Prognostic value of ERBB family mRNA expression in breast carcinomas

The ErbB-driven autocrine growth pathway has been implicated in the development and progression of most common human epithelial malignancies; its blockade is therefore a promising therapeutic strategy, and several candidate drugs are currently undergoing clinical trials. Paradoxically, little is known of the expression pattern of these 4 genes in human tumors, and the clinical significance of the 2 most recently discovered ERBB genes, ERBB3 and ERBB4, is unclear. We used a real-time quantitative RT-PCR assay to quantify ERBB family mRNA copy numbers in a large series of breast tumors from patients with known long-term outcome. ERBB gene expression varied widely, by more than 2 orders of magnitude for ERBB1 and ERBB3, more than 3 orders for ERBB2 and more than 4 orders for ERBB4. We found a positive correlation between ERBB3 and ERBB4 mRNA levels, and a negative correlation between the expression of these 2 latter genes and that of ERBB1. Compared to normal breast tissue, ERBB1 was underexpressed (82.3% of tumors), ERBB2 (16.9%) and ERBB3 (46.2%) were overexpressed and ERBB4 was both underexpressed (24.6%) and overexpressed (29.2%). Links were also found between ERBB status on the one hand and Scarff-Bloom-Richardson (SBR) histopathological grade and estrogen receptor alpha (ERa) status on the other hand. Relapse-free survival (RFS) was shorter among patients with ERBB3-overexpressing tumors (p=0.0092) and longer among those with ERBB4-underexpressing tumors (p=0.0085) relative to patients with normal expression of the respective genes; in contrast, RFS was not significantly influenced by ERBB1 or ERBB2 mRNA status. Only ERBB4 status retained prognostic significance in Cox multivariate regression analysis (p=0.015). Our results point to the involvement of several ErbB-specific ligands (amphiregulin and neuregulin 1) and enzymes or adaptor molecules (PI3K, Src, Shc and Grb7) in the ErbB pathway dysregulation associated with breast cancer. These findings reveal a complex expression pattern of ERBB gene family members in breast tumors and suggest that it is this pattern of expression, rather than the expression of individual family members, that should be taken into account when evaluating antitumoral drugs designed to target these receptors.     

Prognostic value of CCND1 gene status in sporadic breast tumours, as determined by real-time quantitative PCR assays

The CCND1 gene, a key cell-cycle regulator, is often altered in breast cancer, but the mechanisms underlying CCND1 dysregulation and the clinical significance of CCND1 status are unclear. We used real-time quantitative PCR and RT-PCR assays based on fluorescent TaqMan methodology to quantify CCND1 gene amplification and expression in a large series of breast tumours. CCND1 overexpression was observed in 44 (32.8%) of 134 breast tumour RNAs, ranging from 3.3 to 43.7 times the level in normal breast tissues, and correlated significantly with positive oestrogen receptor status (P=0.0003). CCND1 overexpression requires oestrogen receptor integrity and is exacerbated by amplification at 11q13 (the site of the CCND1 gene), owing to an additional gene dosage effect. Our results challenge CCND1 gene as the main 11q13 amplicon selector. The relapse-free survival time of patients with CCND1-amplified tumours was shorter than that of patients without CCND1 alterations, while that of patients with CCND1-unamplified-overexpressed tumours was longer (P=0.011). Only the good prognostic significance of CCND1-unamplified-overexpression status persisted in Cox multivariate regression analysis. This study confirms that CCND1 is an ER-responsive or ER-coactivator gene in breast cancer, and points to the CCND1 gene as a putative molecular marker predictive of hormone responsiveness in breast cancer. Moreover, CCND1 amplification status dichotomizes the CCND1-overexpressing tumors into two groups with opposite outcomes.     

Carbonic anhydrase-9 expression levels and prognosis in human breast cancer: association with treatment outcome

Here, we set out to assess CA9 expression levels by real-time quantitative RT-PCR in breast cancer tissue samples obtained from 253 patients, and correlated those with relapse-free (RFS) survival. The median follow-up time was 75 months (range 2-168 months). CA9 expression was mainly found in high-grade, steroid receptor negative cancer tissues. CA9 levels were not significantly associated with RFS (P=0.926, hazard ratio (HR)=0.99, 95% CI=0.80-1.22) in the total cohort of 253 patients. In multivariate analysis with other clinicopathological factors, CA9 (P=0.018, HR=0.77, 95% CI=0.62-0.96), the interaction of adjuvant chemotherapy with CA9 (P=0.009, HR=1.31, 95% CI=1.07-1.61) and the interaction of adjuvant endocrine therapy with CA9 (P<0.001, HR=1.41, 95% CI=1.20-1.66) all contributed significantly to the final model. These results indicate that patients with low CA9 levels benefit more from adjuvant treatment than do patients with high levels. Thus, the determination of CA9 levels could aid in the selection of patients who will not benefit from adjuvant therapy, and whose prognosis will more likely improve with other treatment modalities.     

Overexpression of TGF-beta by infiltrated granulocytes correlates with the expression of collagen mRNA in pancreatic cancer

Pancreatic cancer is often associated with an intense production of interstitial collagens, known as the desmoplastic reaction. To understand more about desmoplasia in pancreatic cancer, the expression of mRNA for type I and III collagens and potent desmoplastic inducing growth factors transforming growth factor-beta (TGF-beta), connective tissue growth factor (CTGF), acidic and basic fibroblast growth factor (FGF), platelet-derived growth factor (PDGF) A and C and epidermal growth factor (EGF) was analysed by quantitative RT-PCR. Expression of both collagens in 23 frozen primary pancreatic cancer nodules was significantly higher than that in 15 non-neoplastic pancreatic tissues. The expressions of mRNAs for TGF-beta, acidic FGF, basic FGF and PDGF C were likewise higher in surgical cancer nodules, while that of CTGF, PDGF A and EGF were not. Among these growth factors, the expression of TGF-beta mRNA showed the most significant correlation with that of collagens (P<0.0001). By immunohistochemistry, TGF-beta showed faint cytoplasmic staining in cancer cells. In contrast, isolated cells, mainly located on the invasive front surrounding cancerous nests, were prominently and strongly stained. These TGF-beta-positive cells contained a segmented nucleus, were negative for anti-macrophage (CD68) and positive for anti-granulocyte antibodies, indicating their granulocytic nature. In conclusion, TGF-beta seemed to play a major role among the various growth factors in characteristic overproduction of collagens in pancreatic cancer. Moreover, the predominant cells that express TGF-beta were likely to be infiltrated granulocytes (mostly are neutrophils) and not pancreatic cancer cells.     

Prognostic impact of syndecan-1 expression in invasive ductal breast carcinomas

Carcinoma cells lack syndecan-1 expression when they are transiting from an epithelial to a less-differentiated mesenchymal phenotype (epithelial-mesenchymal transition, EMT). Furthermore, a shift of syndecan-1 expression from malignant epithelial cells to reactive stromal cells has also been observed during progression of many carcinomas. Finally, epithelial and/or stromal syndecan-1 expression is of prognostic value in many carcinomas. Because recent results are contradictory in breast carcinomas, we have re-evaluated the prognostic significance of syndecan-1 expression in a cohort of 80 patients with invasive ductal breast carcinomas. The tumours from 80 patients diagnosed with invasive ductal breast carcinomas were used to construct a tissue microarray, which was stained with syndecan-1 by immunohistochemistry. We correlated syndecan-1 expression with clinicopathologic parameters and relapse-free survival (RFS). Exclusive epithelial expression of syndecan-1 is observed in 61.25% of the patients, whereas exclusive stromal expression is observed in 30% of the patients. Only 8.75% of the patients had both stromal and epithelial expressions of syndecan-1. A significant correlation was found between the loss of syndecan-1 epithelial expression and the syndecan-1 stromal expression with high grade of malignancy (P=0.011). The loss of syndecan-1 epithelial expression is correlated with RFS (P=0.001). Using multivariate Cox analysis, loss of epithelial syndecan-1 expression was the only prognostic indicator (P<0.001). We concluded that the loss of syndecan-1 epithelial expression was of strong prognostic value in breast carcinomas.     

Poly(A) RT-PCR measurement of diagnostic genes in pancreatic juice in pancreatic cancer

Background:

The last decade has seen significant progress in understanding the molecular biology of pancreatic adenocarcinoma. There is now an urgent need to translate these molecular techniques to clinical practice in order to improve diagnosis and prediction of response to treatment. The objectives of this study are to utilise poly(A) RT–PCR to measure expression levels of diagnostic Indicator genes, selected from microarray studies, of RNA from intraoperatively sampled pancreatic ductal juice and to correlate these expression levels with those in matched pancreatic tissue resection samples.

Methods:

Intraoperative sampling of pancreatic juice and collection of matched tissue samples was undertaken in patients undergoing pancreaticoduodenectomy for suspected tumour. RNA was isolated and poly(A) PCR and real-time PCR used to measure expression levels of 30 genes. Spearman''s rank correlation test was used to examine the relationship of gene expression between pancreatic juice and tissue.

Results:

Of the 30 Indicator genes measured, just one, ANXA1, showed a significant correlation of expression level between pancreatic juice and tissue samples, whereas three genes, IGFBP3 (P⩽0.035), PSCA (P⩽0.001) and SPINK1 (P⩽0.05), showed significantly different expression between cancerous and benign pancreatic tissue samples.

Conclusions:

These results demonstrate that RNA analysis of pancreatic juice is feasible using the poly(A) cDNA technique, that correlation of gene expression exists between pancreatic juice and tissue for very few genes and that gene expression profiling can distinguish between benign and malignant pancreatic tissue. This indicates possible use of the technique for measurement of Indicator genes in pancreatic tissue for diagnosis of pancreatic cancer from very small tissue samples.     

Prognostic impact of multidrug resistance gene expression on the management of breast cancer in the context of adjuvant therapy based on a series of 171 patients

Study of the prognostic impact of multidrug resistance gene expression in the management of breast cancer in the context of adjuvant therapy. This study involved 171 patients treated by surgery, adjuvant chemotherapy+/-radiotherapy+/-hormonal therapy (mean follow-up: 55 months). We studied the expression of multidrug resistance gene 1 (MDR1), multidrug resistance-associated protein (MRP1), and glutathione-S-transferase P1 (GSTP1) using a standardised, semiquantitative rt-PCR method performed on frozen samples of breast cancer tissue. Patients were classified as presenting low or high levels of expression of these three genes. rt-PCR values were correlated with T stage, N stage, Scarff-Bloom-Richardson (SBR) grade, age and hormonal status. The impact of gene expression levels on 5-year disease-free survival (DFS) and overall survival (OS) was studied by univariate and multivariate Cox analysis. No statistically significant correlation was demonstrated between MDR1, MRP1 and GSTP1 expressions. On univariate analysis, DFS was significantly decreased in a context of low GSTP1 expression (P = 0.0005) and high SBR grade (P = 0.003), size > or = 5 cm (P = 0.038), high T stage (P = 0.013), presence of intravascular embolus (P = 0.034), and >3 N+ (P = 0.05). On multivariate analysis, GSTP1 expression and the presence of ER remained independent prognostic factors for DFS. GSTP1 expression did not affect OS. The levels of MDR1 and MRP1 expression had no significant influence on DFS or OS. GSTP1 expression can be considered to be an independent prognostic factor for DFS in patients receiving adjuvant chemotherapy for breast cancer.     

beta(2)microglobulin mRNA expression levels are prognostic for lymph node metastasis in colorectal cancer patients

Colorectal cancer (CRC) is the fourth most common non-cutaneous malignancy in the United States and the second most frequent cause of cancer-related death. One of the most important determinants of CRC survival is lymph node metastasis. To determine whether molecular markers might be prognostic for lymph node metastases, we measured by quantitative real-time RT-PCR the expression levels of 15 cancer-associated genes in formalin-fixed paraffin-embedded primary tissues derived from stage I-IV CRC patients with (n=20) and without (n=18) nodal metastases. Using the mean of the 15 genes as an internal reference control, we observed that low expression of beta(2)microglobulin (B2M) was a strong prognostic indicator of lymph node metastases (area under the curve (AUC)=0.85; 95% confidence interval (CI)=0.69-0.94). We also observed that the expression ratio of B2M/Spint2 had the highest prognostic accuracy (AUC=0.87; 95% CI=0.71-0.96) of all potential two-gene combinations. Expression values of Spint2 correlated with the mean of the entire gene set at an R(2) value of 0.97, providing evidence that Spint2 serves not as an independent prognostic gene, but rather as a reliable reference control gene. These studies are the first to demonstrate a prognostic role of B2M at the mRNA level and suggest that low B2M expression levels might be useful for identifying patients with lymph node metastasis and/or poor survival.     

Systematic characterisation of GABRP expression in sporadic breast cancer and normal breast tissue

The GABRP gene has been previously identified by in silico analysis of four million ESTs as a candidate gene differentially expressed in breast cancer. GABRP is located on chromosome 5q34 and it encodes the pi-subunit of the gamma-aminobutyric acid (GABA) receptor, a transmembrane protein expressed in the brain and several nonneuronal tissues. Using cDNA dot blot hybridisation (cancer profiling array), quantitative RT-PCR and non-radioisotopic in situ hybridisation (ISH), we have analysed GABRP expression in breast cancer and normal breast tissues as well as in nontumorigenic and tumorigenic breast cell lines. Analysis of the cancer profiling array revealed a more than 2-fold downregulation of GABRP (p < 0.001) in 76% of primary breast carcinomas (n = 50) compared to corresponding normal tissues. Quantitative RT-PCR in a panel of 23 normal human tissues showed that the GABRP expression level was most abundant in the normal breast tissues compared to other human tissues. GABRP downregulation in breast cancer was confirmed by quantitative RT-PCR in cryopreserved breast tumour and normal breast tissue specimens (n = 22), in archival formalin-fixed, paraffin-embedded tissue specimens (n = 32), as well as in breast cancer cell lines (n = 8). Furthermore, a significant downregulation of GABRP was noted in large (pT3-pT4) (p = 0.044) primary breast tumours. Non-radioisotopic ISH showed strong GABRP expression in normal epithelial and benign papilloma breast cells, but no signal could be detected in invasive ductal carcinoma. Altogether, these data suggest that GABRP is progressively down-regulated with tumour-progression, and that it may be useful as a prognostic marker in breast cancer.     

同源异型盒基因HOXD10在乳腺癌组织中的表达及其临床病理意义

背景与目的：乳腺癌是威胁女性健康最常见的恶性肿瘤之一,从分子水平深入研究其癌变机制,对寻找新的标志基因具有重要意义.本研究探讨同源异型盒基因HOXD10基因在乳腺癌发生、发展进程中的可能作用.方法：以18s rRNA基因作为参照,采用荧光实时定量RT-PCR技术,以相对定量方法检测44例乳腺癌组织及16例乳腺良性病变组织中HOXD10 mRNA表达情况.结果：HOXD10在乳腺癌中的表达显著低于乳腺良性病变组织.组织学分级为Ⅲ级的乳腺癌标本中H0XD10 mRNA的表达显著低于组织学Ⅰ、Ⅱ级标本.H0XD10 mRNA的表达与淋巴结转移、肿瘤大小、TNM分期之间相关性不显著.结论：HOXD10基因在乳腺癌中呈低表达甚至表达缺失,由于这种基因可能为乳腺癌发生发展的抑制因素,因此对其我们应给予更多关注.     

c-erbB-4 protein expression in human breast cancer

The Type 1 family of growth factor receptors includes epidermal growth factor receptor (EGFR), c-erbB-2, c-erbB-3 and c-erbB-4. Overexpression of the first two members is associated with poorer prognosis in patients with breast carcinoma. In this study we examined the expression of c-erbB-4 protein using the monoclonal antibody HFR-1. A total of 127 consecutive cases of primary operable invasive breast carcinoma presenting between 1975 and 1977 were studied. All patients were managed by simple mastectomy or conservation surgery with radiotherapy and no adjuvant therapy given. Long-term follow-up was maintained. Routine, formalin-fixed, paraffin-embedded tumour samples were used and sections were stained immunohistochemically using the Duet StreptABC method. Immunoreactivity was classified using a simple semi-quantitative scoring method. Protein expression was generally low but definite positive cytoplasmic, membranous and nuclear reactivity was identified in 58%, 41% and 25% of cases respectively. Expression at all three sites demonstrated significant inverse associations were histological grade. In addition, membrane accentuation correlated inversely with the Nottingham Prognostic Index (NPI), while cytoplasmic reactivity showed a positive association with c-erbB-3 expression. No significant associations were found with disease-free interval or survival. The results of this study demonstrate that higher levels of c-erbB-4 protein expression are associated with a more differentiated histological phenotype in contrast to the other members of the Type 1 family. Larger series with extended follow-up will be required to ascertain definitively the prognostic value of c-erbB-4 expression in breast carcinoma.     

PTEN及bcl-2和p53蛋白表达与乳腺癌预后的关系

目的 :探讨新的抑癌基因PTEN在人乳腺癌组织中的表达、与bcl 2、p5 3的相关性及其与乳腺癌临床病理学特征和预后的关系。方法 :采用免疫组化SP法检测62例浸润性乳腺癌 (包括浸润性导管癌、浸润性小叶癌、髓样癌等 )标本中PTEN、bcl 2及p5 3蛋白的表达。结果 :62例乳腺癌标本中PTEN、bcl 2及p5 3阳性表达率分别为 64 5 % ( 4 0 /62 )、5 6 5 % ( 3 5 /62 )及2 7 4% ( 17/62 ) ;PTEN与bcl 2之间不具有相关性 ,P >0 0 5 ;PTEN与p5 3之间显著相关 ,P <0 0 1。乳腺癌PTEN蛋白表达水平与患者年龄、肿瘤组织学类型、肿瘤大小之间差异无统计学意义 ,P >0 0 5 ;与肿瘤细胞学分级、淋巴结有无转移及预后之间差异有统计学意义 ,P <0 0 5 ;PTEN低表达患者肿瘤细胞学分级高、淋巴结转移阳性率高、5年生存率低。结论 :1)抑癌基因PTEN在乳腺癌中确实存在突变或缺失。其表达水平的高低作为判断乳腺肿瘤细胞增殖活性、侵袭力及转移的重要指标。 2 )PTEN与bcl 2协同表达与预后相关 ,可作为乳腺癌预后评估的有效指标。 3 )PTEN突变或缺失可能与p5 3存在一定的相关性 ,但其作用机制有待进一步研究。