18 samples stained with NapsinA were positive, indicating its potential utility in distinguishing these two entities.\u003c/p\u003e \u003cp\u003eIn our study, HER2 overexpression (3+) was identified in 2 of 29 patients (6.9%), a prevalence notably lower than that reported in gastric adenocarcinoma (approximately 20\u0026ndash;30%).(\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e) Notably, TP53 mutations were absent in HER2-positive patients, implying that HER2 may contribute to tumor progression independently of P53 pathway dysregulation, although the underlying mechanisms warrant further investigation. The heterogeneous expression of HER2, such as focal membranous staining, may impede the efficacy of targeted therapies, mirroring the challenges associated with HER2 heterogeneity in gastric cancer. (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e)Future investigations should assess the clonality of HER2 via multiregional sequencing and examine the therapeutic potential of combining HER2-targeted treatments with PI3K/mTOR inhibitors.\u003c/p\u003e \u003cp\u003eThe PD-L1 positivity rate in immune cells (CPS\u0026thinsp;\u0026ge;\u0026thinsp;1) was 67.9% (19/28). PD-L1-positive patients may respond to immunotherapy, particularly when immunotherapy is combined with radiotherapy or antiangiogenic agents such as bevacizumab. (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e)However, gastric-type adenocarcinoma (GAS) is characterized predominantly by an immune-cold phenotype. Consistent with the sequencing results and immunohistochemical findings, all patients were microsatellite stable (MSS) with a low tumor mutational burden (TMB; mean 2/Mb). A patient with PD-L1 expression (CPS\u0026thinsp;=\u0026thinsp;80) exhibited only a mild-to-moderate response to immunotherapy. Thus, further validation of the predictive value of biomarkers and treatment efficacy is needed.\u003c/p\u003e \u003cp\u003eIn this study, genomic analysis revealed a distinct mutational landscape in GAS. The most frequently mutated genes were TP53 (8/11, 72.7%), KRAS (5/11, 45.5%), SMAD4 (5/11, 45.5%), CDKN2A (4/11, 36.4%), PIK3CA (3/11, 27.3%), and STK11 (2/11, 18.2%). These genes are predominantly involved in key biological processes, including signal transduction pathways, cell cycle regulation networks, and epithelial‒mesenchymal transition. Notably, compared with HPV-associated cervical adenocarcinoma (UEA), endometrial cancer, and cervical squamous cell carcinoma patients, the GAS patients in this study presented mutations in TP53, KRAS, CDKN2A, and PIK3CA. The frequencies of TP53 and KRAS mutations were significantly greater than those reported in the aforementioned three malignancies. Importantly, inactivation of the clinically relevant PTEN gene and mutations in the POLE gene, which are frequently identified in endometrial cancer, were not observed in this cohort. These molecular pathological differences may offer a potential explanation for the distinct aggressive phenotype of GAS.\u003c/p\u003e \u003cp\u003eFurthermore, the high-frequency mutational profile of GAS genes identified in the present study, including KRAS, TP53, SMAD4, and CDKN2A, demonstrated significant molecular homology with the core driver genes of pancreatic cancer. These genes are among the four genes most frequently mutated in pancreatic cancer. Additional analysis revealed partial overlap in the mutation frequencies of TP53 (81.8%) and BRCA2 (9.1%) with the molecular characteristics of cholangiocarcinoma, in which these genes ranked among the ten most frequently mutated genes. Notably, the presence of STK11-inactivating mutations (18.2%) in GAS further supports a molecular pathological association between GAS and tumors associated with Peutz\u0026ndash;Jeghers syndrome.(\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eTherefore, the molecular landscape of gastric-type adenocarcinoma (GAS) is markedly distinct from that of other tumors arising in the female genital tract. Nevertheless, GAS shares certain molecular similarities with gastric and pancreaticobiliary adenocarcinomas. This molecular overlap may provide insights into the aggressive nature of GAS and reinforce its distinct pathogenesis. Notably, none of the patients analyzed in this study exhibited HER2 amplification or a microsatellite instability-high (MSI-H) status. These findings suggest that GAS may exhibit limited sensitivity to trastuzumab or immune checkpoint inhibitors. Thus, further investigations into personalized treatment strategies based on molecular subtyping are essential for optimizing therapeutic approaches for GAS.\u003c/p\u003e \u003cp\u003eThis study constitutes the most extensive clinical and pathological investigation of GAS to date, encompassing 100 cases, a scale that markedly surpasses those reported in previous literature.(\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e)The novelty of this study is primarily reflected in two key aspects. First, it offers the first comprehensive analysis of the immunophenotypic characteristics of GAS. This analysis encompasses markers of gastric differentiation (MUC6, HIK1083, CAIX), intestinal differentiation (CK20, CDX2, SATB2), differentiation from clear cell carcinoma (HNF1β, Napsin A), mismatch repair protein expression, and treatment-related markers (HER2, PD-L1). This approach addresses the limitations of prior studies, which have focused predominantly on a limited number of markers.(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eThis study independently investigated the immunohistochemical characteristics and molecular landscape of GAS rather than analyzing it in conjunction with other cervical adenocarcinoma subtypes. Through a systematic comparison of GAS with other related malignancies, distinct molecular differences were identified (e.g., TP53 mutations and PIK3CA-driven pathways), along with the establishment of disease-specific diagnostic markers. These findings offer compelling evidence to enhance the classification framework for precision pathology.\u003c/p\u003e \u003cp\u003eThis study has several limitations. To ensure the broad inclusion of real-world data, this study incorporated a diverse array of clinical samples, including initial biopsy samples, hysterectomy samples without lymph node dissection, and postoperative consultation cases from external institutions. Although this approach improved the comprehensiveness of the study, it resulted in limitations concerning the completeness of the data on FIGO staging and pathological risk factors.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe presence of abnormal uterine bleeding and vaginal discharge, in conjunction with a negative HPV test, should prompt suspicion for GAS. Histologically, GAS is defined by a gastric-type mucinous epithelium, where varying degrees of differentiation contribute to structural and cytological atypia. A comprehensive panel of immunohistochemical markers, including ER, PR, P53, p16, Ki-67, PAX2, HIK1083, MUC6, PAX8, and CK7, serves as a valuable tool for accurate diagnosis. At the molecular level, GAS is similar to gastric and pancreaticobiliary adenocarcinomas, particularly concerning genetic alterations such as TP53 mutations and PIK3CA-driven pathways. These shared molecular characteristics may provide insight into the aggressive nature of GAS and contribute to an understanding of its distinct pathogenesis.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCA125 Cancer antigen 125\u003c/p\u003e\n\u003cp\u003eCA19-9 Carbohydrate antigen 19-9\u003c/p\u003e\n\u003cp\u003eCAIX Carbonic anhydrase IX\u003c/p\u003e\n\u003cp\u003eCDX2 Caudal type homeobox 2\u003c/p\u003e\n\u003cp\u003eCEA Carcinoembryonic antigen\u003c/p\u003e\n\u003cp\u003eCK7 Cytokeratin 7\u003c/p\u003e\n\u003cp\u003eCK20 Cytokeratin 20\u003c/p\u003e\n\u003cp\u003eCPS Combined Positive Score\u003c/p\u003e\n\u003cp\u003eDNA Deoxyribonucleic acid\u003c/p\u003e\n\u003cp\u003eER Estrogen receptor\u003c/p\u003e\n\u003cp\u003eECA endocervical adenocarcinoma\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFFPE Formalin-fixed paraffin-embedded\u003c/p\u003e\n\u003cp\u003eGAS Gastric-type cervical adenocarcinoma\u003c/p\u003e\n\u003cp\u003eHE hematoxylin‒eosin\u003c/p\u003e\n\u003cp\u003eHNF1\u0026beta; Hepatocyte nuclear factor 1 beta\u003c/p\u003e\n\u003cp\u003eHPF high power field\u003c/p\u003e\n\u003cp\u003eHPV human papillomavirus\u003c/p\u003e\n\u003cp\u003eHPVA HPV-associated adenocarcinoma\u003c/p\u003e\n\u003cp\u003eHPVIA HPV-independent adenocarcinoma\u003c/p\u003e\n\u003cp\u003eLNM lymph node metastasis\u003c/p\u003e\n\u003cp\u003eLVSI Lymphovascular invasion\u003c/p\u003e\n\u003cp\u003eMMR Mismatch repair\u003c/p\u003e\n\u003cp\u003eMuc6 Mucin 6\u003c/p\u003e\n\u003cp\u003eNGS Next generation sequencing\u003c/p\u003e\n\u003cp\u003ep16 Cyclin-dependent kinase inhibitor 2A\u003c/p\u003e\n\u003cp\u003eP53 Tumor protein P53\u003c/p\u003e\n\u003cp\u003ePAX2 Paired box 2\u003c/p\u003e\n\u003cp\u003ePax8 Paired box 8\u003c/p\u003e\n\u003cp\u003ePD-L1 Programmed death-ligand 1\u003c/p\u003e\n\u003cp\u003ePFS progression-free survival\u003c/p\u003e\n\u003cp\u003ePR Progesterone receptor\u003c/p\u003e\n\u003cp\u003eSATB2 Special AT-rich sequence-binding protein 2\u003c/p\u003e\n\u003cp\u003eWHO World Health Organization\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS.G. made substantial contributions to conception and design of study, acquisition of data, drafting of the manuscript and critical revision of the manuscript. Y.S. supervised the study involved in conception and critical revision of the manuscript. All authors read, revise and gave approval of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere was no funding available for this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo datasets were generated or analysed during the current study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe National Cancer Center/Cancer Hospital Ethics Committee, Chinese Academy of Medical Sciences, and Peking Union Medical College approved this study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e1 Department of Pathology, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, China\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAdegoke O, Kulasingam S, Virnig B. Cervical cancer trends in the United States: a 35-year population-based analysis. Journal of women\u0026apos;s health (2002). 2012;21(10):1031-7.\u003c/li\u003e\n\u003cli\u003eSiegel RL, Miller KD, Jemal A. Cancer statistics, 2020. CA: a cancer journal for clinicians. 2020;70(1):7-30.\u003c/li\u003e\n\u003cli\u003eH\u0026ouml;hn AK, Brambs CE, Hiller GGR, May D, Schmoeckel E, Horn L-CJGuF. 2020 WHO classification of female genital tumors. 2021;81(10):1145-53.\u003c/li\u003e\n\u003cli\u003eStolnicu S, Hoang L, Chiu D, Hanko-Bauer O, Terinte C, Pesci A, et al. Clinical Outcomes of HPV-associated and Unassociated Endocervical Adenocarcinomas Categorized by the International Endocervical Adenocarcinoma Criteria and Classification (IECC). Am J Surg Pathol. 2019;43(4):466-74.\u003c/li\u003e\n\u003cli\u003eMikami Y, McCluggage WG. Endocervical glandular lesions exhibiting gastric differentiation: an emerging spectrum of benign, premalignant, and malignant lesions. Advances in anatomic pathology. 2013;20(4):227-37.\u003c/li\u003e\n\u003cli\u003eEhmann S, Sassine D, Straubhar AM, Praiss AM, Aghajanian C, Alektiar KM, et al. Gastric-type adenocarcinoma of the cervix: Clinical outcomes and genomic drivers. Gynecologic oncology. 2022;167(3):458-66.\u003c/li\u003e\n\u003cli\u003eKaramurzin YS, Kiyokawa T, Parkash V, Jotwani AR, Patel P, Pike MC, et al. Gastric-type Endocervical Adenocarcinoma: An Aggressive Tumor With Unusual Metastatic Patterns and Poor Prognosis. Am J Surg Pathol. 2015;39(11):1449-57.\u003c/li\u003e\n\u003cli\u003eO\u0026apos;Neill CJ, McCluggage WG. p16 expression in the female genital tract and its value in diagnosis. Advances in anatomic pathology. 2006;13(1):8-15.\u003c/li\u003e\n\u003cli\u003eNishio S, Mikami Y, Tokunaga H, Yaegashi N, Satoh T, Saito M, et al. Analysis of gastric-type mucinous carcinoma of the uterine cervix - An aggressive tumor with a poor prognosis: A multi-institutional study. Gynecologic oncology. 2019;153(1):13-9.\u003c/li\u003e\n\u003cli\u003eHoughton O, Jamison J, Wilson R, Carson J, McCluggage WG. p16 Immunoreactivity in unusual types of cervical adenocarcinoma does not reflect human papillomavirus infection. Histopathology. 2010;57(3):342-50.\u003c/li\u003e\n\u003cli\u003eLi VD, Li KH, Li JT. TP53 mutations as potential prognostic markers for specific cancers: analysis of data from The Cancer Genome Atlas and the International Agency for Research on Cancer TP53 Database. Journal of cancer research and clinical oncology. 2019;145(3):625-36.\u003c/li\u003e\n\u003cli\u003eKamijo K, Miyamoto T, Oshima S, Asaka S, Shinagawa M, Sato Y, et al. Extensive Pathologic Invasion and Prognostic Implication of Gastric-Type Cervical Adenocarcinoma: A Comparative Analysis With Human Papillomavirus-Associated Adenocarcinoma. Am J Surg Pathol. 2025.\u003c/li\u003e\n\u003cli\u003eLaury AR, Perets R, Piao H, Krane JF, Barletta JA, French C, et al. A comprehensive analysis of PAX8 expression in human epithelial tumors. Am J Surg Pathol. 2011;35(6):816-26.\u003c/li\u003e\n\u003cli\u003eYemelyanova A, Gown AM, Wu LS, Holmes BJ, Ronnett BM, Vang R. PAX8 expression in uterine adenocarcinomas and mesonephric proliferations. International journal of gynecological pathology : official journal of the International Society of Gynecological Pathologists. 2014;33(5):492-9.\u003c/li\u003e\n\u003cli\u003eOrd\u0026oacute;\u0026ntilde;ez NG. Value of PAX2 immunostaining in tumor diagnosis: a review and update. Advances in anatomic pathology. 2012;19(6):401-9.\u003c/li\u003e\n\u003cli\u003eShukla A, Thomas D, Roh MH. PAX8 and PAX2 expression in endocervical adenocarcinoma in situ and high-grade squamous dysplasia. International journal of gynecological pathology : official journal of the International Society of Gynecological Pathologists. 2013;32(1):116-21.\u003c/li\u003e\n\u003cli\u003eRabban JT, McAlhany S, Lerwill MF, Grenert JP, Zaloudek CJ. PAX2 distinguishes benign mesonephric and mullerian glandular lesions of the cervix from endocervical adenocarcinoma, including minimal deviation adenocarcinoma. Am J Surg Pathol. 2010;34(2):137-46.\u003c/li\u003e\n\u003cli\u003eKojima A, Mikami Y, Sudo T, Yamaguchi S, Kusanagi Y, Ito M, et al. Gastric morphology and immunophenotype predict poor outcome in mucinous adenocarcinoma of the uterine cervix. Am J Surg Pathol. 2007;31(5):664-72.\u003c/li\u003e\n\u003cli\u003eMcCluggage WG. Recent Developments in Non-HPV-related Adenocarcinomas of the Lower Female Genital Tract and Their Precursors. Advances in anatomic pathology. 2016;23(1):58-69.\u003c/li\u003e\n\u003cli\u003eMikami Y, Minamiguchi S, Teramoto N, Nagura M, Haga H, Konishi I. Carbonic anhydrase type IX expression in lobular endocervical glandular hyperplasia and gastric-type adenocarcinoma of the uterine cervix. Pathology, research and practice. 2013;209(3):173-8.\u003c/li\u003e\n\u003cli\u003eLiao SY, Rodgers WH, Kauderer J, Darcy KM, Carter R, Susumu N, et al. Endocervical glandular neoplasia associated with lobular endocervical glandular hyperplasia is HPV-independent and correlates with carbonic anhydrase-IX expression: a Gynaecological Oncology Group Study. British journal of cancer. 2013;108(3):613-20.\u003c/li\u003e\n\u003cli\u003eFulgione C, Raffone A, Travaglino A, Arciuolo D, Santoro A, Cianfrini F, et al. Diagnostic accuracy of HIK1083 and MUC6 as immunohistochemical markers of endocervical gastric-type adenocarcinoma: A systematic review and meta-analysis. Pathology, research and practice. 2023;241:154261.\u003c/li\u003e\n\u003cli\u003eCarleton C, Hoang L, Sah S, Kiyokawa T, Karamurzin YS, Talia KL, et al. A Detailed Immunohistochemical Analysis of a Large Series of Cervical and Vaginal Gastric-type Adenocarcinomas. Am J Surg Pathol. 2016;40(5):636-44.\u003c/li\u003e\n\u003cli\u003eNěmejcov\u0026aacute; K, Cibula D, Dundr P. Expression of HNF-1\u0026beta; in cervical carcinomas: an immunohistochemical study of 155 cases. Diagnostic pathology. 2015;10:8.\u003c/li\u003e\n\u003cli\u003eVakiani E. HER2 testing in gastric and gastroesophageal adenocarcinomas. Advances in anatomic pathology. 2015;22(3):194-201.\u003c/li\u003e\n\u003cli\u003eIeni A, Cardia R, Lentini M, Tuccari G. Intratumoral HER2 heterogeneity in early gastric carcinomas: potential bias in therapeutic management. Virchows Archiv : an international journal of pathology. 2019;474(3):401-2.\u003c/li\u003e\n\u003cli\u003eColombo N, Dubot C, Lorusso D, Caceres MV, Hasegawa K, Shapira-Frommer R, et al. Pembrolizumab for Persistent, Recurrent, or Metastatic Cervical Cancer. 2021;385(20):1856-67.\u003c/li\u003e\n\u003cli\u003eBeggs AD, Latchford AR, Vasen HF, Moslein G, Alonso A, Aretz S, et al. Peutz-Jeghers syndrome: a systematic review and recommendations for management. Gut. 2010;59(7):975-86.\u003c/li\u003e\n\u003cli\u003eYang J, Peng Y, Ding Y, Liu Y, Wang Y, Liu Y, et al. The Clinicopathological and Molecular Characteristics of Endocervical Gastric-Type Adenocarcinoma and the Use of Claudin18.2 as a Potential Therapeutic Target. Mod Pathol. 2024;37(10):100569.\u003c/li\u003e\n\u003cli\u003eLu S, Shen D, Zhao Y, Kang N, Wang X. Primary endocervical gastric-type adenocarcinoma: a clinicopathologic and immunohistochemical analysis of 23 cases. Diagnostic pathology. 2019;14(1):72.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"diagnostic-pathology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"dpat","sideBox":"Learn more about [Diagnostic Pathology](http://diagnosticpathology.biomedcentral.com)","snPcode":"13000","submissionUrl":"https://submission.nature.com/new-submission/13000/3","title":"Diagnostic Pathology","twitterHandle":"@OncoBioMed","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"cervical cancer, gastric-type cervical adenocarcinoma, clinical pathological features, molecular features","lastPublishedDoi":"10.21203/rs.3.rs-6295926/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6295926/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective: \u003c/strong\u003eTo investigate the clinicopathological and molecular features, diagnosis, and differential diagnosis of gastric-type cervical adenocarcinoma (GAS).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eA retrospective analysis was conducted on 100 patients diagnosed with GAS at the National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences, from January 2017 to December 2024. Clinicopathological data were collected, histological characteristics and immunohistochemical expression patterns were analyzed, and the relevant literature was reviewed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eThe ages of the 100 patients ranged from 25-73 years, with a median age of 50 years. The most common clinical manifestations included abnormal uterine bleeding and vaginal discharge, and the majority of patients were in FIGO stages II-IV. The GAS tumor glands were irregularly branched or cystically dilated, and the cells lining the glands had eosinophilic cytoplasm, with apical mucin secretion observed and the formation of mucus lakes in the stroma. Highly differentiated GAS may be misdiagnosed as heterogeneous hyperplasia of the cervical glands because of its relatively regular glandular structure. Irregularity of glandular architecture is observed in poorly differentiated areas, with fusion and a back-to-back arrangement occurring alongside an interstitial profibrotic reaction. Immunohistochemical markers, including MUC6, P16, P53, PAX8, ER, and PR, can provide additional diagnostic clues for GAS. Intestinal markers, such as CK7, CK20, CDX2, SATB2, Villin, CA19-9, CEA, and CA125, are not substantially helpful in distinguishing GAS from pancreaticobiliary adenocarcinoma. PAX2 aids in differentiating between GAS and precancerous lesions of benign gastric-type adenopathy. Positive staining for gastric markers, such as MUC6, HIK1083, and CAIX, helps indicate gastric-type differentiation. HNF1β in combination with NapsinA aids in differentiating GAS from clear-cell carcinoma. Patients with HER2 overexpression or PD-L1 expression (CPS ≥1) may benefit from targeted therapy or immunotherapy. Next-generation sequencing (NGS) analysis of 11 cases revealed recurrent somatic mutations in critical oncogenic pathways, including TP53 (72.7%, 8/11), KRAS (45.5%, 5/11), SMAD4 (45.5%, 5/11), CDKN2A (36.4%, 4/11), PIK3CA (27.3%, 3/11), and STK11 (18.2%, 2/11). No statistically significant associations were identified between p53 mutation status and FIGO stage (p=0.32), depth of myometrial invasion (p=0.45), lymphovascular invasion (LVSI) (p=0.67), or lymph node metastasis (LNM) (p=0.89), as determined by Fisher’s exact test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eGAS is a highly malignant, HPV-independent cervical adenocarcinoma characterized by atypical clinical symptoms and complex, varied histology. It can be accurately diagnosed based on histologic features and immunohistochemical staining.\u003c/p\u003e","manuscriptTitle":"Clinical pathological and molecular features of 100 patients with gastric-type cervical adenocarcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-06 10:25:11","doi":"10.21203/rs.3.rs-6295926/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-01T00:27:40+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-26T10:34:10+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-14T00:00:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"103466102708398216484544103429089921162","date":"2025-04-05T08:24:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"194261515460938992337035694358931401901","date":"2025-04-03T02:52:27+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-03T02:29:32+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-26T02:04:55+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-25T04:30:20+00:00","index":"","fulltext":""},{"type":"submitted","content":"Diagnostic Pathology","date":"2025-03-24T13:34:15+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"diagnostic-pathology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"dpat","sideBox":"Learn more about [Diagnostic Pathology](http://diagnosticpathology.biomedcentral.com)","snPcode":"13000","submissionUrl":"https://submission.nature.com/new-submission/13000/3","title":"Diagnostic Pathology","twitterHandle":"@OncoBioMed","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"504b7ace-ad59-4553-9e3c-896c29f15fb0","owner":[],"postedDate":"May 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-06-16T16:00:22+00:00","versionOfRecord":{"articleIdentity":"rs-6295926","link":"https://doi.org/10.1186/s13000-025-01666-7","journal":{"identity":"diagnostic-pathology","isVorOnly":false,"title":"Diagnostic Pathology"},"publishedOn":"2025-06-10 15:57:01","publishedOnDateReadable":"June 10th, 2025"},"versionCreatedAt":"2025-05-06 10:25:11","video":"","vorDoi":"10.1186/s13000-025-01666-7","vorDoiUrl":"https://doi.org/10.1186/s13000-025-01666-7","workflowStages":[]},"version":"v1","identity":"rs-6295926","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6295926","identity":"rs-6295926","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}