Chang Shu Wang

The influence of MMP-14, TIMP-2 and MMP-2 expression on breast cancer prognosis

IntroductionMatrix metalloproteinase (MMP)-2 is very active at degrading extracellular matrix. It is under the influence of an activator, membrane type 1 MMP (MMP-14), and the tissue inhibitor of metalloproteases (TIMP)-2. We hypothesized that the individual expression of these three markers or their balance may help to predict breast cancer prognosis.MethodsMMP-2, MMP-14 and TIMP-2 expression has been evaluated by 35S mRNA in situ hybridization on paraffin material of 539 breast cancers without distant metastasis at diagnosis and with a median follow-up of 9.2 years.ResultsMMP-2 and MMP-14 mRNA was detected primarily in reactive stromal cells whereas TIMP-2 mRNA was expressed by both stromal and cancer cells. Of the three molecules, an adjusted Cox model revealed that high MMP-14 mRNA (≥ 10% cells) alone predicted a significantly shorter overall survival (p = 0.031) when adjusted for clinical factors (tumor size and number of involved lymph nodes). Prognostic significance was lost when further adjusted for Her-2/neu and urokinase-type plasminogen activator (p = 0.284). Furthermore, when all three components were analyzed together, the survival was worst for patients with high MMP-2/high MMP-14/low TIMP-2 (5 year survival = 60%) and best with low MMP-2/low MMP-14/high TIMP-2 (5 year survival = 74%), but the difference did not reach statistical significance (p = 0.3285).ConclusionOf the MMP-14/TIMP-2/MMP-2 complex, MMP-14 was the factor most significantly associated with the outcome of breast cancer and was an independent factor of poor overall survival when adjusted for clinical prognostic factors, but not for certain ancillary markers.

Influence of Smoking and Alcohol Drinking Behaviors on Treatment Outcomes of Patients With Squamous Cell Carcinomas of the Head and Neck

PURPOSE To retrospectively evaluate the prognostic value of smoking and drinking status in patients with head-and-neck squamous cell carcinomas. METHODS AND MATERIALS All patients with all stages and sites were included if complete information was available on baseline smoking and alcohol behavior (never, former, active), disease stage, primary site, radiation dose, sex, and age. Treatment was radiotherapy in 973 patients, postoperative radiotherapy in 469, and chemoradiotherapy in 429. Statistical analysis was performed with Kaplan-Meier and Cox methods. RESULTS Data from 1,871 patients were available. At baseline, 9% of patients never smoked, 40% were former smokers, and 51% were active smokers; 20% never drank, 25% were former drinkers, and 55% were active drinkers. Smoking was associated with inferior local control and survival. For local control, the hazard ratio (HR) of active smokers vs. former smokers was 1.5 (p = 0.0001). For survival, the HRs of former smokers and active smokers vs. those who never smoked were also statistically significant (1.3 and 1.7, respectively, p = 0.000001). Alcohol drinking was associated with local control (p = 0.03), and was associated with survival. For survival, HRs of former and active drinkers compared with those who never drank were, respectively, 1.1 (p = 0.01) and 1.28 (p = 0.001). Adjusted 5-year local control and survival rates for those who never smoked and never drank were 87% and 77%, respectively, and for those who were both active smokers and active drinkers were 72% (p = 0.007) and 52% (p = 0.0009), respectively. CONCLUSION Smoking and drinking at baseline were associated with poor outcomes in these patients.

Establishment and Characterization of a New Cell Line Derived from a Human Primary Breast Carcinoma

A new cell line, designated HDQ-P1, was successfully established from a primary ductal infiltrating mammary carcinoma by using a 3T3 feeder layer lethally irradiated to 60 Gy. The HDQ-P1 cells have been grown in culture for over 115 passages and have a doubling time of 60 hours. Characterization of the cell line was performed. This included morphology by light and transmission electron microscopy, karyotype, growth rate, telomerase expression, tumor antigen expression, xenograft implantation into nude mice, colony formation in soft agar, TP53 sequencing, and gene copy number of C-MYC, C-ERBB-2, and C-H-RAS oncogenes. The epithelial nature of this cell line was confirmed by ultrastructural analysis, expression of cytokeratins, and epithelial membrane antigen. The HDQ-P1 cells possess an extensively rearranged and polyploid karyotype, with an average of 20 recurrent marker chromosomes. Scatchard analysis demonstrated that both primary tumor and HDQ-P1 cells were estrogen- and progesterone-receptor negative. The HDQ-P1 cells had the same expression of human telomerase reverse transcriptase as other established breast cancer cell lines such as MDA-MB-231, SK-BR-3, and MCF-7. Direct DNA sequencing showed a point mutation which yielded to a stop codon at the amino acid 213 in exon 6 of the TP53 gene. A five-fold amplification of C-MYC was observed in HDQ-P1 cells. No amplification of C-ERBB-2 and C-H-RAS genes were observed. This cell line presents unique characteristics and may prove to be a good experimental model for investigating breast cancer biology.

mdr1 mRNA expression by RT-PCR in patients with primary breast cancer submitted to neoadjuvant therapy

Abstractmdr1 expression by reverse transcription and polymerase chain reaction(RT-PCR) has been compared to P-glycoprotein (Pgp) expression byimmunohistochemistry (IHC) and correlated with clinical response toneoadjuvant therapy. RNA has been recovered from glass slide smears offine-needle aspiration from 57 untreated primary breast cancers prior toneoadjuvant chemotherapy (33 cases), hormone therapy (23 cases), or both (1case). Furthermore, mdr1 mRNA has been analyzed in 6 cases after 2 months oftreatment. The neoadjuvant therapy consisted of 4 cycles of adriamycin andcyclophosphamide or tamoxifen. Of 57 tumor specimens, an interpretableresult was obtained in 52 cases, indicating the feasibility of the analysisby RT-PCR with very small tumor specimens. The presence of mdr1 mRNA hasbeen documented in 44/52 (84%) tumor samples with a spectrum ofexpression levels. The expression of mdr1 mRNA was compared withP-glycoprotein (Pgp) expression by IHC using JSB-1, 4E3, and C494 monoclonalantibodies in 48 of the 52 interpretable tumor samples. 12/48 (25%)expressed Pgp by IHC. All tumors expressing Pgp by IHC were also positive byRT-PCR. The results confirm the higher prevalence of mdr1 mRNA compared tothe protein expression. However, mdr1 mRNA expression was found to correlatesignificantly with resistance to neoadjuvant hormone therapy only while Pgpexpression detected by JSB-1 immunostaining only correlated withchemoresistance. The lack of convincing correlation with chemoresistancesuggests that mRNA and Pgp may not be directly or solely responsible forclinical response to drugs. Further studies should focus on theposttranslational modulation of P-glycoprotein and other mechanisms of drugresistance.

Stromelysin-3 expression by mammary tumor-associated fibroblasts under in vitro breast cancer cell induction.

To understand better the influence of the host stromal phenotype on stromal expression of stromelysin‐3 (ST3) in breast cancer, we have investigated ST3 expression by host stromal cells isolated from 9 different primary breast carcinomas. These tumor‐associated fibroblasts were cocultivated with 3 epithelial cancer cell lines of mammary origin (MDA‐MB‐231, SK‐BR‐3 and MCF‐7), as well as with normal human mammary epithelial cells (NME and 184A1) and keratinocytes, using both direct and indirect coculture systems. ST3 expression was demonstrated by both in situ hybridization and immunocytochemistry. The results showed that ST3 expression by stromal cells was cancer‐specific. Indeed, ST3 expression by tumor‐associated stromal cells was induced by 3 malignant cancer cell lines (MDA‐MB‐231, SK‐BR‐3 and MCF‐7), whereas no ST3 was expressed under normal mammary epithelial cell stimulation. ST3 expression was weak or absent in unstimulated tumor‐associated fibroblasts. However, after direct coculture with cancer cells, expression of ST3 transcripts reappeared in 8 of the 9 cases and was observed only in fibroblasts located in close contact with tumor cells. Under similar coculture conditions and using the same cancer cell line stimulation, ST3 expression was, however, quite variable among these 9 cases, reflecting the difference of protease expression observed on the sections of the original tumors. Tumor induction of ST3 expression was much more important by direct cell‐cell contact than by indirect stimulation and was not influenced by the addition of basic fibroblast growth factor (bFGF) and anti‐bFGF to the culture medium. Our results suggest that the host stromal cell phenotype may significantly influence host stromal cell protease expression under cancer cell stimulation.

Cathepsin D expression by cancer and stromal cells in breast cancer: an immunohistochemical study of 1348 cases

This study was aimed at investigating the influence of cathepsin D (CD) expression by cancer cells and stromal cells on breast cancer prognosis. This is a study of 1348 node‐positive (NPBC) and node‐negative (NNBC) breast cancers diagnosed between 1980 and 1986 and with a minimum follow‐up of 5.2 years. CD expression was assessed by immunohistochemistry on archival material using a polyclonal antibody. The expression by cancer and stromal cells was assessed separately and correlated with distant metastasis free (DMFS) and overall survival (OS). Cancer cells expressed CD (more than 10% cells expressing CD) in 38.9% of cases and reactive stromal cells in 43.6%. CD expression by reactive stromal cells, and not cancer cells, correlated with several factors of poor prognosis by cancer cells. A strong association was also found with expression of other proteases (stromelysin‐3, gelatinase A, and urokinase Plasminogen Activator) by these same reactive stromal cells. CD expression by cancer cells did not predict DMFS or OS but, by univariate analysis, CD expression by reactive stromal cells was associated with earlier recurrence and shorter survival in NNBC (p = 0.0425) and NPBC patients submitted to adjuvant chemotherapy (p = 0.0234). However, CD expression by reactive stromal cells remained a significant predictor of recurrence by multivariate analyses only in a subgroup of NPBC submitted to adjuvant chemotherapy. Overall, those data support the concept that proteases produced by reactive stromal cells are under cancer cell stimulation and that CD by stromal cells, and not cancer cells, influences the prognosis, but only in a subgroup of patients with breast cancer.

Expression of cathepsin D, stromelysin-3, and urokinase by reactive stromal cells on breast carcinoma prognosis

Current literature suggests that several proteases act in a cascade to mediate remodeling of the extracellular matrix and favor cancer progression. Others and the authors of this study recently identified cathepsin D, stromelysin‐3, and urokinase plasminogen activator (uPA) expression by reactive stromal cells as significant factors of poor prognosis in breast carcinoma. The authors evaluated the joint effect of protease expression on cancer aggressiveness.

Selective culture of epithelial cells from primary breast carcinomas using irradiated 3T3 cells as feeder layer

The main drawback of the selective culture of human mammary epithelial cells from primary breast cancer is the overgrowth of tumor-associated stromal fibroblasts. This drawback may be overcome by using, in primary culture, lethally irradiated 3T3 cells which act as a feeder layer to maintain tumor-derived epithelial cell proliferation. These 3T3 cells, exposed to 60 Gy at confluence, form a specific cellular substrate which constitutes an obstacle to fibroblast attachment. Enzyme-disaggregated breast cells from six primary breast carcinomas were cocultured over lethally irradiated but living 3T3 cells. The method led to the purification of tumor-derived epithelial cells from all six cancer samples, and long-term culture was obtained in one. The epithelial nature of these purified tumor-derived epithelial cells was demonstrated by their general morphology and by the expression of cytokeratins and Epithelial Membrane Antigen. These results confirm the stimulatory effect of a this stromal feeder layer on breast epithelial cell growth and show that this stromal feeder layer can also control the fibroblast overgrowth. Our results provide an alternative approach in the selective culture of epithelial cells from primary breast carcinoma.

Effect of Pretreatment Anemia on Treatment Outcome of Concurrent Radiochemotherapy in Patients With Head and Neck Cancer

PURPOSE To investigate the effect of anemia on outcome of treatment with radiochemotherapy in patients with head-and-neck cancer. METHODS AND MATERIALS The data of 196 patients with Stage II-IV head-and-neck cancer treated with concomitant cisplatin-based radiochemotherapy were retrospectively reviewed. Anemia was defined according to World Health Organization criteria as hemoglobin <130 g/L in men and <120 g/L in women. RESULTS Fifty-three patients were classified as anemic, 143 as nonanemic. The 3-year local control rate of anemic and nonanemic patients was 72% and 85%, respectively (p = 0.01). The 3-year overall survival rate of anemic and nonanemic patients was 52% and 77%, respectively (p = 0.004). In multivariate analysis, anemia was the most significant predictor of local control (hazard ratio, 0.37, p = 0.009) and survival (hazard ratio, 0.47, p = 0.007). A dose-effect relationship was also found for local control (p = .04) and survival (0.04) when grouping by hemoglobin concentration: <120, 120-140, and >140 g/L. CONCLUSIONS Anemia was strongly associated with local control and survival in this cohort of patients with head-and-neck cancer receiving radiochemotherapy.

Production of bioengineered cancer tissue constructs in vitro : epithelium–mesenchyme heterotypic interactions

SummaryA few models have been established to study cancer cells in vitro. However, the cellular interactions have rarely been studied specifically using bioengineered cancer constructs combining human carcinoma cells and tumor-associated fibroblasts. We developed an in vitro model of tridimensional bioengineered cancer tissue constructs (bCTC) by seeding mammary epithelial cancer cells or normal keratinocytes over a mesenchymal layer containing tumor-derived fibroblastic cells or normal skin fibroblasts. After the introduction of epithelial cells, each construct was cultured for another 10 d. Histologic analyses showed that carcinoma cell lines could invade the subjacent mesenchymal layer and that the capacity to migrate was related to the invasive potential of cancer cells and the type of fibroblasts used, while noninvasive populations did not. Of the tested epithelial cells, MDA-MB-231 and, to a lesser degree, HDQ-P1 cell lines were invasive, and the invasion was deeper into the mesenchymal component containing tumor-derived fibroblasts. However, with normal skin fibroblasts, the mesenchymal layer was degraded twice faster than with tumor-derived fibroblastic cells. MDA-MB-231 cells and normal keratinocytes induced the highest level of gelatinase B, and the level was lowest with the MCF-7 cell line. The activated form of gelatinase B was, however, induced to the highest levels in the keratinocyte-seeded bCTC containing tumor-derived but not normal fibroblasts. MDA-MB-231 was the only epithelial cancer cell line whose activity of gelatinase A was reduced when cocultured with tumor-derived fibroblasts but not under normal fibroblast stimulation. Finally, a 50/48-kDa gelatinase band has been observed in bCTCs with noninvasive epithelial cells only. Our study demonstrates the selective secretion of gelatinases according to the phenotype of the cells seeded in the various bCTCs.