USP18 promotes colon adenocarcinoma progression via targeting the ERK-MNK signaling pathway.

BACKGROUND: Colorectal cancer is the third most common malignancy worldwide and is one of the leading causes of cancer-related mortality. Ubiquitin-specific peptidase 18 (USP18) protein has been reported to exert different tumor-related effects in distinct tumor types. Here, we initially investigated the expression and signaling pathways of USP18 in colon adenocarcinoma (COAD). METHODS: A quantitative real-time PCR was conducted to evaluate the mRNA level of USP18 in cultured cells. Immunohistochemical staining was used to explore the protein expression of USP18 in clinical COAD samples. Specific knockdown was achieved by transient transfection of small interfering RNAs into SW480 and HT29 cells using Lipo3000. Cell conting kit-8 assay, transwell assay and matrigel-transwell assays were conducted to evaluate proliferation, migration and invasion capacities, respectively. Western blotting was performed to analyze downstream signaling pathways. A chi-squared test and univariate and multivariate analyses were used to evaluate the clinical data. Xenografts from mice model were assessed to validate the in vitro findings. RESULTS: Higher USP18 level was identified in COAD tissues and was positively correlated with advanced tumor stage. High USP18 protein expression indicated poorer prognosis of COAD patients. Silencing USP18 suppressed COAD cell proliferation and invasion via destabilizing extracellular signal-regulated kinase (ERK) protein and suppressing ERK downstream pathways. Simultaneously silencing interferon-stimulated gene 15 (ISG15) with USP18 can partially rescue the tumor cell viability, indicating its involvement in USP18 signaling. The oncogenic effects of USP18 were also confirmed in mice models. CONCLUSIONS: USP18 plays oncogenic effects in colon adenocarcinoma via ISG15-ERK pathways. High USP18 expression indicates poor clinical outcomes for colon adenocarcinoma patients.

Feline lung-digit syndrome: A differential diagnosis for shifting, waxing and waning lameness in a cat.

The clinical presentation, cytologic findings, radiographic findings, and postmortem assessment of a cat with primary pulmonary adenocarcinoma with multiple digital metastasis are described. An unusual shifting, waxing and waning pattern of lameness, suspected to be an early manifestation of digital metastasis before any gross lesions were visible, was documented. Initial cytologic finding of a lung nodule was equivocal for diagnosis of neoplasia despite being strongly suspicious. Palliative management was short-lived, with rapid progression culminating in widespread metastasis to multiple digits, muscles, and other organs. The diagnosis of pulmonary adenocarcinoma was confirmed via necropsy and histopathology. Key clinical message: This case report highlights that feline lung-digit syndrome is an important differential diagnosis for an acute, waxing and waning, shifting leg lameness in an older cat. This pattern of lameness should raise the index of suspicion for an underlying primary lung neoplasm, and thoracic imaging (radiographs) should be considered.

Syndrome pulmonaire-digital félin : un diagnostic différentiel des boiteries changeantes, croissantes et décroissantes chez un chatLa présentation clinique, les résultats cytologiques, les résultats radiographiques et l'évaluation post mortem d'un chat atteint d'adénocarcinome pulmonaire primaire avec métastases numériques multiples sont décrits. Un schéma inhabituel de boiterie, variable, croissante et décroissante, suspecté d'être une manifestation précoce de métastases digitales avant que des lésions macroscopiques ne soient visibles, a été documenté. La découverte cytologique initiale d'un nodule pulmonaire était équivoque pour le diagnostic de néoplasie bien qu'elle soit fortement suspecte. La prise en charge palliative a été de courte durée, avec une progression rapide aboutissant à des métastases généralisées à plusieurs doigts, muscles et autres organes. Le diagnostic d'adénocarcinome pulmonaire a été confirmé par autopsie et histopathologie.Message clinique clé :Ce rapport de cas souligne que le syndrome pulmonaire-digital félin est un diagnostic différentiel important pour une boiterie aiguë, croissante et décroissante et mobile des pattes chez un chat ágé. Ce type de boiterie devrait faire suspecter une tumeur primaire du poumon sous-jacente, et une imagerie thoracique (radiographies) devrait être envisagée.(Traduit par Dr Serge Messier).

Single-cell analysis of treatment-resistant prostate cancer: Implications of cell state changes for cell surface antigen-targeted therapies.

Targeting cell surface molecules using radioligand and antibody-based therapies has yielded considerable success across cancers. However, it remains unclear how the expression of putative lineage markers, particularly cell surface molecules, varies in the process of lineage plasticity, wherein tumor cells alter their identity and acquire new oncogenic properties. A notable example of lineage plasticity is the transformation of prostate adenocarcinoma (PRAD) to neuroendocrine prostate cancer (NEPC)-a growing resistance mechanism that results in the loss of responsiveness to androgen blockade and portends dismal patient survival. To understand how lineage markers vary across the evolution of lineage plasticity in prostate cancer, we applied single-cell analyses to 21 human prostate tumor biopsies and two genetically engineered mouse models, together with tissue microarray analysis on 131 tumor samples. Not only did we observe a higher degree of phenotypic heterogeneity in castrate-resistant PRAD and NEPC than previously anticipated but also found that the expression of molecules targeted therapeutically, namely PSMA, STEAP1, STEAP2, TROP2, CEACAM5, and DLL3, varied within a subset of gene-regulatory networks (GRNs). We also noted that NEPC and small cell lung cancer subtypes shared a set of GRNs, indicative of conserved biologic pathways that may be exploited therapeutically across tumor types. While this extreme level of transcriptional heterogeneity, particularly in cell surface marker expression, may mitigate the durability of clinical responses to current and future antigen-directed therapies, its delineation may yield signatures for patient selection in clinical trials, potentially across distinct cancer types.

Clinicopathological Features of 26 Intraoral Polymorphous Adenocarcinomas from a Single Brazilian Institution.

PURPOSE: This study describes a large, well-documented case series of salivary gland polymorphous adenocarcinomas (PAC) from a single Brazilian center. METHODS: Demographic data, clinical presentation, histopathological and immunohistochemical features from 26 cases of PAC were analyzed and discussed in detail. RESULTS: Most patients were females (n = 21), with a ratio of 1:4.2 (male: female) with a mean age of 58.8 years (ranging from 36 to 84 years). The most common clinical presentation was a fibrocollagenous, firm nodular lesion, with a mean size of 2.46 cm (ranging from 0.5 to 3 cm). Most lesions occurred on the palate (n = 16), followed by buccal mucosa (n = 3), upper lip (n = 3), buccal vestibule (n = 2) and alveolar ridge (n = 1). Histologically, various growth patterns were observed, including tubular, solid, cribriform, papillary, and cystic. Additionally, glomeruloid slit-like structures, mucous, and clear cells were noted. Surface papillary epithelial hyperplasia was observed in a few cases. Nine cases exhibited myxoid and collagenous areas, while two cases showed fusiform areas and another case demonstrated squamous differentiation. Clear cell predominance was noted in two cases, and peri- and intraneural invasion was seen in eight cases. Immunohistochemical analysis revealed positivity for S-100, p63 and CK7, and negativity for p40 in all cases. The Ki-67 proliferation index was markedly low in most cases, with a mean of 2.5%. CONCLUSION: We have provided a broad, detailed description of the clinical and microscopic features of PAC in a large, Brazilian cohort. These findings, in a resource-limited area, may be quite useful for establishing a proper diagnosis.

γδ T cells in human colon adenocarcinomas comprise mainly Vδ1, Vδ2, and Vδ3 cells with distinct phenotype and function.

Γδ T cell infiltration into tumours usually correlates with improved patient outcome, but both tumour-promoting and tumoricidal effects of γδ T cells have been documented. Human γδ T cells can be divided into functionally distinct subsets based on T cell receptor (TCR) Vδ usage. Still, the contribution of these different subsets to tumour immunity remains elusive. Here, we provide a detailed γδ T cell profiling in colon tumours, using mass and flow cytometry, mRNA quantification, and TCR sequencing. δ chain usage in both the macroscopically unaffected colon mucosa and tumours varied considerably between patients, with substantial fractions of Vδ1, Vδ2, and non-Vδ1 Vδ2 cells. Sequencing of the Vδ complementarity-determining region 3 showed that almost all non-Vδ1 Vδ2 cells used Vδ3 and that tumour-infiltrating γδ clonotypes were unique for every patient. Non-Vδ1Vδ2 cells from colon tumours expressed several activation markers but few NK cell receptors and exhaustion markers. In addition, mRNA analyses showed that non-Vδ1 Vδ2 cells expressed several genes for proteins with tumour-promoting functions, such as neutrophil-recruiting chemokines, Galectin 3, and transforming growth factor-beta induced. In summary, our results show a large variation in γδ T cell subsets between individual tumours, and that Vδ3 cells make up a substantial proportion of γδ T cells in colon tumours. We suggest that individual γδ T cell composition in colon tumours may contribute to the balance between favourable and adverse immune responses, and thereby also patient outcome.

Immune cells and checkpoints in pancreatic adenocarcinoma: Association with clinical and pathological characteristics.

INTRODUCTION: Pancreatic adenocarcinoma is an extremely aggressive neoplasm, with many challenges to be overcome in order to achieve a truly effective treatment. It is characterized by a mostly immunosuppressed environment, with dysfunctional immune cells and active immunoinhibitory pathways that favor tumor evasion and progression. Thus, the study and understanding of the tumor microenvironment and the various cells subtypes and their functional capacities are essential to achieve more effective treatments, especially with the use of new immunotherapeutics. METHODS: Seventy cases of pancreatic adenocarcinoma divided into two groups 43 with resectable disease and 27 with unresectable disease were analyzed using immunohistochemical methods regarding the expression of programmed cell death ligand 1 (PD-L1), programmed cell death ligand 2 (PD-L2), and human leukocyte antigen G (HLA-G) molecules as well as the populations of CD4+ and CD8+ T lymphocytes, regulatory T cells (Tregs), and M2 macrophages (MM2). Several statistical tests, including multivariate analyses, were performed to examine how those immune cells and immunoinhibitory molecules impact the evolution and prognosis of pancreatic adenocarcinoma. RESULTS: CD8+ T lymphocytes and M2 macrophages predominated in the group operated on, and PD-L2 expression predominated in the unresectable group. PD-L2 was associated with T stage, lymph node metastasis, and clinical staging, while in survival analysis, PD-L2 and HLA-G were associated with a shorter survival. In the inoperable cases, Tregs cells, MM2, PD-L1, PD-L2, and HLA-G were positively correlated. CONCLUSIONS: PD-L2 and HLA-G expression correlated with worse survival in the cases studied. Tumor microenvironment was characterized by a tolerant and immunosuppressed pattern, mainly in unresectable lesions, where a broad positive influence was observed between immunoinhibitory cells and immune checkpoint proteins expressed by tumor cells.

Diffuse-Type Histology Is Prognostic for All Siewert Types of Gastroesophageal Adenocarcinoma.

PURPOSE: The optimal treatment for gastroesophageal junction adenocarcinoma (GEJA) remains controversial. We evaluated the treatment patterns and outcomes of patients with locally advanced GEJA according to the histological type. MATERIALS AND METHODS: We conducted a single-institution retrospective cohort study of patients with locally advanced GEJA who underwent curative-intent surgical resection between 2010 and 2020. Perioperative therapies as well as clinicopathologic, surgical, and survival data were collected. The results of endoscopy and histopathological examinations were assessed for Siewert and Lauren classifications. RESULTS: Among the 58 patients included in this study, 44 (76%) were clinical stage III, and all received neoadjuvant therapy (72% chemoradiation, 41% chemotherapy, 14% both chemoradiation and chemotherapy). Tumor locations were evenly distributed by Siewert Classification (33% Siewert-I, 40% Siewert-II, and 28% Siewert-III). Esophagogastrectomy (EG) was performed for 47 (81%) patients and total gastrectomy (TG) for 11 (19%) patients. All TG patients received D2 lymphadenectomy compared to 10 (21%) EG patients. Histopathological examination showed the presence of 64% intestinal-type and 36% diffuse-type histology. The frequencies of diffuse-type histology were similar among Siewert groups (37% Siewert-I, 36% Siewert-II, and 33% Siewert-III). Regardless of Siewert type and compared to intestinal-type, diffuse histology was associated with increased intraabdominal recurrence rates (P=0.03) and decreased overall survival (hazard ratio, 2.33; P=0.02). With a median follow-up of 31.2 months, 29 (50%) patients had a recurrence, and the median overall survival was 50.5 months. CONCLUSIONS: Present in equal proportions among Siewert types of esophageal and gastric cancer, a diffuse-type histology was associated with high intraabdominal recurrence rates and poor survival. Histopathological evaluation should be considered in addition to anatomic location in the determination of multimodal GEJA treatment strategies.

Colon cancer diagnosis by means of explainable deep learning.

Early detection of the adenocarcinoma cancer in colon tissue by means of explainable deep learning, by classifying histological images and providing visual explainability on model prediction. Considering that in recent years, deep learning techniques have emerged as powerful techniques in medical image analysis, offering unprecedented accuracy and efficiency, in this paper we propose a method to automatically detect the presence of cancerous cells in colon tissue images. Various deep learning architectures are considered, with the aim of considering the best one in terms of quantitative and qualitative results. As a matter of fact, we consider qualitative results by taking into account the so-called prediction explainability, by providing a way to highlight on the tissue images the areas that from the model point of view are related to the presence of colon cancer. The experimental analysis, performed on 10,000 colon issue images, showed the effectiveness of the proposed method by obtaining an accuracy equal to 0.99. The experimental analysis shows that the proposed method can be successfully exploited for colon cancer detection and localisation from tissue images.

Mesonephric-type adenocarcinomas of the ovary: prevalence, diagnostic reproducibility, outcome, and value of PAX2.

Mesonephric-type (or -like) adenocarcinomas (MAs) of the ovary are an uncommon and aggressive histotype. They appear to arise through transdifferentiation from Müllerian lesions creating diagnostic challenges. Thus, we aimed to develop a histologic and immunohistochemical (IHC) approach to optimize the identification of MA over its histologic mimics, such as ovarian endometrioid carcinoma (EC). First, we screened 1,537 ovarian epithelial neoplasms with a four-marker IHC panel of GATA3, TTF1, ER, and PR followed by a morphological review of EC to identify MA in retrospective cohorts. Interobserver reproducibility for the distinction of MA versus EC was assessed in 66 cases initially without and subsequently with IHC information (four-marker panel). Expression of PAX2, CD10, and calretinin was evaluated separately, and survival analyses were performed. We identified 23 MAs from which 22 were among 385 cases initially reported as EC (5.7%) and 1 as clear cell carcinoma. The interobserver reproducibility increased from fair to substantial (κ = 0.376-0.727) with the integration of the four-marker IHC panel. PAX2 was the single most sensitive and specific marker to distinguish MA from EC and could be used as a first-line marker together with ER/PR and GATA3/TTF1. Patients with MA had significantly increased risk of earlier death from disease (hazard ratio = 3.08; 95% CI, 1.62-5.85; p < 0.0001) compared with patients with EC, when adjusted for age, stage, and p53 status. A diagnosis of MA has prognostic implications for stage I disease, and due to the subtlety of morphological features in some tumors, a low threshold for ancillary testing is recommended.

[Intraperitoneal injection of human-derived anti-c-Met single-chain antibody inhibits the growth of transplanted tumours in A549 lung adenocarcinoma-loaded mice].

Objective To verify the anti-tumor effect of the mesenchymal-epithelial transition single-chain antibody (Met scFv) on subcutaneously transplanted tumors in nude mice. Methods A tumor model was established in nude mice by subcutaneous injection of A549 lung adenocarcinoma cells. Once the tumors were formed, IRDye680 LT N-hydroxysuccinimide (NHS) ester-labeled Met scFv was administered intraperitoneally. Real-time monitoring was conducted using a small animal imager to observe the dynamic distribution of the antibody in tumor-bearing mice. The affinity between c-Met and the antibody in tumor cells was detected. Tumor volume changes were observed and the tumor growth curve were plotted following regular tail vein injections of Met scFv. Immunohistochemical staining was employed to determine whether Met scFv could effectively bind to the c-Met antigen in tumor tissues. Results The distribution of Met scFv in nude mice showed that it was primarily located in the peritoneal cavity within the first 3 hours. After approximately 48 hours, fluorescent signals began to accumulate in the tumor tissue. Immunohistochemical staining of the tumors revealed high expression of c-Met in the tumor tissues; regular tail vein injections of Met scFv significantly slowed down the growth of tumors in mice. Conclusion Met scFv specifically recognizes tumor cells in vivo and exhibites significant anti-tumor activity.

Evaluating the prognostic value of tumor deposits in non-metastatic lymph node-positive colon adenocarcinoma using Cox regression and machine learning.

BACKGROUND: The 8th AJCC TNM staging for non-metastatic lymph node-positive colon adenocarcinoma patients(NMLP-CA) stages solely by lymph node status, irrespective of the positivity of tumor deposits (TD). This study uses machine learning and Cox regression to predict the prognostic value of tumor deposits in NMLP-CA. METHODS: Patient data from the SEER registry (2010-2019) was used to develop CSS nomograms based on prognostic factors identified via multivariate Cox regression. Model performance was evaluated by c-index, dynamic calibration, and Schmid score. Shapley additive explanations (SHAP) were used to explain the selected models. RESULTS: The study included 16,548 NMLP-CA patients, randomized 7:3 into training (n = 11,584) and test (n = 4964) sets. Multivariate Cox analysis identified TD, age, marital status, primary site, grade, pT stage, and pN stage as prognostic for cancer-specific survival (CSS). In the test set, the gradient boosting machine (GBM) model achieved the best C-index (0.733) for CSS prediction, while the Cox model and GAMBoost model optimized dynamic calibration(6.473) and Schmid score (0.285), respectively. TD ranked among the top 3 most important features in the models, with increasing predictive significance over time. CONCLUSIONS: Positive tumor deposit status confers worse prognosis in NMLP-CA patients. Tumor deposits may confer higher TNM staging. Furthermore, TD could play a more significant role in the staging system.

Ascending colon cancer metastasized to the right testicle: a case report.

BACKGROUND: Testicular metastasis from malignant solid tumors is extremely rare. It is usually found by chance during autopsy or pathological examination of testicular specimens. Therefore, we consider it necessary to report our patient's case of testicular metastasis from colon cancer. CASE PRESENTATION: We report a 61-year-old Han Chinese male patient who presented to our clinic with progressive painless swelling of the right testicle for 2 years. Positron emission tomography-computed tomography scans showed increased 18F-fluorodeoxyglucose metabolism in the right testicle, possibly owing to distant metastasis. His previous medical history suggested that he had undergone laparoscopic-assisted right hemicolectomy for ascending colon cancer 4 years ago. Considering the ascending colon cancer metastasis to the right testicle, we performed a right radical testicular resection through an inguinal approach. Postoperative histological examination showed intestinal metastatic adenocarcinoma. CONCLUSION: Colon cancer metastasis to the testes is uncommon. The clinical and imaging manifestations of this tumor are nonspecific, so the diagnosis relies on postoperative pathology. If testicular metastasis is found, treatment principles for advanced colon cancer should be followed.

Immunohistochemical Expression of Human Epidermal Growth Factor Receptor 2 and Ki67 in Apocrine Gland Anal Sac Adenocarcinoma.

Apocrine gland anal sac adenocarcinoma is an aggressive neoplasm, and surgery remains the treatment of choice, although it is controversial in advanced cases. The prognostic factors are not well established. Human Epidermal Growth Factor Receptor 2 (HER2) is a membrane protein related to tumorigenesis, whereas Ki67 is a nuclear protein related to cell proliferation. Both are potential prognostic markers and therapeutic targets. This study aimed to evaluate the expression of HER2 and Ki67 markers in canine apocrine gland anal sac adenocarcinoma. The tumor samples were divided into four groups: largest tumor diameter less than 2.5 cm, largest tumor diameter greater than 2.5 cm, metastatic lymph nodes, and control group of non-neoplastic anal sacs. Each contained 10 samples. Immunohistochemistry was performed to verify the expression of HER2 and Ki67 markers. Positive HER2 staining was observed in 45% of the neoplastic cases and negative HER2 staining in 100% of the control group. The Ki67 expression had a median of 25% in all groups, except for the control group, which had a median of 8%. The HER2 and Ki67 expression was present in apocrine gland anal sac adenocarcinoma, making them potential therapeutic targets. However, it was not possible to determine the clinical value of either marker.

Overexpression of TSPAN8 in consensus molecular subtype 3 colorectal cancer.

BACKGROUND: Recently, consensus molecular subtypes (CMSs) have been proposed as a robust transcriptome-based classification system for colorectal cancer (CRC). Tetraspanins (TSPANs) are transmembrane proteins. They have been associated with the development of numerous malignancies, including CRC, through their role as "master organizers" for multi-molecular membrane complexes. No previous study has investigated the correlation between TSPANs and CMS classification. Herein, we investigated the expression of TSPANs in patient-derived primary CRC tissues and their CMS classifications. METHODS: RNA samples were derived from primary CRC tissues (n = 100 patients diagnosed with colorectal adenocarcinoma) and subjected to RNA sequencing for transcriptome-based CMS classification and TSPAN-relevant analyses. Immunohistochemistry (IHC) and immunofluorescence (IF) stains were conducted to observe the protein expression level. To evaluate the relative biological pathways, gene-set enrichment analysis was performed. RESULTS: Of the highly expressed TSPAN genes in CRC tissues (TSPAN8, TSPAN29, and TSPAN30), TSPAN8 was notably overexpressed in CMS3-classified primary tissues. The overexpression of TSPAN8 protein in CMS3 CRC was also observed by IHC and IF staining. As a result of gene-set enrichment analysis, TSPAN8 may potentially play a role in organizing signaling complexes for kinase-based metabolic deregulation in CMS3 CRC. CONCLUSIONS: The present study reports the overexpression of TSPAN8 in CMS3 CRC. This study proposes TSPAN8 as a subtype-specific biomarker for CMS3 CRC. This finding provides a foundation for future CMS-based studies of CRC, a complex disease and the second leading cause of cancer mortality worldwide.

Fucoidan from Lessonia trabeculata Induces Apoptosis through Caspase Dependent and Caspase-Independent Activation in 4T1 Breast Adenocarcinoma In Vitro.

Experiments conducted on triple-negative breast cancer have shown that fucoidan from Lessonia trabeculata (FLt) exhibits cytotoxic and antitumor properties. However, further research is necessary to gain a complete understanding of its bioactivity and level of cytotoxicity. The cytotoxic effect of FLt was determined by the 2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Apoptosis was analyzed using annexin V and caspase 3/7 staining kit and DNA fragmentation. In addition, transcriptional expression of antiapoptotic (Bcl-2 and XIAP) and proapoptotic (caspase 8, caspase 9, and AIF) genes were analyzed in TNBC 4T1 cells. After 72 h of culture, the IC50 for FLt was 561 µg/mL, while doxorubicin (Dox) had an IC50 of 0.04 µg/mL. In addition, assays for FLt + Dox were performed. Annexin V and caspase 3/7 revealed that FLt induces early and late-stage apoptosis. DNA fragmentation results support necrotic death of 4T1 cells. Similarly, transcripts that prevent cell death were decreased, while transcripts that promote cell death were increased. This study showed that FLt induces apoptosis by both caspase-dependent and caspase-independent mechanisms. These findings suggest that FLt may have potential applications in breast cancer treatment. Further research will provide more information to elucidate the mechanism of action of FLt.

Transforming tumoroids derived from ALK-positive pulmonary adenocarcinoma to squamous cell carcinoma in vivo.

Approximately 3-5% of non-small cell lung cancers (NSCLC) harbor ALK fusion genes and may be responsive to anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors. There are only a few reports on cell lines with EML4-ALK variant 3 (v3) and tumoroids that can be subject to long-term culture (> 3 months). In this study, we established tumoroids (PDT-LUAD#119) from a patient with lung cancer harboring EML4-ALK that could be cultured for 12 months. Whole-exome sequencing and RNA sequencing analyses revealed TP53 mutations and an EML4-ALK v3 mutation. PDT-LUAD#119 lung tumoroids were sensitive to the ALK tyrosine kinase inhibitors (ALK TKIs) crizotinib, alectinib, entrectinib, and lorlatinib, similar to NCI-H3122 cells harboring EML4-ALK variant 1 (v1). Unexpectedly, clear squamous cell carcinoma and solid adenocarcinoma were observed in xenografts from PDT-LUAD#119 lung tumoroids, indicating adenosquamous carcinoma. Immunostaining revealed that the squamous cell carcinoma was ALK positive, suggesting a squamous transformation of the adenocarcinoma. Besides providing a novel cancer model to support basic research on ALK-positive lung cancer, PDT-LUAD#119 lung tumoroids will help elucidate the pathogenesis of adenosquamous carcinoma.