High-sensitivity PCR method for detecting BRAF V600E mutations in metastatic colorectal cancer using LNA/DNA chimeras to block wild-type alleles.

The response to epidermal growth factor receptor (EGFR)-targeted therapy in metastatic colorectal cancer (mCRC) is variable because of intra-tumor heterogeneity at the genetic level, and consequently, it is important to develop sensitive and selective assays to predict patient responses to therapy. Low-abundance BRAF V600E mutations are associated with poor response to treatment with EGFR inhibitors. We developed a method for the detection of BRAF V600E mutations in mCRC using real-time wild-type blocking PCR (WTB-PCR), in which a chimera composed of locked nucleic acids and DNA is incorporated to amplify the mutant allele at high efficiency while simultaneously inhibiting the amplification of wild-type alleles. Mixing experiments showed that this method is exquisitely sensitive, with detection of the mutated allele at a mutant/wild-type ratio of 1:10,000. To demonstrate the applicability of this approach for mCRC patients, we assessed the V600E mutations in 50 clinical cases of mCRC by real-time WTB-PCR. The percentage of patients with V600E mutation as determined by WTB-PCR (16%, 8/50) was higher than by traditional PCR (10%, 5/50), suggesting an increased sensitivity for WTB-PCR. By calculating the ΔC q for real-time traditional PCR, which amplifies all BRAF alleles, versus WTB-PCR, which selectively amplifies mutant BRAF, we demonstrated that among the V600E-positive mCRC patient samples, the percentage of BRAF DNA with the V600E mutation ranged from 0.05 to 52.32%. In conclusion, WTB-PCR provides a rapid, simple, and low-cost method to detect trace amounts of mutated BRAF V600E gene.

Primary pulmonary hyalinizing clear cell carcinoma with fusions of both EWSR1::CREM and IRF2::NTRK3: report of a case with an aggressive behavior.

Primary pulmonary hyalinizing clear cell carcinoma (HCCC) is a rare salivary gland-type tumor newly recognized in recent years, with approximately 21 cases reported to date in the English literature, which constitutes a challenge in pathology diagnosis, particularly in small biopsy specimens. Here, we present a case of pulmonary HCCC diagnosed by computed tomography-guided percutaneous lung biopsy in a 70-year-old man's right lower lung. Although the morphology and immunophenotype of the tumor suggested the diagnosis of mucoepidermoid carcinoma, fluorescence in situ hybridization failed to reveal the rearrangement of MAML2 gene, which is characteristic of mucoepidermoid carcinoma. Instead, further molecular genetic testing showed that the tumor harbored a rare EWSR1::CREM fusion combined with a previously unreported IRF2::NTRK3 fusion. Pulmonary HCCC is commonly regarded as a low-grade malignant tumor with an indolent course, but this case has a different biological behavior, presenting extensive dissemination and metastases at the time of diagnosis, which expands our understanding of the prognosis of this tumor. The patient has had five cycles of combination chemotherapy and has been alive with the tumor for eight months.

Neutralizing IL-8 potentiates immune checkpoint blockade efficacy for glioma.

Malignant gliomas are largely refractory to immune checkpoint blockade (ICB) therapy. To explore the underlying immune regulators, we examine the microenvironment in glioma and find that tumor-infiltrating T cells are mainly confined to the perivascular cuffs and express high levels of CCR5, CXCR3, and programmed cell death protein 1 (PD-1). Combined analysis of T cell clustering with T cell receptor (TCR) clone expansion shows that potential tumor-killing T cells are mainly categorized into pre-exhausted/exhausted and effector CD8+ T subsets, as well as cytotoxic CD4+ T subsets. Notably, a distinct subpopulation of CD4+ T cells exhibits innate-like features with preferential interleukin-8 (IL-8) expression. With IL-8-humanized mouse strain, we demonstrate that IL-8-producing CD4+ T, myeloid, and tumor cells orchestrate myeloid-derived suppressor cell infiltration and angiogenesis, which results in enhanced tumor growth but reduced ICB efficacy. Antibody-mediated IL-8 blockade or the inhibition of its receptor, CXCR1/2, unleashes anti-PD-1-mediated antitumor immunity. Our findings thus highlight IL-8 as a combinational immunotherapy target for glioma.

Expert Consensus on Pathological Diagnosis of Intrahepatic Cholangiocarcinoma (2022 version).

Intrahepatic cholangiocarcinoma (iCCA) can originate from the large bile duct group (segment bile ducts and area bile ducts), small bile duct group (septal bile ducts and interlobular bile ducts), and terminal bile duct group (bile ductules and canals of Hering) of the intrahepatic biliary tree, which can be histopathological corresponding to large duct type iCCA, small duct type iCCA and iCCA with ductal plate malformation pattern, and cholangiolocarcinoma, respectively. The challenge in pathological diagnosis of above subtypes of iCCA falls in the distinction of cellular morphologies, tissue structures, growth patterns, invasive behaviors, immunophenotypes, molecular mutations, and surgical prognoses. For these reasons, this expert consensus provides nine recommendations as a reference for standardizing and refining the diagnosis of pathological subtypes of iCCA, mainly based on the 5th edition of the World Health Organization Classification of Tumours of the Digestive System.

Perivascular Epithelioid Cell Tumor of Urinary Bladder.

OBJECTIVES: To review the clinicopathologic features of perivascular epithelioid cell tumor (PEComa) of the urinary bladder. METHODS: Seven cases of bladder PEComa were studied by light microscopy, immunohistochemistry, and fluorescence in situ hybridization (FISH). RESULTS: In our 7 cases, 5 patients were female and 2 were male, with ages between 26 and 78 years. Patients presented with hematuria and recurrent abdominal discomfort as the main clinical symptoms. Microscopically, the epithelioid and spindle-shaped tumor cells with clear to granular eosinophilic cytoplasm were arranged in fascicular, acinar, or nested patterns. The tumor cells were positive for HMB45, melan-A, and SMA, but no TFE3 gene rearrangement was detected in any of the 7 samples by FISH. The analysis of all 35 cases from the literature and ours showed a patient age range from 16 to 78 years (mean age, 39 years), a male-to-female ratio of 1:1.3, maximal tumor diameters from 0.6 to 18.8 cm (mean, 4.5 cm). With a mean follow-up of 27 months, the recurrence, metastasis, and mortality rates were 10.7%, 10.7%, and 7.1%, respectively. CONCLUSIONS: Bladder PEComa is extremely rare, remains a diagnostic challenge, and needs more attention. Strengthening the understanding of this tumor will improve diagnostic accuracy.

A standardized pathological proposal for evaluating microvascular invasion of hepatocellular carcinoma: a multicenter study by LCPGC.

BACKGROUND AND AIMS: Microvascular invasion (MVI) is a key pathological factor that severely affects the postoperative prognosis of patients with hepatocellular carcinoma (HCC). However, no MVI classification schemes based on standardized gross sampling protocols of HCC are available at present. METHODS: 119 HCC specimens were sampled at multiple sites (3-, 7-, and 13 points) for the optimum MVI detection rate. 16,144 resected HCCs were graded as M0, M1 or M2 by adopting three-tiered MVI grading (MVI-TTG) scheme based on the seven-point sampling protocol (SPSP). Survival analyses were performed on 2573 patients to explore the advantages of MVI-TTG. RESULTS: The MVI detection rate determined by SPSP was significantly higher than that determined by the 3-point sampling method (34.5% vs. 47.1%, p = 0.048), but was similar to that determined by the 13-point sampling method (47.1% vs. 51.3%, p = 0.517). Among 16,144 resected HCCs, the proportions of M0, M1 and M2 specimens according to SPSP were 53.4%, 26.2% and 20.4%, respectively. Postoperative survival analysis in 2573 HCC patients showed that the 3-year recurrence rates in M0, M1 and M2 MVI groups were 62.5%, 71.6% and 86.1%, respectively (p < 0.001), and the corresponding 3-year overall survival (OS) rates were 94.1%, 87.5% and 67.0%, respectively (p < 0.001). M1 grade was associated with early recurrence, while M2 grade was associated with both early and late recurrence. MVI-TTG had a larger area under the curve and net benefit rate than the two-tiered MVI grading scheme for predicting time to recurrence and OS. CONCLUSIONS: SPSP is a practical method to balance the efficacy of sampling numbers and MVI detection rates. MVI-TTG based on SPSP is a better prognostic predictor than the two-tiered MVI scheme. The combined use of SPSP and MVI-TTG is recommended for the routine pathological diagnosis of HCC.

Sensitive and selective detections of codon 12 and 13 KRAS mutations in a single tube using modified wild-type blocker.

It was hypothesized that in the WTB-PCR system, the greater number of cycles, associated with the thermodynamic driving force of DNA polymerase resulted in artificial introduction of mutant nucleotides in amplicons. In the current study, universal WTB-PCR was developed to overcome these limitations, in which two strategies were used: phosphorothioate modifications were made at the 5'-termini bases of the WTB oligonucleotides, and amplification of referenced internal positive controller (RIPC) fragments was performed. The results showed that universal WTB-PCR could detect single-copy KRAS mutant alleles with higher selectivity (i.e., 0.01%), and with greater ability to eliminate non-specific amplification of KRAS wild-type alleles in amounts up to 200 ng. Moreover, the introduction of referenced internal positive controller (RIPC) fragments prevented false-negative results caused by inadequate amounts of input sample DNA, and allowed for quantitative analysis of the mutation levels in each FFPE sample. In clinical application in 50 samples of FFPE tissue sections from mCRC patients, 70% (35/50) showed various mutations at codons 12 and 13 of KRAS genes; 30% (15/50) could be detected by traditional PCR without WTB oligonucleotides. In conclusion, universal WTB-PCR is a rapid, simple and low-cost method for detection of low-abundance KRAS mutations in mCRC patients.

Nodal follicular dendritic cell sarcoma with four atypical histomorphologic features: an unusual case report.

BACKGROUND: Follicular dendritic cell sarcoma (FDCS) is a rare malignancy. In addition to the classical histopathologic features, it has also some special morphological variants that can present a challenge in the diagnosis of this disease. CASE PRESENTATION: A 45-year-old male who presented with a left supraclavicular mass was given a final diagnosis of FDCS after lymph node biopsy. The specimen obtained during radical resection revealed five different morphologies, including the classical histological appearance and atypical areas resembling desmoplastic infiltrative carcinoma, anaplastic large cell lymphoma (ALCL), hemangiopericytoma and classical Hodgkin's lymphoma (CHL). Immunohistochemistry was notable for positive CD21 and CD23 expression across all morphologies. Given the atypical appearance and location, the specimen was initially misdiagnosed as a metastatic carcinoma based on histology alone at an outside institution. The patient eventually underwent surgical resection followed by adjuvant chemotherapy and radiation. Despite treatment, the disease progressed, and the patient passed away 36 months after surgery. CONCLUSIONS: This unusual case of FDCS contains four types of atypical histomorphologies within a single tumor specimen, including those resembling ALCL and hemangiopericytoma which are described here for the first time. Our report further expands the histopathologic spectrum of FDCS and may help assist in the diagnosis of other such challenging cases.

Mediastinal follicular dendritic cell sarcoma: a rare, potentially under-recognized, and often misdiagnosed disease.

BACKGROUND: Mediastinal follicular dendritic cell sarcoma (FDCS) is extremely rare. Due to potential under-recognization of this disease, it happens to be misdiagnosed, especially on core needle biopsy. We report 3 cases of mediastinal FDCS and provide a literature review to improve better understanding of the tumor and to reduce misdiagnosis. METHODS: Three cases of mediastinal FDCS in our clinic practice were studied, including their core needle biopsy and resected specimens, and those cases reported previously in English literature were retrieved and analyzed. RESULTS: The core needle biopsy of case 1 showed a tumor reminiscent of classical Hodgkin's lymphoma (CHL), while the resected mass was finally diagnosed with FDCS combined with hyaline-vascular Castleman's disease. Both the biopsy and resected tissue of case 2 were constitutive of the clear epithelioid cells with marked atypia. In both cases, definitive diagnoses were not made on core needle biopsy. In case 3, there were some areas morphologically similar to CHL, and some areas contained ovoid to spindle-shaped tumor cells with fascicular pattern. The analysis of 43 cases of mediastinal FDCS showed the age of patients were from 16 to 76 years old, the male to female ratio was 1.5:1, the maximal tumor diameters were 3-17 cm. 18 cases were underwent preoperative biopsy, whereas 15 (83.3%) of which were misdiagnosed initially, often as lymphoma. 32 patients had available follow-up data, the rates of recurrence, metastasis, and mortality were 12.5, 18.8 and 28.1%, respectively. Current limited data suggested no statistical differences between adverse prognosis and gender, age, tumor size, necrosis, or different therapeutics, respectively. CONCLUSIONS: Mediastinal FDCS is a rare malignancy that has yet not been fully understood and been often misdiagnosed, particularly when making a diagnosis on core needle biopsy. Increased awareness of this enigmatic tumor is crucial to avoid diagnostic pitfalls.

Primary follicular dendritic cell sarcoma of the urinary bladder: the first case report and potential diagnostic pitfalls.

BACKGROUND: Extranodal follicular dendritic cell sarcoma (FDCS) is a very rare malignancy with a variable clinical course. It is often not considered and has the potential to result in a misdiagnosis of other common sarcomas or sarcomatoid carcinomas. This is particularly true with the preoperative biopsy specimen, in which the tissue sample is often small. CASE PRESENTATION: A case of FDCS in a 63-year-old woman, arising in the urinary bladder, a previously unreported site, is described. The patient presented with the typical clinical symptoms of a bladder cancer, and the morphology of the tumor was similar to a lymphoepithelioma-like carcinoma, ultimately resulting in it being misdiagnosed. The patient received radical cystectomy, without further radiotherapy or chemotherapy. Two years after operation, a metastatic tumor to the lung was found. The mass of the right main bronchus lumen was frozen and resected through bronchoscopy, and radiotherapy was performed. The patient has lived with the tumor since then. CONCLUSIONS: This paper presents the first FDCS occurring in the urinary bladder with metastasis to the lung and emphasizes potential diagnostic pitfalls.

Improved detection of BRAF V600E using allele-specific PCR coupled with external and internal controllers.

Although traditional allele-specific PCR (tAS-PCR) is a common screening method for BRAF V600E mutations, its lower amplification specificity and mutation selectivity have limited its clinical applications. We hypothesize that these limitations are associated with the weaker specificities of allele-specific primers and the thermodynamic driving forces of DNA polymerase. We used three strategies to circumvent these limitations, namely, modifying allele-specific primers, introducing a competitive external allele-specific controller (i.e., cAS-PCR), and introducing a referenced internal positive controller in the cAS-PCR (i.e., rcAS-PCR). The amplification sensitivities and specificities were influenced by the position of the artificially introduced mismatched nucleotide in the allele-specific primers. Moreover, both cAS-PCR and rcAS-PCR could detect single-copy BRAF V600E alleles with higher mutation selectivity (0.1%) than tAS-PCR. In addition, cAS-PCR eliminated false-negative results caused by various PCR inhibitors that might be present in the DNA solutions. The rcAS-PCR could also be employed to avoid the false-negative results caused by low-abundance input templates in cAS-PCR. In conclusion, rcAS-PCR provides a rapid, simple, and low-cost method for detecting low levels of the mutated BRAF V600E gene.

[Characteristic and clinicopathologic significance of lymphangiogenesis in colorectal cancer].

OBJECTIVE: To investigate the distribution patterns and proliferative activity of lymphatic vessels in colorectal carcinomas (CRC) and their relationship with tumor metastasis and disease prognosis. METHODS: The microlymphatic density (MLD) and microvascular density in tumoral and non-tumoral areas of 96 cases of CRC were evaluated by immunohistochemistry, using monoclonal antibodies for podoplanin and CD34 respectively. The Ki-67 expression of the lymphatic and blood vessels was detected by double-labeling immunohistochemistry. The relationship between MLD and clinicopathologic features and prognosis was analyzed. RESULTS: The lymph vessels at central and superficia1 portions of CRC often had a reticular architecture with numerous tiny and ill-defined lumina, while those at the tumor borders had large and open lumina. The MLD at tumor borders (51.2 +/- 25.5) was significantly higher than that in normal colorectal mucosa (29.4 +/- 9.0) and other portions of CRC (P < 0.01). The Ki-67 labeling index of the lymphatic lining cells at tumor borders (0.23 +/- 0.17) was significantly higher than that in other portions of CRC (P < 0.05). The MLD significantly correlated with lymphatic involvement by tumor cells, regional lymph node metastasis and distant metastasis (P < 0.01). The 5-year survival rate was also significantly lower in patients with high MLD (P < 0.05). CONCLUSIONS: Neolymphatic vessels are commonly seen in CRC, especially at tumor borders. High MLD at tumor borders is associated with metastasis. The detection of MLD at tumor borders may thus be useful in predicting lymph node metastasis and prognosis in patients with CPC.

Single-Tubed Wild-Type Blocking Quantitative PCR Detection Assay for the Sensitive Detection of Codon 12 and 13 KRAS Mutations.

The high degree of intra-tumor heterogeneity has meant that it is important to develop sensitive and selective assays to detect low-abundance KRAS mutations in metastatic colorectal carcinoma (mCRC) patients. As a major potential source of tumor DNA in the aforementioned genotyping assays, it was necessary to conduct an analysis on both the quality and quantity of DNA extracted from formalin-fixed paraffin-embedded (FFPE). Therefore, four commercial FFPE DNA extraction kits were initially compared with respect to their ability to facilitate extraction of amplifiable DNA. The results showed that TrimGen kits showed the greatest performance in relation to the quality and quantity of extracted FFPE DNA solutions. Using DNA extracted by TrimGen kits as a template for tumor genotyping, a real-time wild-type blocking PCR (WTB-PCR) assay was subsequently developed to detect the aforementioned KRAS mutations in mCRC patients. The results showed that WTB-PCR facilitated the detection of mutated alleles at a ratio of 1:10,000 (i.e. 0.01%) wild-type alleles. When the assay was subsequently used to test 49 mCRC patients, the results showed that the mutation detection levels of the WTB-PCR assay (61.8%; 30/49) were significantly higher than that of traditional PCR (38.8%; 19/49). Following the use of the real-time WTB-PCR assay, the ΔCq method was used to quantitatively analyze the mutation levels associated with KRAS in each FFPE sample. The results showed that the mutant levels ranged from 53.74 to 0.12% in the patients analyzed. In conclusion, the current real-time WTB-PCR is a rapid, simple, and low-cost method that permits the detection of trace amounts of the mutated KRAS gene.

[The significance of beta-catenin and matrix metalloproteinase-7 expression in colorectal adenoma and carcinoma].

OBJECTIVE: To investigate the relationship between beta-catenin and matrix metalloproteinase-7 (MMP-7) expression and development/biologic behavior of human colorectal cancer. METHODS: Immunohistochemical study for beta-catenin and MMP-7 was carried out on colorectal adenoma-carcinoma tissue microarrays and results analyzed. RESULTS: The nuclear beta-catenin expression rate was 35.9% in adenoma with malignant transformation, significantly higher than that in adenoma (16.7%) and carcinoma (19.7%) (both P < 0.05). The cytoplasmic and nuclear beta-catenin expression rate in adenoma with severe dysplasia was significantly higher than that in adenoma with mild dysplasia (both P < 0.05). The nuclear beta-catenin expression rate in adenocarcinomas of the ulcerative type, with lymph node metastasis and in the late tumor stages were all significantly higher than that in adenocarcinomas of the polypoid type, with negative lymph node and in the early tumor stages (P < 0.05 or P < 0.01). The MMP-7 expression rate in adenocarcinoma (69.2%) was significantly higher than that in normal colorectal mucosa (15.0%), adenoma (35.0%) and adenoma with malignant transformation (46.2%, P < 0.05 or P < 0.01). The MMP-7 expression rate in ulcerative type adenocarcinoma with lymph node metastasis and in late tumor stages was significantly higher than that in polypoid type adenocarcinoma with negative lymph node and in early tumor stages (all P < 0.05). The cytoplasmic and nuclear beta-catenin expression was thus in positive correlation with the expression of MMP-7 (both P < 0.01). CONCLUSIONS: The cytoplasmic and nuclear beta-catenin expression, probably an early event, was related to the development of colorectal cancer. beta-catenin may enhance the degradative function of the target gene MMP-7 through nuclear translocation and may further facilitate local invasion and metastasis by the colorectal cancer cells.

Endogenous axon guiding chemorepulsant semaphorin-3F inhibits the growth and metastasis of colorectal carcinoma.

PURPOSE: To elucidate the role of Semaphorin-3F (SEMA3F), originally described as an axon guiding chemorepulsant implicated in nerve development, in the progression of colorectal carcinoma. EXPERIMENTAL DESIGN: SEMA3F and its receptor NRP2 were examined in 72 cases of human colorectal carcinoma specimens and cell lines LoVo, SW480, and SW620 with immunohistochemistry and Western blotting. SEMA3F mRNA expression in the frozen tissue specimens and cell lines was examined with quantitative reverse transcriptase-PCR. Confocal laser scanning microscopy was used for detection of cellular localization of the proteins by immunofluorescent staining. MTT assay, flow cytometry, cell adhesion and migration, and xenografts were used to evaluate biological significance of SEMA3F. RESULTS: SEMA3F was significantly reduced in colorectal carcinoma tissues and cell lines. Overexpression of SEMA3F resulted in reduced proliferation, adhesion to fibronectin, and migratory capability as well as reduced S-phase population and integrin αvß3 expression of SW480 colon cancer cells. In addition, SEMA3F-overexpressing cells exhibited diminished tumorigenesis when transplanted orthotopically in nude mice and reduced liver metastases. Moreover, transfection of siRNA targeting SEMA3F in colon cancer cells increased their tumorigenicity in vivo. CONCLUSIONS: Endogenous SEMA3F acts as a suppressor of the growth and metastasis of human colorectal cancer cells.

A synthetic MD-2 mimetic peptide attenuates lipopolysaccharide-induced inflammatory responses in vivo and in vitro.

Myeloid differentiation protein-2 (MD-2), a secreted glycoprotein that binds to both lipopolysaccharide (LPS) and toll like receptor 4 (TLR4), contributes to the fine ligand recognition and signaling activation on LPS-induced inflammation. Here we synthesized a novel MD-2 mimetic peptide (MDMP), derived from the putative LPS-binding domain and TLR4-binding domain of MD-2, and found that MDMP dose-dependently bound to LPS and inhibited LPS-activated Limulus amebocyte lysate (LAL). Pretreatment with MDMP dampened LPS-induced inflammatory responses in RAW264.7 cells, including down-regulation of TLR4-MD-2 complex on the cell surface, suppression of LPS binding to the cells, inhibition of mitogen-activated protein kinase (MAPKs) and nuclear factor kappa B (NF-kappaB) activation, reduction of tumor necrosis factor-alpha (TNF-alpha) production. Further, in vivo pretreatment with MDMP markedly protected against LPS-induced acute lung injury and liver injury, as indicated by the notable reduction of lethality, inflammatory responses and TNF-alpha production. These results demonstrate that MDMP attenuates LPS-induced inflammatory responses in vivo and in vitro, and suggests that MDMP may be useful in the treatment of inflammation associated with LPS.

Extranodal follicular dendritic cell sarcoma of the pharyngeal region: a potential diagnostic pitfall, with literature review.

Extranodal follicular dendritic cell sarcoma (FDCS) of the pharyngeal region is a rare malignant tumor recognized in recent years, with approximately 37 cases so far reported in the literature. It is often not considered at the initial evaluation and may be misdiagnosed in a small biopsy specimen. We report 4 cases of extranodal FDCS, 2 cases in the nasopharynx that were diagnosed as undifferentiated carcinomas because they were characterized by syncytial epithelial cells with sheet or nest-like distribution and 2 cases in the tonsil and soft palate that were characterized by vaguely concentric whorls consisting of spindle to ovoid cells. The latter case was diagnosed as ectopic meningioma. The analysis of all cases from the literature and ours shows that 58% (21/36) of the cases are misdiagnosed initially, often as undifferentiated carcinoma or meningioma, which the differential diagnoses should be mostly focused on. With a median follow-up of 27 months, the recurrence, metastasis, and mortality rates are 23%, 21%, and 3%, respectively, suggesting that extranodal FDCS of the pharyngeal region remains a low-grade sarcoma. Radical surgery is recommended, whereas there is no evidence to support adjuvant therapy.

[Clinical significance of E-cadherin and proliferating cell nuclear antigen expression in adenoid cystic carcinoma].

BACKGROUND & OBJECTIVE: The recurrence and metastasis after treatment of adenoid cystic carcinoma was usually observed in the clinical practice. There is no good indicator to evaluate the prognosis of ACC at present. This study was designed to investigate the relationship between the expression of cell adhesion molecules E-cadherin (E-cad) and proliferating cell nuclear antigen (PCNA) and prognosis in the patients with ACC. METHODS: The expression of E-cad and PCNA in 34 patients with ACC were detected by immunohistochemical staining. RESULTS: The 5-year survival rate of the patients was significantly lower in solid type of ACC than in cribriform or tubular type (P < 0.01). Clinical stages were significantly related to local recurrence and/or distant metastasis of the tumor (P < 0.01) and to 5-year survival rate of the patients with ACC (P < 0.01). Absent or low E-cad expression (50.0%) was observed more frequently in ACC with local recurrence and/or distant metastasis and with less than 5-year survival period (P < 0.05). The rate of local recurrence and/or distant metastasis of the tumor was significantly higher in high PCNA expression group (P < 0.005) than in low PCNA expression group, while the 5-year survival rate of the former was significantly lower (P < 0.005). The expression of E-cad was in high correlation with that of PCNA (P < 0.005). CONCLUSIONS: E-cad, PCNA expression, and clinical stages could be regarded as effective indices for evaluating the prognosis of ACC. E-cad may cooperate with PCNA in the malignant development of ACC.