Serum IgA as a Prognostic Beacon: Anticipating Systemic Therapy Efficacy in Metastatic Clear Cell Renal Cell Carcinoma.

OBJECTIVE: Immunotherapy-tyrosine kinase inhibitor (IO-TKI) therapy has revolutionized the treatment landscape for metastatic clear cell renal cell carcinoma (mccRCC); however, the absence of effective biomarkers poses a challenge in predicting the efficacy of these regimens. This study aims to explore the predictive and prognostic value of serum immunoglobulin A (IgA) in mccRCC patients undergoing IO-TKI therapy. METHODS: Ninety-six mccRCC patients treated with IO-TKI therapy from 2019 to 2023 were enrolled and serum IgA levels were assessed at the pretreatment baseline and after 3 months of treatment. RESULTS: Notably, baseline levels of IgA showed no correlation with the objective response rate. However, patients achieving complete or partial responses exhibited a remarkable decrease in IgA levels, while those with stable or progressive disease displayed an increase in IgA levels after 3 months of treatment. Furthermore, the dynamic alteration in IgA levels after 3 months of treatment demonstrated predictive value for both progression-free survival (PFS) and overall survival (OS). The time-dependent receiver operating characteristic curves exhibited outstanding performance in predicting PFS (AUC 0.793) and OS (AUC 0.738). CONCLUSION: Taken together, this study demonstrates that dynamic alteration of serum IgA after 3 months of treatment was significantly correlated with prognosis and therapeutic efficacy in mccRCC patients.

Prognosis and Clinicopathological Characters of Adult TFEB-Altered Renal Cell Carcinoma: A Single Center Experience of 18 Cases.

INTRODUCTION: TFEB-altered renal cell carcinoma (RCC) is a rare entity characterized by the rearrangement of the TFEB gene or TFEB amplified. The therapeutic implications and long-term survival of TFEB-altered RCC remain unclear, especially for metastatic cases. MATERIALS AND METHODS: The current study initially enrolled 7604 consecutive RCC patients at our center and a total of 248 patients were selected for FISH and immunohistochemistry (IHC) analysis. Eventually, eighteen TFEB-altered RCC patients were identified. We then reported the clinical, morphological, IHC, and radiological features of these cases. RESULTS: The median age at initial diagnosis was 45 years, ranging from 18 years to 66 years. The majority of the TFEB-altered RCC patients were male (61.1%), with localized disease (T1-2N0M0, 77.8%). The median split TFEB fluorescent signal was 24%, ranging from 15%-80%. The morphological characteristics of TFEB-altered RCC were variable, with acinar, papillary, solid, or nest patterns. IHC and magnetic resonance imaging features of TFEB-altered RCC were nonspecific. Nine patients with localized disease received partial nephrectomy and five patients with localized disease received radical nephrectomy. During the median follow-up of 67 months, no signs of recurrence or metastasis were found in these patients. Two patients had distant metastasis and received axitinib plus PD-1 immunotherapy. One of them died at 40-month follow-up and another still alive at 88-month follow-up. CONCLUSION: TFEB-altered RCC is an extremely rare variant, exhibited mixed morphological characteristics. The radiological feature lack specificity, resembling clear cell RCC or papillary RCC. Genetic analyses including FISH analysis is crucial in the diagnosis of TFEB-altered RCC. For localized TFEB-altered RCC, both radical nephrectomy and partial nephrectomy conferred satisfactory prognosis. For metastatic TFEB-altered RCC, immunotherapy-based drug combinations could be a promising treatment strategy.

Dynamic alteration and prognostic significance of tumor-associated CD68+ and CD68+ PD-L1- macrophages in muscle-invasive bladder cancer treated with neoadjuvant chemotherapy.

BACKGROUND: The current study aimed to investigate the dynamic alteration and prognostic significance of tumor-infiltrating lymphocytes (TILs), tumor-associated macrophages (TAMs), and PD-L1 status of immune cells in muscle-invasive bladder cancer (MIBC) treated with neoadjuvant chemotherapy (NAC). METHODS: Multiplex immunofluorescence staining was performed to examine CD68+ TAM, CD4+ T cell, CD8+ T cell, FOXP3+ Treg cell, and PD-L1 expression in paired MIBC tissues (n = 54) before and after NAC. Patients were then divided into definite responders (DR), (≤pT1) and incomplete responders (IR). RESULTS: There was no significant difference between DR and IR cohorts for the immune cell infiltration levels at the baseline status. Tobacco history was identified to be associated with worse NAC efficacy. CD68+ (stroma area: p = 0.025; tumor area: p = 0.028; total area: p = 0.013) and CD68+ PD-L1- (stroma area: p = 0.035; tumor area: p = 0.013 total area: p = 0.014) TAMs infiltration levels decreased significantly after NAC, while there was no significant difference of CD68+ PD-L1+ and TILs. The infiltration of CD68+ (p = 0.033), CD68+ PD-L1- (p = 0.033), and CD68+ PD-L1+ (p < 0.001) TAMs in stroma area were significantly associated with poorer disease-free survival rate (DFS) of MIBC patients. CONCLUSION: CD68+ and CD68+ PD-L1- TAMs infiltration levels decreased significantly after NAC and pre-treatment TAM infiltration levels were independent prognostic factors for MIBC patients. While there was no sufficient evidence demonstrating that pre-treatment TILs or TAMs could predict response to NAC in MIBC patients.

Disease Management of Clinical Complete Responders to Neoadjuvant Chemotherapy of Muscle-Invasive Bladder Cancer: A Review of Literature.

Muscle-invasive bladder cancer (MIBC) is an aggressive disease requiring active management. Neoadjuvant chemotherapy (NAC) followed by radical cystectomy (RC) is considered the standard treatment paradigm for MIBC patients, which could result in significant perioperative mortality and morbidity, as well as the significant alteration of the quality of life (QOL). Notably, multimodal bladder-preserving treatment strategies have been recommended for highly selected patients. Pathologic complete response (pCR) after NAC is a powerful prognostic indicator of survival for patients with MIBC. Clinical complete response (cCR) is then introduced as a complementary endpoint for pCR to assess disease status preoperatively. Bladder preservation strategy for patients who achieve cCR following NAC is emerging as a new treatment concept. However, the efficiency of the conservative strategy remains controversial. In this state-of-the-art review, we discuss the advantages and limitations of cCR and the feasibility and safety of bladder preservation strategy in highly selected MIBC patients who achieve cCR following NAC. We conclude that a conservative strategy can be considered a reasonable alternative to RC in carefully selected cCR MIBC patients, leading to acceptable oncological outcomes.

Survival Analysis of Lymphoepithelioma-Like Carcinoma of the Urinary Bladder and the Effect of Surgical Treatment Modalities on Prognosis.

Purpose: This study aimed to investigate the prognostic factors of patients with lymphoepithelioma-like carcinoma of the urinary bladder (LELCB) and explore the value of surgical treatment. Methods: Data of patients with LELCB were extracted from the Surveillance, Epidemiology, and End Results (SEER) database. The multivariate analysis was performed using the stepwise Cox proportional hazards regression model and conditional inference tree method to identify significant prognosticators of overall survival (OS) from the parameters such as age, gender, lymph node involvement, tumor extent, radiation, chemotherapy, and surgery type. Literature review (LR) was performed, and eligible cases were used to validate prognostic classification using the Kaplan-Meier method with log-rank tests. Results: Sixty patients with a median age of 69.5 years were identified from the SEER database and 91 patients through LR. The Cox analysis identified age, gender, lymph node involvement, and surgical approach as independent prognosticators of OS. Based on the nomogram scores, patients were stratified into three prognostic groups: (I) patients younger than 70 years; (II) patients older than 70 years, who received bladder-sparing therapy (BST); and (III) patients older than 70 years undergoing radical cystectomy (RC). Patients in group II had the worst outcomes in terms of OS compared with patients in groups I and III (p < 0.001 and p = 0.03, respectively). A similar survival pattern was found in the LR cohort. Conclusion: The nomogram provided individualized prognostic quantification of OS in patients with LELCB. BST could yield favorable outcomes when treating LELCB, especially for younger patients, whereas older patients might derive more survival benefit from RC.

[Diagnosis and Treatment of 126 Cases of Chromophobe Renal Cell Carcinoma].

Objective To investigate the clinicopathological features and prognosis of chromophobe renal cell carcinoma(ChRCC). Methods The clinical and pathological data of 126 patients with ChRCC treated in Cancer Hospital of Chinese Academy of Medical Sciences were retrospectively analyzed. Results The patients included 64 males and 62 females,with the age of 22-80 years(median of 52 years).The tumor was located on the right side in 70 cases and on the left side in 56 cases.Ultrasound,CT or magnetic resonance imaging(MRI)were performed.Of the 110 cases receiving ultrasound examination,63,23,13,10,and 1 cases showed hypoecho,hyperecho,isoecho,uneven or mixed echo,and dark hypoecho,respectively.Color Doppler flow imaging showed no blood flow signal in 42 cases and low blood flow signal in 60 cases out of 68 cases with blood flow signal.Among the 54 cases receiving CT,50 cases showed equal density or low density and 4 cases showed high density with clear boundary.The enhanced scanning showed mild to moderate uniform or non-uniform reinforcement,mostly below the renal parenchyma,and still showed reinforcement in the delayed period.Among the 97 cases receiving MRI,96 cases showed hypo-or isointense signals and 1 case showed hyperintense signal in T1 weighted images;71 cases showed hyper-or isointense signals and 26 cases showed hypo-or isointense signals in T2 weighted images;93 cases showed hyperintense signals with obvious limited diffusion and 4 cases showed unobvious limited diffusion in diffusion weighted images.Mild to moderate uniform or non-uniform reinforcement was observed in most of the enhanced scans.All the 126 patients underwent surgical treatment,including 64 cases of nephron sparing surgery and 62 cases of radical surgery.Pathological examinations confirmed ChRCC for all the patients,including 91 cases of T1N0M0,15 cases of T2N0M0,and 20 cases of T3N0M0.The immunohistochemical assay demonstrated the positive expression rate of 48.2%(54/112)for CD10,92.3%(96/104)for CD117,8.0%(9/112)for vimentin,85.6%(95/111)for CK7,and 97.6%(83/85)for colloidal iron.Conclusions ChRCC is less common,with low level of malignancy and good prognosis.Since the clinical symptoms of ChRCC are not typical,MRI is an important means of imaging differential diagnosis,and the disease can be confirmed depending on pathological diagnosis.Surgery is the preferred treatment method,and currently there is no standard treatment regimen for metastatic patients.

Impact of Cytoreductive Nephrectomy on Survival in Patients with Metastatic Renal Cell Carcinoma Treated by Targeted Therapy.

BACKGROUND: The metastatic renal cell carcinoma (mRCC) patients treated with upfront cytoreductive nephrectomy combined with α-interferon yields additional overall survival (OS) benefits. It is unclear whether mRCC patients treated with vascular endothelial growth factor receptor-tyrosine kinase inhibitor (VEGFR-TKI) will benefit from such cytoreductive nephrectomy either. The aim of the study was to identify variables for selection of patients who would benefit from upfront cytoreductive nephrectomy for mRCC treated with VEGFR-TKI. METHODS: Clinical data on 74 patients enrolled in 5 clinical trials conducted in Cancer Hospital (Institute), Chinese Academy of Medical Sciences from January 2006 to January 2014 were reviewed retrospectively. The survival analysis was performed by the Kaplan-Meier method. Comparisons between patient groups were performed by Chi-square test. A Cox regression model was adopted for analysis of multiple factors affecting survival, with a significance level of α = 0.05. RESULTS: Fifty-one patients underwent cytoreductive nephrectomy followed by targeted therapy (cytoreductive nephrectomy group) and 23 patients were treated with targeted therapy alone (noncytoreductive nephrectomy group). The median OS was 32.2 months and 23.0 months in cytoreductive nephrectomy and noncytoreductive nephrectomy groups, respectively (P = 0.041). Age ≤45 years (P = 0.002), a low or high body mass index (BMI <19 or >30 kg/m2) (P = 0.008), a serum lactate dehydrogenase (LDH) concentration >1.5 × upper limit of normal (P = 0.025), a serum calcium concentration >10 mg/ml (P = 0.034), and 3 or more metastatic sites (P = 0.023) were independent preoperative risk factors for survival. The patients only with 0-2 risk factors benefited from upfront cytoreductive nephrectomy in terms of OS when compared with the patients treated with targeted therapy alone (40.0 months vs. 23.2 months, P = 0.042), while those with more than 2 risk factors did not. CONCLUSIONS: Five risk factors (age, BMI, LDH, serum calcium, and number of metastatic sites) seemed to be helpful for selecting patients who would benefit from undergoing upfront cytoreductive nephrectomy.

Fascin and CK4 as biomarkers for esophageal squamous cell carcinoma.

BACKGROUND: Several studies have suggested that fascin, cytokeratin 14 and cytokeratin 4 may have significant roles as biomarkers for the progression and survival of esophageal squamous cell carcinoma (ESCC). MATERIALS AND METHODS: This study performed immunohistochemistry in tissue microarrays, profiling premalignant lesions and invasive tumors. RESULTS: Fascin increased across the following states as follows: normal-appearing epithelium (26%) to dysplasia (46%) to ESCC (68%), while CK4 was undetectable in ESCC (0%) compared to normal-appearing epithelium (45%) or dysplasia (41%). CK14 was elevated and invariant in expression. In regression analyses, compared to normal-appearing epithelium, higher fascin expression was associated with a 36% increased risk of dysplasia (odds ratio=1.36) and a 56% increased risk of invasive ESCC (odds ratio=1.56). CONCLUSION: Expression of fascin is up-regulated in the transformation from normal-appearing epithelium, through dysplasia, into invasive carcinoma. Expression of CK4, CK14 and fascin did not correlate with patient survival. Fascin has a potential role as an early detection biomarker and CK4 as a tumor marker in ESCC.

Global gene expression profiling and validation in esophageal squamous cell carcinoma and its association with clinical phenotypes.

PURPOSE: Esophageal squamous cell carcinoma (ESCC) is an aggressive tumor with poor prognosis. Understanding molecular changes in ESCC will enable identification of molecular subtypes and provide potential targets for early detection and therapy. EXPERIMENTAL DESIGN: We followed up a previous array study with additional discovery and confirmatory studies in new ESCC cases by using alternative methods. We profiled global gene expression for discovery and confirmation, and validated selected dysregulated genes with additional RNA and protein studies. RESULTS: A total of 159 genes showed differences with extreme statistical significance (P < E-15) and 2-fold differences or more in magnitude (tumor/normal RNA expression ratio, N = 53 cases), including 116 upregulated and 43 downregulated genes. Of 41 genes dysregulated in our prior array study, all but one showed the same fold change directional pattern in new array studies, including 29 with 2-fold changes or more. Alternative RNA expression methods validated array results: more than two thirds of 51 new cases examined by real-time PCR (RT-PCR) showed 2-fold differences or more for all seven genes assessed. Immunohistochemical protein expression results in 275 cases which were concordant with RNA for five of six genes. CONCLUSION: We identified an expanded panel of genes dysregulated in ESCC and confirmed previously identified differentially expressed genes. Microarray-based gene expression results were confirmed by RT-PCR and protein expression studies. These dysregulated genes will facilitate molecular categorization of tumor subtypes and identification of their risk factors, and serve as potential targets for early detection, outcome prediction, and therapy.

Biomarkers of apoptosis and survival in esophageal squamous cell carcinoma.

BACKGROUND: Cancer of the esophagus is a deadly malignancy, and development of biomarkers that predict survival is an urgent need. The apoptotic pathways have been hypothesized as important in progression of esophageal squamous cell carcinoma (ESCC). We investigated a panel of proteins that regulate apoptosis as candidate of biomarkers of prognosis in ESCC. METHODS: Tissue microarray (TMA) including 313 surgically-resected cases of ESCC specimens was built for immunohistochemical interrogation. We evaluated seven genes in the FasL-Fas apoptotic pathway - FasL, Fas, FAS-associated death domain protein (FADD), phosphorylated-FADD, and caspase 8 and 10, and the antiapoptotic protein bcl-2. We studied pathway integrity and relations to risk and clinical factors, and determined the prognostic significance of each marker. RESULTS: Five markers showed strong inter-marker correlations (r > or = 0.28, p < 0.001), including FasL, Fas, FADD, and caspases 8 and 10. FasL and FADD also showed modest correlations with one or more cancer risk factors, but none of the markers was significantly associated with either tumor stage or lymph node metastasis, the only two clinical factors that predicted survival in these ESCC cases. Multivariate-adjusted proportional hazard regression models showed no association between protein expression and risk of death for any of the seven markers examined. CONCLUSION: Individual biomarkers in the apoptosis pathway do not appear to predict survival of patients with ESCC.

Overexpression of CDC25B and LAMC2 mRNA and protein in esophageal squamous cell carcinomas and premalignant lesions in subjects from a high-risk population in China.

Molecular events associated with the initiation and progression of esophageal squamous cell carcinoma (ESCC) remain poorly understood but likely hold the key to effective early detection approaches for this almost invariably fatal cancer. CDC25B and LAMC2 are two promising early detection candidates emerging from new molecular studies of ESCC. To further elucidate the role of these two genes in esophageal carcinogenesis, we did a series of studies to (a) confirm RNA overexpression, (b) establish the prevalence of protein overexpression, (c) relate protein overexpression to survival, and (d) explore their potential as early detection biomarkers. Results of these studies indicated that CDC25B mRNA was overexpressed (>/=2-fold overexpression in tumor compared with normal) in 64% of the 73 ESCC cases evaluated, whereas LAMC2 mRNA was overexpressed in 89% of cases. CDC25B protein expression was categorized as positive in 59% (144 of 243) of ESCC cases on a tumor tissue microarray, and nonnegative LAMC2 patterns of protein expression were observed in 82% (225 of 275) of cases. Multivariate-adjusted proportional hazard regression models showed no association between CDC25B protein expression score and risk of death [hazard ratio (HR) for each unit increase in expression score, 1.00; P = 0.90]; however, several of the LAMC2 protein expression patterns strongly predicted survival. Using the cytoplasmic pattern as the reference (the pattern with the lowest mortality), cases with a diffuse pattern had a 254% increased risk of death (HR, 3.52; P = 0.007), cases with no LAMC2 expression had a 169% increased risk of death (HR, 2.69; P = 0.009), and cases with a peripheral pattern had a 130% greater risk of death (HR, 2.30; P = 0.02). CDC25B protein expression scores in subjects with esophageal biopsies diagnosed as normal (n = 35), dysplastic (n = 23), or ESCC (n = 32) increased significantly with morphologic progression. For LAMC2, all normal and dysplastic patients had a continuous pattern of protein expression, whereas all ESCCs showed alternative, noncontinuous patterns. This series of studies showed that both CDC25B and LAMC2 overexpress RNA and protein in a significant majority of ESCC cases. The strong relation of LAMC2 pattern of protein expression to survival suggests a role in prognosis, whereas the association of CDC25B with morphologic progression indicates a potential role as an early detection marker.

Tissue microarray analysis reveals a tight correlation between protein expression pattern and progression of esophageal squamous cell carcinoma.

BACKGROUND: The development of esophageal squamous cell carcinoma (ESCC) progresses a multistage process, collectively known as precursor lesions, also called dysplasia (DYS) and carcinoma in situ (CIS), subsequent invasive lesions and final metastasis. In this study, we are interested in investigating the expression of a variety of functional classes of proteins in ESCC and its precursor lesions and characterizing the correlation of these proteins with ESCC malignant progression. METHODS: Fas, FADD, caspase 8, CDC25B, fascin, CK14, CK4, annexin I, laminin-5gamma2 and SPARC were analyzed using immunohistochemistry on tissue microarray containing 205 ESCC and 173 adjacent precursor lesions as well as corresponding normal mucosa. To confirm the immunohistochemical results, three proteins, fascin, CK14 and laminin-5gamma2, which were overexpressed in ESCC on tissue microarray, were detected in 12 ESCC cell lines by Western blot assay. RESULTS: In ESCC and its precursor lesions, FADD, CDC25B, fascin, CK14, laminin-5gamma2 and SPARC were overexpressed, while Fas, caspase 8, CK4 and annexin I were underexpressed. The abnormalities of these proteins could be classified into different groups in relation to the stages of ESCC development. They were "early" corresponding to mild and moderate DYS with overexpression of fascin, FADD and CDC25B and underexpression of Fas, caspase 8, CK4 and annexin I, "intermediate" to severe DYS and CIS with overexpression of FADD and CK14, and "late" to invasive lesions (ESCC) and to advanced pTNM stage ESCC lesions with overexpression of CK14, laminin-5gamma2 and SPARC. CONCLUSION: Analyzing the protein expression patterns of Fas, FADD, caspase 8, CDC25B, fascin, CK14, CK4, annexin I, laminin-5gamma2 and SPARC would be valuable to develop rational strategies for early detection of lesions at risk in advance as well as for prevention and treatment of ESCC.

Detection of chromosomal alterations in bladder transitional cell carcinomas from Northern China by comparative genomic hybridization.

To identify chromosome alterations in Chinese bladder cancer, forty-six transitional cell carcinomas of the bladder were analyzed by comparative genomic hybridization. Frequent gains of DNA copy number were observed on 1p (13/46), 1q (13/46), 5p (8/46), 6p (9/46), 7p (7/46), 8q (12/46), 11q (8/46), 17q (11/46), 19q (7/46), 20q (8/46) and Yq (8/46), with minimal overlapping regions at 1p32-pter (10/46), 1q21-q24 (12/46), 5p (8/46), 6p22-p23 (7/46), 7p11.2-p14 (7/46), 8q22-q24 (12/46), 11q13-q14 (8/46), 17q22-qter (11/46), 19q11-13.2 (7/46), 20q11-q13.2 (8/46) and Yq11 (8/46). Losses were predominantly found on 2q (16/46), 5q (8/46), 8p (7/46), 9p (8/46), 9q (13/46), 11p (7/46), 13q (7/46), 17p (12/46), 18q (7/46), Xp (18/46) and Xq (19/46), with smallest overlapping regions at 2q32-qter (16/46), 5q12-q31 (8/46), 8p12-pter (7/46), 9p21-pter (10/46), 9q (13/46), 11p (7/46), 13q13-q22 (7/46), 17p (12/46), 18q21-qter (7/46), Xp (18/46) and Xq (19/46). There were significantly higher frequencies of gains of 1q21-q24 and 17q22-qter in moderately differentiated tumors as compared with those in well-differentiated tumors, indicating a possible association of these two abnormalities with the dedifferentiation of tumor cells. Gains of 1p32-pter, 5p, 6p22-p23, 11q13-q14, 17q22-qter and losses of 2q32-qter, 9q, 17p were more frequent in pT1 as compared with those in pTa carcinomas. Gains at 1q21-q24, 7p11.2-p14, 8q22-q24, 19q, 20q11-q13.2 and losses at 5q12-q31, 8p12-pter, 9p21-pter, 11p, 13q13-q22 and 18q21-qter were unique to pT1 and higher stage tumors, suggesting that genes responsible for the invasion and progression of bladder cancer might be located at these chromosomal regions. In multiple tumors from the same patients, consistent alterations such as gains of 8q, 11q13-q14, 12q13-q15, 13q12, 20q and losses of 2q32-qter, 8p, 9, 11p, 11q21-qter, 13q13-qter, X were detected. These abnormalities were possibly earlier events, which might play a critical role during the genesis of the tumors. Further detailed studies to the recurrent aberration regions may lead to the identification of oncogenes and tumor suppressor genes involved in the development and progression of Chinese bladder cancer.

Comprehensive characterization of annexin I alterations in esophageal squamous cell carcinoma.

PURPOSE: The purpose is to characterize alterations of the annexin I gene, its mRNA, and protein expression in esophageal squamous cell carcinoma. EXPERIMENTAL DESIGN: Fifty-six cases of esophageal squamous cell carcinoma were analyzed using four microsatellite markers flanking the annexin I gene (9q11-q21) to identify loss of heterozygosity. In addition, we performed (a) single-strand conformation polymorphism and DNA sequencing along the entire promoter sequence and coding region to identify mutations, (b) real-time quantitative reverse transcription-PCR of RNA from frozen esophageal squamous cell carcinoma tissue (n = 37) and in situ hybridization (n = 5) on selected cases to assess mRNA expression, and (c) immunohistochemistry (n = 44) to evaluate protein expression. The prevalence of the allelic variants identified in the first 56 patients was refined in 80 additional esophageal squamous cell carcinoma patients and 232 healthy individuals. RESULTS: Forty-six of 56 (82%) esophageal squamous cell carcinoma patients showed loss of an allele at one or more of the four microsatellite markers; however, only one (silent) mutation was seen. Two intragenic variants were identified with high frequency of allelic loss (A58G, 64%; L109L, 69%). Thirty of 37 (81%) esophageal squamous cell carcinoma patients showed reduced annexin I mRNA expression, which was confirmed by in situ hybridization, whereas annexin I protein expression was reduced in 79% of poorly differentiated tumor cell foci but in only 5% of well-differentiated tumor foci, although allelic loss on chromosome 9 was found in both tumor grades. CONCLUSIONS: Allelic loss of annexin I occurs frequently, whereas somatic mutations are rare, suggesting that annexin I is not inactivated in esophageal squamous cell carcinoma via a two-hit mechanism. A decrease in annexin I protein expression was confirmed, consistent with a quantitative decrease in mRNA expression, and appeared to be related to tumor cell differentiation. We conclude that annexin I is not the tumor suppressor gene corresponding to the high levels of loss of heterozygosity observed on chromosome 9 in esophageal squamous cell carcinoma; however, dysregulation of mRNA and protein levels is associated with this tumor type.