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Clinical Chemistry 52: 1070-1079, 2006. First published April 20, 2006; 10.1373/clinchem.2005.065599
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(Clinical Chemistry. 2006;52:1070-1079.)
© 2006 American Association for Clinical Chemistry, Inc.

Cancer Diagnostics

Quantitative Reverse Transcription-PCR Assay for Detection of mRNA Encoding Full-Length Human Tissue Kallikrein 7: Prognostic Relevance of KLK7 mRNA Expression in Breast Cancer 3

Leon Holzscheiter1, Julia C. Biermann1, Matthias Kotzsch2, Panagiotis Prezas3, Juliane Farthmann1, Gustavo Baretton2, Thomas Luther2,4, Vivianne C.G. Tjan-Heijnen5, Maroulio Talieri3, Manfred Schmitt1, Fred C.G.J. Sweep6, Paul N. Span6 and Viktor Magdolen1,a

1 Clinical Research Unit, Department of Obstetrics and Gynecology, Technical University of Munich, Munich, Germany.
2 Institute of Pathology, Technical University of Dresden, Dresden, Germany.
3 G Papanicolaou Research Center-Saint Savas Hospital, Athens, Greece.
4 Medical Laboratory Unit, Bautzen, Germany.
Departments of5 Medical Oncology and 6 Chemical Endocrinology, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands.

aAddress correspondence to this author at: Klinische Forschergruppe der Frauenklinik der Technischen Universität München, Klinikum rechts der Isar, Ismaninger Strasse 22, D-81675 München, Germany. Fax 49-89-4140-7410; e-mail viktor.magdolen{at}lrz.tum.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: The human tissue kallikrein gene family (KLK1 to KLK15) encodes a group of 15 serine proteases (hK1 to hK15), several of which have been implicated in cancer-related processes.

Methods: We established a specific quantitative reverse transcription-PCR assay for full-length KLK7 mRNA that excluded amplification of the exon 2 deletion splice variant (the latter does not encode a functional protease), and evaluated full-length KLK7 mRNA expression [normalized to human glucose-6-phosphate dehydrogenase (h-G6PDH)] in tumor tissue specimens from 155 breast cancer patients.

Results: High KLK7 mRNA expression (continuous) was significantly associated with a better patient outcome according to both univariate (P = 0.005) and multivariate (P = 0.046) Cox survival analysis. Separation of patients by optimized dichotomization revealed a significantly better prognosis for patients with high KLK7 mRNA status (n = 89) compared with patients with low KLK7 mRNA status (n = 66) [univariate hazard ratio (HR) = 0.45 (P = 0.001); multivariate HR = 0.50 (P = 0.005)]. In the subgroup of patients not receiving adjuvant treatment (n = 69), KLK7 mRNA status was a significant prognosticator [univariate HR = 0.29 (P = 0.002); multivariate HR = 0.40 (P = 0.034)]. This subgroup was least influenced by postoperative treatment and thus best showed the impact of KLK7 expression on the natural course of breast cancer disease.

Conclusion: Expression of full-length KLK7 mRNA may represent a new prognostic marker in breast cancer disease.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Breast cancer is the most commonly diagnosed cancer in women (1), but only a few reliable prognostic markers have been developed for this disease. Human tissue kallikreins (hKs) 1 are serine proteases with diverse physiologic functions. Fifteen human tissue kallikrein (KLK) genes have been identified (2)(3). Under physiologic conditions, KLK genes exhibit differential expression in many tissues. Many human tissue kallikreins are associated with malignancies, such as prostate-specific antigen (hK3), which is associated with prostate cancer and is used as a serum marker to identify and monitor patients with the disease (4)(5). Other members of the family (hK2, hK6, and hK10) are emerging tumor markers for prostate, ovarian, and breast cancer (6)(7). Tissue kallikrein genes have large numbers of splice variants, which may have diagnostic or prognostic significance for certain types of malignancies (8).

Human stratum corneum chymotryptic enzyme (hK7; HSCCE) (9)(10), one of the few tissue kallikreins for which a physiologic function has been proposed, catalyzes the degradation of intercellular cohesive structures in the outermost layer of the skin and contributes to the cell shedding process at the skin surface (11). Recent reports have linked high KLK7 2 expression to a significantly poorer prognosis in ovarian and breast cancer, indicating that KLK7 may be a potent prognostic marker for ovarian and breast cancer (12)(13)(14).

The KLK7 gene consists of 6 exons, 5 of which harbor coding regions. The amino acids of the invariant catalytic triad of serine proteases (His57, Asp102, and Ser195) are encoded by exon 3, 4, and 6, respectively. Two variants of the noncoding 5'-terminal exon 1 have been identified. Two of the 3 described mRNA variants of human KLK7 (8)(10)(13) encode full-length hK7 because these mRNAs encompass either of the 2 alternative noncoding 5'-terminal exons 1 and all of the coding exons. Previous analyses of KLK7 expression in breast and ovarian cancer were nonselective studies of all KLK7 mRNA forms (14). To investigate whether a more specific analysis of full-length KLK7 mRNA would lead to the same results and conclusions, we developed a quantitative reverse transcription-PCR (QPCR) assay for the measurement of full-length KLK7 mRNA.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
study population
We used tissue specimens from a population-based cohort of 176 primary breast cancer patients who underwent breast cancer surgery in participating hospitals of the Comprehensive Cancer Center East in The Netherlands and the University Medical Center Dresden in Germany. After exclusion of 21 cDNA samples (11.9%) considered to be of poor quality because of low housekeeping gene concentrations (see the section on the housekeeping gene below), tissue specimens from 155 breast cancer patients remained for KLK7 expression analysis. The study adhered to the respective national regulations on ethics issues and was approved by the local ethics committees. After surgical resection of the primary tumor, representative areas of the tumor tissues were selected macroscopically by a pathologist and immediately snap-frozen in liquid nitrogen. Histologic grades of the tumors were determined according to Bloom–Richardson criteria, and tumor stage was classified according to the TNM classification system.

Patients who had received neoadjuvant treatment, had had a previous diagnosis of cancer, or had a carcinoma in situ, as well as patients with recurrent disease within 1 month after surgery or with distant metastases at the time of diagnosis were excluded from the study. Patient age at diagnosis ranged from 31 to 86 years with a median of 59 years. Follow-up information was available for all patients; the median follow-up was 61 months, and the maximum follow-up period was 120 months. During that time, 67 patients (43.2%) had a recurrence (17 locoregional relapses, 46 distant metastases, and 4 both) and 47 patients (30.3%) died.

Patients underwent modified radical mastectomy or breast-preserving lumpectomy combined with axillary lymph node dissection. Postoperative locoregional radiotherapy of the breast was carried out after incomplete resection or after breast-preserving treatment. Depending on the degree of nodal involvement, axillary or supraclavicular regions were irradiated. Lymph node metastases were detected in 87 patients (56.1%), and were not detected in 68 patients (43.9%). Adjuvant systemic treatment was administered according to respective consensus recommendations at the time: 69 patients received no further treatment; 49 patients were treated with endocrine therapy; 28 were given chemotherapy alone; and 9 patients received both chemotherapy and endocrine therapy after tumor resection. Of 68 patients without involvement of axillary lymph nodes, 59 (86.8%) did not receive adjuvant systemic treatment.

In addition, expression of full-length KLK7 was analyzed in tumor-free tissue from 10 breast cancer patients (provided by the Institute of Pathology, Technical University of Dresden, Germany).

quantification of KLK7 MRNA by QPCR
We isolated total RNA from tissue samples with the RNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol (15)(16)(17). Reverse transcription was performed with the Reverse Transcription System (Promega) or the cDNA-Cycle Kit (Invitrogen) as described previously (15)(16)(17). For quantification of KLK7 mRNA, we established a highly sensitive QPCR assay applying the LightCycler technology (Roche Diagnostics; software Ver. 3.5). We aimed at establishing a QPCR assay also suitable for quantification of mRNA concentrations in formalin-fixed, paraffin-embedded tissue specimens despite the extensive RNA fragmentation that may occur in tissues preserved in this way (generally <300 bases in length). For this, the amplicon length was minimized as far as possible, considering that the LightCycler technology uses 2 primers for amplification and 2 primers for detection. On the basis of the cDNA sequence of KLK7 (GenBank accession no. L33404), we designed the following gene-specific primers (amplicon length, 108 bp): forward primer, 5'-GGA AAC TGC AGG AGA AGA AGC-3'; reverse primer, 5'-TGG AGC TGA TTG CCA CTG A-3'.

Real-time monitoring of PCR was performed with hybridization probes with the following sequences: 5'-GGT GGG AGC CTC TTG CAC ATG G-fluorescein-3'; 5'-LC Red640-CGC CAT CAA TAA TCT TGT CAC CCT G-phosphate-3'. We obtained primers and hybridization probes from TIB Molbiol. Reverse transcription-PCR was performed with an optimized master mixture containing 4.5 mM MgCl2, 0.5 µM each primer, and 0.2 µM each hybridization probe in a total volume of 20 µL. The amplification program started with pre-denaturation at 95 °C for 10 min, followed by 45 cycles of amplification (denaturation for 10 s at 95 °C, annealing for 10 s at 64 °C, and elongation for 6 s at 72 °C). To verify the results of the QPCR measurements, randomly selected samples were run on 1.5% agarose gels and sequenced after purification.

We generated 5-log-range calibration curves for each PCR run by use of 8 glass capillaries coated with defined numbers of linearized plasmid molecules carrying the KLK7 cDNA (pRcRSV-KLK7). The plasmid molecule numbers had been exactly determined by HPLC calibration (Roboscreen) (18). The glass capillaries were coated with 10, 20, 50, 100, 1000, 5000, 10 000, and 100 000 copies, respectively.

housekeeping gene
We selected human glucose-6-phosphate dehydrogenase (h-G6PDH) for normalization of the data because it is an appropriate housekeeping gene for breast cancer studies (19). The h-G6PDH Housekeeping Gene Set (Roche Diagnostics) was used according to the manufacturer’s protocol (amplicon length, 123 bp). All cDNA samples displaying <10 000 h-G6PDH molecules were considered to be of nonoptimal quality (19) and were excluded from further analyses (21 of 176 cDNA samples; 11.9%). All further calculations and statistical analyses were carried out with the relative mRNA expression ratios (zmol KLK7/amol h-G6PDH).

generation of cell lines overexpressing KLK7 MRNA
Human full-length cDNA encoding hK7 was obtained by reverse transcription-PCR with mRNA from ovarian cancer tissue as a template. Subsequently, the cDNA fragment was directionally subcloned into the plasmid pRcRSV (Invitrogen) with 5' HindIII and 3' XbaI restriction sites introduced into the cDNA by PCR. The correct sequence of the cDNA was verified by sequencing.

Two human breast cancer cell lines, MDA-231 (provided by the Institute of Experimental Oncology and Therapy Research, Technical University of Munich, Germany) and aMCF-7 (an Adriamycin-resistant subline of MCF-7 provided by the Max-Delbrueck-Center of Molecular Medicine, Berlin-Buch, Germany), as well as the human ovarian cancer cell line OVMZ-6 (Staedtische Kliniken Frankfurt a.M.-Hoechst, Frankfurt, Germany) were transfected with the pRcRSV-based expression plasmid harboring KLK7 or with the vector alone, and were then examined for KLK7 expression (MDA-231+RSV, MDA-231+KLK7, aMCF-7+RSV, aMCF-7+KLK7, OVMZ-6+RSV, OVMZ-6+KLK7). In addition, we used an immortalized keratinocyte cell line, HaCaT (provided by the Department of Dermatology, Technical University of Dresden, Germany), for expression analysis.

Cell lines were cultured at 37 °C in DMEM (Invitrogen) in a humidified atmosphere of 5% CO2 and 95% air, supplemented with 100 mL/L fetal calf serum (Invitrogen), 100 kIU/L penicillin–100 mg/L streptomycin (Biochrom), 0.55 mmol/L arginine–0.27 mmol/L asparagine (Sigma), and 10 mmol/L HEPES (Invitrogen). After 2 days, cells were harvested from monolayer dishes (19). Total RNA from the cells was extracted with Trizol Reagent (Invitrogen), and cDNA was synthesized from ~1 µg of isolated RNA by use of the AMV cDNA First Strand Synthesis Kit (Roche Diagnostics). cDNAs from the cell lines were diluted 5-fold and stored at –20 °C.

statistical analysis
We studied the association between KLK7 expression and clinical or histomorphologic features with the t-test and one-way ANOVA under conditions of gaussian distribution. We analyzed disease-free survival time (DFS), defined as the time from surgery until diagnosis of recurrent disease or death (combined DFS, in accordance with the CALGB and NSABP B-27 study protocols). The associations of KLK7 mRNA expression and other clinical and histomorphologic factors with DFS were analyzed by use of Cox univariate and multivariate proportional hazard regression models (20). Survival curves were generated by univariate Kaplan–Meier estimation with the log-rank regression model (21). P values ≤0.05 were considered to be statistically significant. Calculations were performed with the SPSS 12.0 statistical software package.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
development of a QPCR assay for KLK7 MRNA
To quantify KLK7 mRNA, we established a highly sensitive real-time QPCR assay, applying LightCycler technology (Fig. 1 ). The KLK7 gene consists of 6 exons (E1–E6). One major alternatively spliced KLK7 mRNA has been described that lacks exon 2 (13). That mRNA encodes a protein without the pre-/proregion and, most importantly, also lacks the N-terminal sequence of mature hK7, including His57 of the catalytic triad. Thus, the encoded shortened protein cannot display serine protease activity. Furthermore, in the mRNA splice variant, the open reading frame with homology to hK7 is preceded by 4 out-of-frame ATG codons, making it unlikely that the nonfunctional hK7-like protein is translated at all. In our assay, a forward primer directed mainly against exon 2 and partially overlapping the unique boundary between exons 2 and 3 was chosen for specific amplification of full-length KLK7 mRNA (Fig. 1A ). The reverse primer lies within exon 3. Previously, PCR assays have been applied by Kyriakopoulou et al. (12) and Talieri et al.(14) to analyze possible associations between KLK7 expression and clinical or histomorphologic variables in ovarian and breast cancer, respectively. These assays, in contrast to the newly developed QPCR assay, detect both full-length KLK7 mRNA and the short splice variant (Fig. 1A ).


Figure 1
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  		Figure 1. QPCR assay for KLK7.

(A), the KLK7 gene consists of 6 exons (E1–E6), 5 of which harbor coding regions ( Figure 1, noncoding regions; {square}, coding regions). Two alternative noncoding E1 exons have been described; both E1–E6 mRNA forms encode full-length hK7 (8)(10)(13). The amino acids of the invariant catalytic triad of serine proteases (His57, Asp102, and Ser195) are encoded by 3 different exons: E3, E4, and E6, respectively. In addition to the KLK7 mRNAs encoding full-length hK7, an alternatively spliced variant has been described that lacks exon 2 (13). Previously, PCR assays have been applied by Kyriakopoulou et al. (12), Talieri et al.(14), and Planque et al. (25). These assays—in contrast to our newly developed QPCR assay—detect both full-length KLK7 mRNA and the short splice variant, which encodes a nonfunctional serine protease. (B), LightCycler PCR calibration curves for quantification of KLK7 mRNA. The plots of detected vs theoretical KLK7 molecule numbers were generated from 33 independent PCR runs. The correlation coefficient (r) for the values is 0.99 (P <0.0001).

Because the 5' amplification primer overlaps the exon 2/3 boundary, amplification of (possibly contaminating) genomic KLK7 DNA is excluded [exons 2 and 3 are separated by a 412-bp intron in the genomic DNA (8)]. To generate calibration curves, we used glass capillaries coated with defined numbers of the plasmid pRcRSV-KLK7. A plot of measured vs theoretical KLK7 molecule numbers in 33 independent PCR runs for the capillaries coated with 10 to 100 000 copies is shown in Fig. 1BUp .

quantification of KLK7 MRNA in cell lines
We evaluated the performance of the QPCR assay by assessing KLK7 expression in transfected cell lines. Two breast cancer cell lines (MDA-231 and aMCF-7) and the ovarian cancer cell line OVMZ-6 were stably transfected with a pRcRSV-based expression plasmid encoding KLK7 or with the vector alone. We also analyzed KLK7 mRNA expression in the keratinocyte cell line HaCaT because human epidermis is known to express high amounts of KLK7 (9). As can be seen in Table 1 , the HaCaT cells expressed distinct amounts of KLK7 mRNA, whereas in the 3 analyzed tumor cell lines, there was no or only little KLK7 mRNA detectable. After transfection with a KLK7 expression vector, KLK7 mRNA expression was strongly increased (Table 1 ).


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  		Table 1. Quantification of KLK7 mRNA in cell lines.1

KLK7 MRNA expression in tumor-free breast tissue
We assayed for expression of full-length KLK7 in tumor-free tissue samples from 10 breast cancer patients. KLK7 mRNA was detectable in all but 1 specimen, with a median expression of 15.3 (zmol KLK7/amol h-G6PDH) and an interquartile range of 2.3–25.2. This result indicates that KLK7 is widely expressed in nonmalignant breast tissue.

KLK7 MRNA expression in breast cancer tissue specimens and its association with clinical and histomorphologic indices
We determined the expression of full-length KLK7 mRNA in 155 breast cancer tissue specimens with a median expression of 2.6 (zmol KLK7/amol h-G6PDH) and an interquartile range of 0.7–10.1. Only 5 samples (3.2%) were negative for full-length KLK7 mRNA expression. We found that the KLK7 values followed a log-normal distribution, as the distribution of the log-transformed KLK7 values [10 x log(zmol KLK7/amol h-G6PDH)] did not significantly deviate from a gaussian distribution (Lilliefors-corrected Kolmogorov–Smirnov test after logarithmic transformation, P ≥0.200). All further analyses were performed with log-transformed values. The median of the log-transformed KLK7 values was 4.1 (interquartile range, –1.5 to 10.1) and a mean (SD) of 4.1 (9.4).

The relationship between KLK7 expression and clinical or histomorphologic variables, including menopausal status, lymph node (LN) status, tumor size (pT), grade (Bloom–Richardson), estrogen receptor (ER) status, progesterone receptor (PgR) status, and adjuvant treatment, was analyzed by use of the parametric t-test and one-way ANOVA, respectively. As shown in Table 2 , we observed significantly higher KLK7 expression in ER-negative tumors (P = 0.001). Apart from that, there were no significant associations between KLK7 expression and the other clinical or histomorphologic variables (Table 2 ).


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  		Table 2. Relationship between KLK7 mRNA concentrations (log-normal distribution) and clinical or histomorphologic variables in tumor tissue specimens from 155 breast cancer patients.

association of KLK7 MRNA expression with dfs
Univariate Cox survival analysis revealed that high KLK7 expression (continuous) was significantly associated with prolonged DFS of the breast cancer patients [hazard ratio (HR) = 0.97; 95% confidence interval, 0.94–0.99; P = 0.005; Table 3 ]. Hence, the risk of relapse or death decreased by ~3% per unit of log-transformed KLK7 expression.


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  		Table 3. Characterization of breast cancer specimens in relation to DFS of the patients by univariate Cox regression analysis (n = 155).1

Because we found that KLK7 expression as a continuous variable was a significant prognosticator, we defined an optimal cutoff value of 2.5 [10 x log(zmol KLK7/amol h-G6PDH)] by optimized dichotomization (within the 30th–70th percentile range), based on the ability of KLK7 to predict DFS. This cutoff value classified 66 (42.6%) of the 155 patients as being KLK7 mRNA status low and 89 (57.4%) of the 155 patients as being KLK7 mRNA status high.

The results of the univariate Cox survival analysis for KLK7 expression (continuous), KLK7 mRNA status, and the clinical and histomorphologic variables are summarized in Table 3Up . KLK7 expression (continuous) and KLK7 mRNA status, along with LN status, tumor grade, and adjuvant treatment, were significant prognostic factors for DFS. Positive LN status was strongly linked to poorer prognosis with an HR of 2.09 (P = 0.003). Adjuvant treatment was associated with a shorter DFS through its association with features indicating an unfavorable prognosis, such as axillary LN involvement at diagnosis or larger size of the primary tumor. Patients with KLK7 mRNA status high had a significantly lower risk (HR = 0.45) of relapse or death (P = 0.001) than patients with KLK7 mRNA status low. Univariate Kaplan–Meier survival analysis confirmed the significantly poorer outcome of patients with KLK7 mRNA status low compared with patients with KLK7 mRNA status high (Fig. 2 ; log-rank test, P <0.001).


Figure 2
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  		Figure 2. Prognostic significance of KLK7 mRNA expression in breast cancer patients (n = 155).

Univariate Kaplan–Meier survival analysis revealed that breast cancer patients with KLK7 mRNA status low had an increased risk of relapse or death (combined DFS).

When KLK7 expression (continuous) was included with all clinical and histomorphologic variables (menopausal status, LN status, tumor size and grade, ER status, PgR status, adjuvant treatment, and age) in a multivariate Cox regression model, KLK7 expression (continuous) remained an independent significant predictor of DFS in this collective of breast cancer patients (HR = 0.97; P = 0.046; Table 4 ). A separate multivariate model including KLK7 mRNA status with all clinical and histomorphologic variables also indicated that KLK7 mRNA status is an independent significant prognostic factor (HR = 0.50; P = 0.005; Table 4 ).


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  		Table 4. Characterization of breast cancer specimens in relation to DFS of the patients by separate multivariate Cox regression models for KLK7 expression (continuous) and KLK7 mRNA status (n = 155).1

To determine the impact of KLK7 expression (continuous) and KLK7 mRNA status on the natural course of breast cancer disease, we excluded all patients receiving adjuvant treatment and performed univariate and multivariate Cox survival analyses. The subgroup of patients not receiving adjuvant treatment (n = 69) was least influenced by postoperative treatment and thus best reflected the natural course of the disease. The analysis revealed that high KLK7 expression (continuous) as well as KLK7 mRNA status high were significantly associated with prolonged DFS in these patients in both univariate [KLK7 continuous, HR = 0.92 (P = 0.001); KLK7 mRNA status, HR = 0.29 (P = 0.002)] and multivariate [KLK7 continuous, HR = 0.94 (P = 0.013); KLK7 mRNA status, HR = 0.40 (P = 0.034)] Cox regression analysis (Table 5 ). The Kaplan–Meier survival plot in Fig. 3 depicts the prognostic relevance of the KLK7 mRNA status in patients not receiving adjuvant treatment (log rank test, P = 0.001). Thus, KLK7 expression (continuous) and KLK7 mRNA status appear to be clinically relevant prognostic factors associated with a favorable disease outcome.


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  		Table 5. Characterization of the patients not receiving adjuvant treatment vs the subgroup receiving adjuvant treatment in relation to DFS by univariate and multivariate Cox regression analyses.1


Figure 3
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  		Figure 3. Subgroup survival analysis.

Patients were grouped according to adjuvant treatment. KLK7 mRNA status high in patients not receiving adjuvant treatment was associated with a significantly prolonged DFS.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
hK7 plays an important role in the terminal differentiation of squamous skin epithelia (22)(23)(24), and KLK7 gene expression has also been observed in the breasts, ovaries, kidneys, central nervous system, salivary glands, and lungs (10)(13)(14)(25)(26). KLK7/hK7 is frequently overexpressed in ovarian and other, mainly hormone-dependent, tumors, and hK7 may contribute to tumor cell growth and metastasis (10)(27)(28)(29). High concentrations of KLK7 mRNA were associated with shorter disease-free and overall survival of ovarian cancer patients (12). Similarly, increased KLK7 expression was found to be an indicator of unfavorable outcome in breast cancer patients (14). However, Yousef et al.(30) found no overexpression of the 15 tissue kallikreins in breast cancer tissue compared with healthy breast tissue and down-regulation of the expression of at least 4 tissue kallikrein genes (KLK5, KLK6, KLK8, and KLK10) in breast cancer tissue.

In contrast to a previous study of breast cancer patients (14), we found that higher amounts of KLK7 expression were associated with a decreased risk of relapse or death according to univariate and multivariate Cox regression analysis. This discrepancy may be related to the different assay formats used. In the previous study performed by Talieri et al. (14), more than one half of all samples (54.3%) were classified as negative according to a qualitative PCR assay. In the present study, we used a quantitative assay and found that only 5 of 155 samples (3.2%) were negative for KLK7 expression. We conclude that the qualitative PCR assay used in the previous study is not as sensitive as the assay used in our study.

We found a similar inconsistency when we analyzed KLK7 mRNA expression in tumor-free breast tissue. Whereas no KLK7 mRNA was detected in nonmalignant breast tissue in the previous study (14), we observed expression of the KLK7 gene in 9 of 10 analyzed samples. The latter observation, however, is in good agreement with previous RNA expression and protein quantification studies in nonmalignant breast tissue (10)(26).

The design of the present QPCR assay may also have led to discrepancies between the 2 studies. In our assay, the forward primer targets the splice junction between exons 2 and 3 of the KLK7 mRNA (with all but the 3' terminal nucleotide binding to exon 2 sequences), and the reverse primer is directed against a sequence in exon 3. Thus, only full-length KLK7 mRNA transcripts (encoding a functional serine protease) are amplified. In contrast, the primers used by Talieri et al. (14) for amplification of mRNA derived from breast cancer tissue and the primers used by Kyriakopoulou et al. (12) for detection of mRNA derived from ovarian cancer tissue are directed against exon 4 and exon 5, respectively (Fig. 1AUp ). Hence, these assays amplify both mRNA encoding full-length hK7 and the variant lacking exon 2. Alternative splicing is a frequent event within the human KLK gene locus. The transcriptome covering the 15 KLK genes comprises at least 82 mRNA forms (8). Several of these forms are cancer-specific and are therefore potential cancer markers (29). Thus, the combined amplification of full-length KLK7 mRNA and its strongly expressed tumor-associated mRNA splice variant may lead to different results than selective amplification of KLK7 full-length mRNA alone.

High expression of KLK7 mRNA tends to occur in tumor tissue of lung cancer patients without nodal or distant metastases, whereas most metastatic tumors express low concentrations of this mRNA (25). The same trend is seen in the present study; lower KLK7 expression was seen in tumor samples from LN-positive patients than from LN-negative patients, although the difference only approached statistical significance (P = 0.059; Table 2Up ).

Differences in patient treatment may also explain the different results. In contrast to the present study, the Greek study (14) included patients who had received neoadjuvant chemotherapy. Whereas most of the patients in the Greek study group (87 of 92; 94.6%) were treated with adjuvant systemic therapy (endocrine and/or chemotherapy), 44.5% of the patients in the present study (69 of 155) did not receive any adjuvant systemic treatment. There were only slight differences between the 2 studies in the distribution of the patients concerning nodal status and hormone receptor status.

KLK7 is up-regulated in breast cancer cells by estrogens and glucocorticoids (10). Positive KLK7 expression has been inversely correlated to tumor grade and PgR status (14). We detected no significant relationship between tumor grade and KLK7 expression. PgR status also showed no significant association with KLK7 expression, but higher KLK7 values were significantly linked to a negative ER status.

In conclusion, our results indicate that functions of proteases in malignancies are not limited to promotion of tumor growth or facilitation of metastasis. Certain proteases, such as matrix metalloproteinase-9 in node-negative breast cancer, were previously shown to be indicators of favorable patient outcome (31). Similarly, high expression of full-length KLK7 mRNA (and, consequently, high concentrations of the protease hK7) may be a favorable prognostic marker in breast cancer.


   Acknowledgments
 
This work was funded in part by the Dr. Mildred Scheel Stiftung für Krebsforschung, the Graduiertenkolleg 333 of the Deutsche Forschungsgemeinschaft, and by the Kommission Klinische Forschung der Technischen Universität München.


   Footnotes
 
1 Nonstandard abbreviations: hK, human tissue kallikrein; hK7, human stratum corneum chymotryptic enzyme (HSCCE); QPCR, quantitative reverse transcription-PCR; h-G6PDH, human glucose-6-phosphate dehydrogenase; DFS, disease-free survival; LN, lymph node; ER, estrogen receptor; PgR, progesterone receptor; and HR, hazard ratio.

2 Human genes: KLK7, kallikrein 7 (chymotryptic, stratum corneum).

3 Report from the PathoBiology Group of the European Organisation for Research and Treatment of Cancer.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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