CK19、Galectin-3、HBME-1、p27及CyclinD1在甲状腺乳头状癌中的表达及意义

背景与目的:甲状腺乳头状癌的诊断有时会很困难,而淋巴结转移是甲状腺乳头状癌局部复发的主要原因并影响预后.本研究探讨CK19、Galectin-3、HBME-1、p27及CyclinD1在甲状腺乳头状癌中的表达特点和诊断价值以及p27、CyclinD1与乳头状癌淋巴结转移间的关系.方法:收集2005年1月至2008年12月间在我院手术的50例甲状腺良性病变患者 (结节性甲状腺肿40例,甲状腺腺瘤10例)及50例甲状腺乳头状癌(经典型40例,滤泡型10例) 患者的手术标本,用免疫组化EnViSion法检测CK19、HBME-1、Galectin-3、p27及CyclinD1的表达.结果:细胞质或细胞膜均有CK19、Galectin-3和HBME-1三种标志物表达,但p27和CyclinD1则在细胞核表达.CK19、HBME-1、Galectin-3在甲状腺乳头状癌中的表达阳性率分别为100%、96%和100%,而在良性病变中则为10%、10%和6%,两者比较差异有显著性(P<0.01).p27在甲状腺乳头状癌的表达阳性率为14%,而在良性病变中则为84%,两者比较差异有显著性(P<0.01).CyclinD1在甲状腺良、恶性病变中的表达阳性率分别为68%和78%,两者间差异无显著性(P>0.05).在甲状腺乳头状癌患者中,CK19、Galectin-3和HBME-1共同表达者占98%(49/50),其中伴p27低表达者占90%(45/50),而在良性病变者中则分别为2%(1/50)和0%(0/50),前两者与后者之间相比,差异有显著性(P<0.01).p27低表达与CyclinD1表达(转移组阳性率72.7%,未转移组71.4%)与甲状腺乳头状癌淋巴结转移间无显著相关性(P>0.05).结论:联合检测CK19、Galectin-3和HBME-1对甲状腺乳头状癌的诊断与鉴别诊断具有重要价值,而p27的低表达与CyclinD1表达对甲状腺乳头状癌的淋巴结转移无提示作用.     

Defining a molecular phenotype for benign and malignant parathyroid tumors

BACKGROUND:

It is frequently difficult to establish histologically whether a parathyroid tumor is a parathyroid carcinoma, parathyromatosis, or an atypical adenoma. The authors asked whether these tumors have a distinctive molecular profile, whether benign tumors could be distinguished from malignant tumors, and whether parathyromatosis is a low‐grade parathyroid carcinoma or is benign tissue that can invade other organs.

METHODS:

Samples of parathyroid carcinoma, atypical adenoma, parathyromatosis, parathyroid adenoma, and hyperplasia were obtained for tissue microarray studies. The molecular expression of genes involved in parathyroid tumor progression (HRPT2 [“parafibromin”], galectin‐3, Ki‐67, Rb, p27, and mdm‐2) was investigated by immunohistochemistry.

RESULTS:

Complete loss of parafibromin expression was seen in 5 of 16 (31.3%) parathyroid carcinomas; all parathyromatosis, atypical adenomas, adenomas, and hyperplasia stained positive for parafibromin. Loss of Rb expression was seen in 5 (33.3%) of 15 parathyroid carcinomas and 1 (7.1%) of 14 parathyroid hyperplasias; all parathyromatosis, atypical adenomas, and adenomas stained positive. Galectin‐3 stained strongly positive in 14 (93.3%) of 15 parathyroid carcinomas, and positive in 3 (18.7%) of 16 cases of parathyromatosis, 2 (100%) of 2 atypical adenomas, 1 (5.6%) of 18 adenomas, and 2 (14.3%) of 14 hyperplasias. The Ki‐67 proliferative index was high in 9 (60%) of 15 parathyroid carcinomas, 1 (6.7%) of 15 cases of parathyromatosis, 1 (5.6%) of 18 adenomas, and no atypical adenomas or hyperplasia. P27 and mdm‐2 protein expression did not differ appreciably among the tumor types.

CONCLUSIONS:

No single diagnostic marker currently determines whether a parathyroid tumor is a parathyroid carcinoma, but loss of parafibromin and Rb expression, and overexpression of galectin‐3, generally distinguish parathyroid carcinoma from other parathyroid tumors. Parathyromatosis does not appear to be a low‐grade parathyroid carcinoma. Cancer 2009. © 2009 American Cancer Society.     

Fine-needle aspirations of papillary carcinoma with oncocytic features: an expanded cytologic and histologic profile

BACKGROUND:

Although a wide variety of papillary carcinomas of the thyroid can have abundant granular cytoplasm and may be difficult to distinguish from Hürthle cell lesions in fine‐needle aspirations (FNAs), the literature on these tumors is limited. The author described 18 cases with a spectrum of cytologic appearances.

METHODS:

A series of 7089 FNAs was correlated with 1331 subsequent resection specimens. Cases in which the original cytologic and histologic diagnoses included the differential diagnosis of papillary carcinoma or Hürthle cell lesions were identified.

RESULTS:

A total of 18 (1.3% of cases with resection) cases were identified. On review, 3 cases had classic features of papillary carcinoma, including nuclear crowding, along with a moderate amount of granular cytoplasm. Four cases had a population of cells that mimicked repair and/or cyst‐lining cells with almost no other epithelial cells. In 2 of those 4 cases, the cells were extremely large, and in 2 other cases, they could not be distinguished from typical cyst‐lining cells. The remaining 11 cases had cells with overlapping features including pale to granular chromatin, small to medium nucleoli either centrally or eccentrically, occasional grooves, and rare intranuclear inclusions. Typical Hürthle cells also were commonly present. Nuclear crowding was not present, and the cells were in sheets, follicles, or appeared alone. No papillae were identified. On resection, 7 cases were follicular variants of papillary carcinoma, 2 cases occurred in the setting of Hashimoto thyroiditis, and 2 cases had features of the tall‐cell variant.

CONCLUSIONS:

The author concluded that a subset of papillary carcinomas of the thyroid were difficult to distinguish from Hürthle cell lesions or repair and/or cyst‐lining cells because of the presence of abundant granular cytoplasm and a lack of nuclear crowding. These tumors were often follicular or cystic variants of papillary carcinoma. Cancer (Cancer Cytopathol) 2011;. © 2011 American Cancer Society.     

Microsatellite instability in thyroid tumours and tumour-like lesions

Fifty-one thyroid tumours and tumour-like lesions were analysed for instability at ten dinucleotide microsatellite loci and at two coding mononucleotide repeats within the transforming growth factor β (TGF-β) type II receptor (TβRII) and insulin-like growth factor II (IGF-II) receptor (IGFIIR) genes respectively. Microsatellite instability (MI) was detected in 11 out of 51 cases (21.5%), including six (11.7%) with MI at one or two loci and five (9.8%) with Ml at three or more loci (RER⁺ phenotype). No mutations in the TβRII and IGFIIR repeats were observed. The overall frequency of MI did not significantly vary in relation to age, gender, benign versus malignant status and tumour size. However, widespread MI was significantly more frequent in follicular adenomas and carcinomas than in papillary and Hürthle cell tumours: three out of nine tumours of follicular type (33.3%) resulted in replication error positive (RER⁺), versus 1 out of 29 papillary carcinomas (3.4%, P = 0.01), and zero out of eight Hürthle cell neoplasms. Regional lymph node metastases were present in five MI-negative primary cancers and resulted in MI-positive in two cases. © 1999 Cancer Research Campaign     

MicroRNA expression profiling is a potential diagnostic tool for thyroid cancer

BACKGROUND:

Approximately 30% of fine‐needle aspiration (FNA) biopsies of thyroid nodules are indeterminate or nondiagnostic. Recent studies suggest microRNA (miRNA, miR) is differentially expressed in malignant tumors and may have a role in carcinogenesis, including thyroid cancer. The authors therefore tested the hypothesis that miRNA expression analysis would identify putative markers that could distinguish benign from malignant thyroid neoplasms that are often indeterminate on FNA biopsy.

METHODS:

A miRNA array was used to identify differentially expressed genes (5‐fold higher or lower) in pooled normal, malignant, and benign thyroid tissue samples. Real‐time quantitative polymerase chain reaction was used to confirm miRNA array expression data in 104 tissue samples (7 normal thyroid, 14 hyperplastic nodule, 12 follicular variant of papillary thyroid cancer, 8 papillary thyroid cancer, 15 follicular adenoma, 12 follicular carcinoma, 12 Hurthle cell adenoma, 20 Hurthle cell carcinoma, and 4 anaplastic carcinoma cases), and 125 indeterminate clinical FNA samples. The diagnostic accuracy of differentially expressed genes was determined by analyzing receiver operating characteristics.

RESULTS:

Ten miRNAs showed >5‐fold expression difference between benign and malignant thyroid neoplasms on miRNA array analysis. Four of the 10 miRNAs were validated to be significantly differentially expressed between benign and malignant thyroid neoplasms by quantitative polymerase chain reaction (P < .002): miR‐100, miR‐125b, miR‐138, and miR‐768‐3p were overexpressed in malignant samples of follicular origin (P < .001), and in Hurthle cell carcinoma samples alone (P < .01). Only miR‐125b was significantly overexpressed in follicular carcinoma samples (P < .05). The accuracy for distinguishing benign from malignant thyroid neoplasms was 79% overall, 98% for Hurthle cell neoplasms, and 71% for follicular neoplasms. The miR‐138 was overexpressed in the FNA samples (P = .04) that were malignant on final pathology with an accuracy of 75%.

CONCLUSIONS:

MicroRNA expression differs for normal, benign, and malignant thyroid tissue. Expression analysis of differentially expressed miRNA could help distinguish benign from malignant thyroid neoplasms that are indeterminate on thyroid FNA biopsy. Cancer 2011. © 2011 American Cancer Society.     

Expression of galectin-3 in fine-needle aspirates as a diagnostic marker differentiating benign from malignant thyroid neoplasms.

BACKGROUND: Galectin-3 is a beta-galactoside-binding protein that has been reported to be expressed preferentially in thyroid malignancies. The current study was designed to substantiate this finding further and to establish a presurgical diagnostic modality of differentiating between benign and malignant thyroid neoplasms by analyzing galectin-3 expression in fine-needle aspirates. METHODS: The expression of galectin-3 was examined immunohistochemically in total of 172 specimens: 45 primary and 20 metastatic papillary carcinomas, 8 primary and 2 metastatic follicular carcinomas, 5 primary and 3 metastatic anaplastic carcinomas, 3 primary medullary carcinomas, 25 follicular adenomas, 3 goiters, and 58 adjacent normal thyroid tissue. Alternatively, epithelial cells were isolated from the fine- needle aspirates of 14 thyroid nodules and subjected to immunoblotting analysis of galectin-3. RESULTS: Immunohistochemical analysis revealed that all thyroid malignancies of follicular cell origin (including papillary, follicular, and anaplastic carcinomas) showed high and diffuse expression of galectin-3, whereas one of the three medullary carcinomas of parafollicular cell origin displayed weaker and focal expression of galectin-3. In contrast, neither benign thyroid adenomas, goiters, nor normal thyroid tissues expressed galectin-3. Immunoblot analysis of the isolated epithelial cells detected galectin-3 in nine thyroid nodules that were proven histologically to be malignant ( eight papillary carcinomas and one follicular carcinoma) after surgical intervention, whereas galectin-3 was not detected in five nodules proven to be benign follicular adenomas. CONCLUSIONS: Galectin-3 serves as a marker of thyroid malignancy of follicular cell origin. Analysis of galectin-3 expression in fine-needle aspirates enhances the differential diagnostic accuracy between benign and malignant thyroid neoplasms.     

Challenge in the Pathological Diagnosis of the Follicular- Patterned Thyroid Lesions

Background: The follicular-patterned thyroid lesions (FPTLs) include hyperplastic nodules (HN), follicular adenoma (FA), non-invasive follicular neoplasm with papillary-like nuclear features (NIFTP), follicular carcinoma (FC), and the follicular variant of papillary carcinoma (FVPTC). Sometimes the pathologists cannot accurately separate these lesions from each others on a histological basis. Aims: To evaluate the utility of immunohistochemistry in the diagnosis of FPTLs. Materials and methods: Immunohistochemical analysis, incorporating 83 cases of histologically confirmed FPTLs out of which 20 carcinomas, 51 benign FPTLs (38 HN and 13 FA), and 12NIFTP were separated from each others using four immunostains (HBME-1, CK19, Galectin-3, and CD56). Results: We found statistically significantly more frequent expression of HBME-1, CK19, Galectin-3 proteins in carcinomas as compared to benign FPTLs (p = <0.01). HBME-1 and Galectin-3 were the most sensitive markers for the diagnosis of malignant FPTLs (75%). Galectin-3 was the most specific marker for the diagnosis of carcinoma (90.3%). Conclusions: The histomorphological features remain the cornerstone of the diagnosis of FPTN. Although HBME-1, Galectin-3, and CK19 immunostains have some diagnostic value in the separation of malignant from benign FPTLs, they are variably expressed in the benign and malignant FPTLs. No single immunostain has sufficient sensitivity and specificity and therefore their diagnostic use is controversial. Future studies are mandated to find more reliable markers that can separate between benign and malignant FPTLs.     

Identification of borderline thyroid tumors by gene expression array analysis

BACKGROUND:

A subset of follicular lesions of the thyroid is encapsulated similar to follicular adenomas but with partial nuclear features suggestive of papillary thyroid carcinoma (PTC), raising the possibility of biologically borderline tumors.

METHODS:

Gene expression profiling and advanced significance analyses were performed on 50 histologically unequivocal benign and malignant tumors, and a list of 61 differentially expressed genes was generated. By using this 61‐gene list, unsupervised hierarchical and K‐means cluster analyses were performed on 40 additional tumors, including 15 histologically borderline tumors, 11 benign tumors, and 14 PTCs.

RESULTS:

Analysis revealed 3 distinct tumor groups—benign, malignant, and intermediate. Tumors in the intermediate group (n = 15) were mostly histologic borderline tumors and had an expression profile overlapping with the benign and malignant groups. Twenty‐seven genes were expressed differentially between the benign and intermediate groups, including the cyclic AMP response element‐binding protein/p300‐interactivator with glutamic acid/aspartic acid‐rich carboxy‐terminal domain 1 or CITED1 gene and the fibroblast growth factor receptor 2 or FGFR2 gene. Fourteen genes were expressed differentially between the intermediate group and malignant tumors, notably overexpression of the met proto‐oncogene and of the high‐mobility group adenine/thymine‐hook 2 or HMGA2 gene in malignancies. Mutations of the v‐raf murine sarcoma viral oncogene homolog B1 or BRAF gene were identified in 4 of 14 malignant tumors but not in benign or intermediate tumors. Patients who had either histologically or molecularly borderline tumors did not have metastasis or recurrences.

CONCLUSIONS:

Gene expression profiling supported the finding that encapsulated thyroid follicular lesions with partial nuclear features of PTC are biologically borderline tumors that are distinct molecularly from benign and malignant tumors. Cancer 2009. © 2009 American Cancer Society.     

CD26 (dipeptidyl peptidase IV/DPP IV) as a novel molecular marker for differentiated thyroid carcinoma

In this report we show that CD26 (dipeptidyl peptidase IV/DPP IV) is a novel molecular marker for differentiated thyroid carcinoma. Northern-blot analysis of 22 various thyroid tissues revealed that CD26 is a more specific marker of differentiated thyroid carcinoma than 3 proto-oncogenes previously reported to increase mRNA expression in thyroid carcinomas: c-met, c-erbB-2 and EGF-R. A comparative study of 3 CD26 assays, Northern blotting, immunohistochemical staining and activity staining clearly showed that CD26 enzyme activity staining is the most specific assay for differentiated thyroid carcinoma, yet the easiest to perform. Activity staining of 216 thyroid tissues detected CD26 in all 52 papillary carcinomas and all 5 follicular carcinomas, while all 58 cases of Graves' disease were CD26-negative. Among benign neoplasms, 54 of 55 adenomatous goiters and 29 of 33 follicular adenomas were CD26-negative. Staining intensity of the enzyme activity was relative to the degree of CD26 mRNA expression. Southern-blot study showed no gene amplification or major translocation of the CD26 gene in 7 papillary carcinomas examined. Based on this study, ectopic expression of CD26 in differentiated thyroid carcinomas is thought to be mainly caused by increased CD26 mRNA expression. In conclusion, CD26 activity staining is a simple, specific assay which should be added to the usual pathological examinations in order to distinguish differentiated thyroid carcinomas from benign thyroid diseases. © 1995 Wiley-Liss, Inc.     

甲状腺良恶性乳头状病变的鉴别诊断

目的探讨CK19、galectin-3和TPO在甲状腺乳头状癌与甲状腺良性乳头状增生中的表达及鉴别诊断意义。方法 采用免疫组织化学S-P法检测53例甲状腺乳头状病变中CK19、galectin-3和TPO的表达情况。其中乳头状癌32例, 良性乳头状增生21例。结果 CK19、galectin-3在甲状腺乳头状癌中阳性表达率分别为97%和94%, 而在甲状腺乳头状增生中的表达率分别为19%和14%, CK19、galectin-3在甲状腺乳头状癌与良性乳头状增生中的表达差异有统计学意义(P＜0.05);TPO在甲状腺乳头状癌与良性乳头状增生中的表达率分别为6%和95%, 其差异亦有显著性(P＜0.05)。结论 CK19、galectin-3和TPO可作为鉴别甲状腺乳头状癌与良性乳头状增生的重要标记物, 联合应用效果更好。     

Immune‐checkpoint molecules on regulatory T‐cells as a potential therapeutic target in head and neck squamous cell cancers

Immune‐checkpoint inhibitors improve the survival of head and neck squamous cell carcinoma (HNSCC) patients. Although recent studies have demonstrated that the tumor immune microenvironment (TIME) has critical roles in immunotherapy, the precise mechanisms involved are unclear. Therefore, further investigations of TIME are required for the improvement of immunotherapy. The frequency of effector regulatory T‐cells (eTregs) and the expression of immune‐checkpoint molecules (ICM) on eTregs and conventional T‐cells (Tconvs) both in peripheral blood lymphocytes (PBL) and tumor‐infiltrating lymphocytes (TIL) from HNSCC patients were analyzed by flow cytometry and their distributions were evaluated by multi‐color immunofluorescence microscopy. High frequency eTreg infiltration into HNSCC tissues was observed and high expressions of CD25, FOXP3, stimulatory‐ICM (4‐1BB, ICOS, OX40 and GITR) and inhibitory‐ICM (programmed cell death‐1 [PD‐1] and cytotoxic T‐lymphocyte‐associated protein‐4 [CTLA‐4]) were found on invasive eTregs. In contrast, the expression of stimulatory‐ICM on Tconvs was low and the expression of inhibitory‐ICM was high. In addition, ICM‐ligands (programmed cell death‐1 [PD‐L1], galectin‐9 and CEACAM‐1) were frequently expressed on cancer cells. PD‐L1 and galectin‐9 were also expressed on macrophages. PD‐1⁺ T‐cells interacted with PD‐L1⁺ cancer cells or PD‐L1⁺ macrophages. This suggested that in TIL, eTregs are highly activated, but Tconvs are exhausted or inactivated by eTregs and immune‐checkpoint systems, and ICM and eTregs are strongly involved in the creation of an immunosuppressive environment in HNSCC tissues. These suggested eTreg targeting drugs are expected to be a combination partner with immune‐checkpoint inhibitors that will improve immunotherapy of HNSCC.     

Cytokeratin expression profiles in thyroid carcinomas

Aims: The aim of this study was to establish the cytokeratin expression profile of different types of thyroid carcinoma. Materials and Methods: The expression of cytokeratins (CKs) 1, 4, 6, 7, 10/13, 18, 19 and 20 in 153 thyroid carcinomas were examined by immunohistochemistry. Results: All papillary carcinomas (n=86) and follicular carcinomas (n=19) showed expression of CK7 and CK18. The staining was often diffuse. CK19 staining was expressed in all papillary carcinomas and the staining was often diffuse. The staining was noted in 68% of follicular carcinomas and the staining was often focal. No difference in the expression was noted between the minimally invasive and widely invasive follicular carcinomas. Poorly differentiated carcinomas (n=10) showed CK7, CK18, CK19 expression in 60%, 60% and 40%, respectively. Anaplastic carcinomas (n=25) expressed CK7 in 84%, CK18 in 80%, CK19 in 76% and CK10/13 in 16%. Medullary carcinomas (n=13) showed CK7 expression in 100%, CK18 in 85% and CK19 in 77%. None of the medullary carcinomas showed diffuse positivity to CK19. All the thyroid carcinomas were negative for CKs 1, 4, 6 and 20. Conclusions: Cytokeratin expression profile for each type of thyroid carcinoma was established.     

Randomised controlled trials and population-based observational research: partners in the evolution of medical evidence

Background:

This study aimed to identify novel biomarkers for thyroid carcinoma diagnosis and prognosis.

Methods:

We have constructed a human single-chain variable fragment (scFv) antibody library that was selected against tumour thyroid cells using the BRASIL method (biopanning and rapid analysis of selective interactive ligands) and phage display technology.

Results:

One highly reactive clone, scFv-C1, with specific binding to papillary thyroid tumour proteins was confirmed by ELISA, which was further tested against a tissue microarray that comprised of 229 thyroid tissues, including: 110 carcinomas (38 papillary thyroid carcinomas (PTCs), 42 follicular carcinomas, 30 follicular variants of PTC), 18 normal thyroid tissues, 49 nodular goitres (NG) and 52 follicular adenomas. The scFv-C1 was able to distinguish carcinomas from benign lesions (P=0.0001) and reacted preferentially against T1 and T2 tumour stages (P=0.0108). We have further identified an OTU domain-containing protein 1, DUBA-7 deubiquitinating enzyme as the scFv-binding antigen using two-dimensional polyacrylamide gel electrophoresis and mass spectrometry.

Conclusions:

The strategy of screening and identifying a cell-surface-binding antibody against thyroid tissues was highly effective and resulted in a useful biomarker that recognises malignancy among thyroid nodules and may help identify lower-risk cases that can benefit from less-aggressive management.     

Prognostic value of arginase‐II expression and regulatory T‐cell infiltration in head and neck squamous cell carcinoma

Tumor‐infiltrating lymphocytes are present in a variety of tumors and play a central role in antitumor immune responses. Nevertheless, most cancers progress probably because tumors are only weakly immunogenic and develop multiple immunosuppressive mechanisms. In the present study, on head and neck squamous cell carcinoma, we found high intraepithelial infiltration of regulatory FOXP3⁺ T cells, and relatively high levels of BDCA2⁺ and FOXP3⁺ cells in stromal (peripheral) regions of the tumors. Tumor‐infiltrating (intraepithelial) FOXP3⁺ T cells were significantly more frequent in patients with oropharynx and oral cavity squamous cell carcinoma and in patients without lymph node metastasis. Furthermore, arginase‐II (ARG2) was expressed by 60%, inducible nitric oxide synthetase by 9%, cyclooxygenase‐2 by 43%, and B‐cell lymphoma 2 (BCL2) by 26% of tumors. Interestingly, the absence of ARG2 expression, enhanced stromal infiltration of CD11c⁺ myeloid dendritic cells, and high numbers of FOXP3⁺ T cells were each significantly associated with prolonged overall survival, and the latter two parameters were also confirmed by multivariate analysis. For disease‐free survival, multivariate analysis revealed significant negative correlations with BCL2 and ARG2 expression by tumor cells. These findings shed new light on mechanisms of cancer progression, and provide rationales for therapeutic inhibition of immunosuppressive mechanisms in head and neck squamous cell carcinoma.     

Contribution of Na+,HCO3−‐cotransport to cellular pH control in human breast cancer: A role for the breast cancer susceptibility locus NBCn1 (SLC4A7)

Genome‐wide association studies recently linked the locus for Na⁺,HCO₃^?‐cotransporter NBCn1 (SLC4A7) to breast cancer susceptibility, yet functional insights have been lacking. To determine whether NBCn1, by transporting HCO₃^? into cells, may dispose of acid produced during high metabolic activity, we studied the expression of NBCn1 and the functional impact of Na⁺,HCO₃^?‐cotransport in human breast cancer. We found that the plasmalemmal density of NBCn1 was 20–30% higher in primary breast carcinomas and metastases compared to matched normal breast tissue. The increase in NBCn1 density was similar in magnitude to that observed for Na⁺/H⁺‐exchanger NHE1 (SLC9A1), a transporter previously implicated in cell migration, proliferation and malignancy. In primary breast carcinomas, the apparent molecular weight for NBCn1 was increased compared to normal tissue. Using pH‐sensitive fluorophores, we showed that Na⁺,HCO₃^?‐cotransport is the predominant mechanism of acid extrusion and is inhibited 34 ± 9% by 200 μM 4,4′‐diisothiocyanatostilbene‐2,2′‐disulfonic acid in human primary breast carcinomas. At intracellular pH (pH_i) levels >6.6, CO₂/HCO₃^?‐dependent mechanisms accounted for >90% of total net acid extrusion. Na⁺/H⁺‐exchange activity was prominent only at lower pH_i‐values. Furthermore, steady‐state pH_i was 0.35 ± 0.06 units lower in the absence than in the presence of CO₂/HCO₃^?. In conclusion, expression of NBCn1 is upregulated in human primary breast carcinomas and metastases compared to normal breast tissue. Na⁺,HCO₃^?‐cotransport is a major determinant of pH_i in breast cancer and the modest DIDS‐sensitivity is consistent with NBCn1 being predominantly responsible. Hence, our results suggest a major pathophysiological role for NBCn1 that may be clinically relevant.