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1
Laboratoire de Génétique Moléculaire, Faculté des Sciences Pharmaceutiques et Biologiques de Paris, 4 Avenue de l'Observatoire, F-75006 Paris, France.
2
Laboratoire d'Oncogénétique, Centre
René Huguenin, F-92211 St-Cloud, France.
a Author for correspondence. Fax 33 1 44 07 17 54; e-mail mvidaud{at}teaser.fr
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: We have developed and validated a real-time quantitative reverse transcription (RT)-PCR assay, based on fluorescent TaqMan methodology, to quantify ERBB2 gene expression at the mRNA level in breast tumors. This recently developed method of nucleic acid quantification in homogeneous solutions has the potential for a wide dynamic range, interlaboratory agreement, and high-throughput capacity without tedious post-PCR processing. The ERBB2 mRNA signal was normalized to the signal for TATA box-binding protein mRNA.
Results: The dynamic range was >1000-fold. The relationship
between Ct and log starting concentration was linear
(r2 
0.99). The mean (SD) normalized expression
of ERBB2 in healthy breast tissue was 0.95 (0.37).
Overexpression (>5 SD above mean for healthy breast) of the
ERBB2 gene was observed (at 3.2- to 135-fold) in
23 (17%) of 134 breast tumor RNA samples. As expected,
ERBB2 overexpression was present in all tumors with
ERBB2 gene amplification but was uncommon and at a low
ratio (<5) in breast cancers without gene amplification.
Conclusions: This new simple, rapid, semi-automated assay is a major alternative to fluorescence in situ hybridization and immunochemistry for gene alteration analysis in human tumors and may be a powerful tool for large randomized, prospective cooperative group trials and to support future ERBB2-based biological and gene therapy approaches.© 1999 American Association for Clinical Chemistry
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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These promising new clinical perspectives call for a sensitive, accurate, and rapid method to screen breast cancer patients for amplification/overexpression of the ERBB2 gene (9). Such a method should be simple, widely applicable, and reproducible. The results should be subject to specific quality controls and provide the possibility for determining the CV. Whenever possible, values should be continuous rather than dichotomous because cutoff values are often arbitrary and suboptimal. Two principal methods are used: fluorescence in situ hybridization (FISH)1 for the detection of gene amplification and immunohistochemistry for the detection of gene expression at the protein level. Although these two techniques can measure alterations on an individual cell basis, they are complex and subject to considerable variations in the hands of different teams, posing problems for reproducibility and widespread use (10)(11)(12). Moreover, FISH does not assess gene expression and cannot identify cases in which the gene product is overexpressed in the absence of gene amplification.
We developed a real-time quantitative reverse transcription (RT)-PCR assay based on TaqMan methodology to quantify ERBB2 mRNA in homogeneous total RNA solutions obtained from tumor samples (13). This real-time RT-PCR method has a major advantage over current quantitative PCR methods (14) as well as over FISH and immunohistochemistry, having a high-throughput capacity because no post-PCR manipulations are required. Moreover, the target input copy number is measured during the exponential phase of the reaction (when none of the reaction components is rate-limiting), leading to more accurate estimation of the sample concentration.
TaqMan methodology is based on the 5' nuclease assay first described by Holland et al. (15), which uses the 5'-3' exonuclease activity of Taq polymerase to cleave a dual-labeled probe annealed to a target sequence during amplification. Release of a fluorogenic tag from the 5' end of the probe is proportional to the target concentration and can be measured in "real time" during PCR amplification by a sequence detector (ABI PRISM 7700 Sequence Detection System; Perkin-Elmer Applied Biosystems).
We used this technique to measure ERBB2 gene expression at the mRNA level in a series of 134 unilateral invasive primary breast tumors. We also determined whether overexpression of the ERBB2 gene correlated with DNA amplification at the ERBB2 locus, determined previously by a real-time PCR method (16).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The patients (mean age, 58.3 years; range, 34–91 years) met the
following criteria: primary unilateral nonmetastatic breast carcinoma
on which complete clinical, histological, and biological data were
available; and no radiotherapy or chemotherapy before surgery. The main
prognostic factors are presented in Table 1
. The median follow-up was 8.2 years (range, 1.0–15.9 years).
Forty-eight patients relapsed; the distribution of first
relapse events was as follows: 14 local and/or regional
recurrences, 30 metastases, and 4 both.
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To help validate the kinetic quantitative RT-PCR method, we also analyzed five breast tumor cell lines obtained from the American Tissue Type Culture Collection (SK-BR-3, T-47D, BT-20, HBL-100, and MCF7).
Specimens of adjacent healthy breast tissue from 10 of the breast cancer patients and healthy breast tissue from 10 women undergoing cosmetic breast surgery were used as sources of normal RNA. Total RNA from a pool of six healthy human breast tissue samples was also purchased from Clontech.
evaluation of "classical" prognostic factors
The histological type and steroid-hormone receptor status of each
tumor as well as the number of positive axillary nodes were established
at the time of surgery. The malignancy of infiltrating carcinomas was
scored according to the histoprognostic system of Bloom and Richardson
(17). Estrogen and progesterone receptor status was assayed
as described by the European Organization for Research and Treatment of
Cancer (18), with a detection limit of 10 fmol/mg cytosolic
protein.
real-time rt-pcr
Theoretical basis.
The reactions are characterized by the
point during cycling when amplification of the PCR product is first
detected rather than the amount of PCR product accumulated after a
fixed number of cycles. The higher the starting quantity of the target
molecule, the earlier a significant increase in fluorescence is
observed. The parameter Ct (threshold cycle) is
defined as the fractional cycle number at which the fluorescence
generated by cleavage of the probe passes a fixed threshold above
baseline. The ERBB2 target message in unknown samples is
quantified by measuring Ct and by using a
calibration curve to determine the starting target message quantity.
The precise amount of total RNA added to each reaction mix (based on absorbance) and its quality (i.e., lack of extensive degradation) are both difficult to assess. We therefore also quantified transcripts of the gene coding for the TATA box-binding protein (TBP, a component of the DNA-binding protein complex TFIID) as the endogenous RNA control, and each sample was normalized on the basis of its TBP content. We selected the TBP gene as an endogenous control because the prevalence of its transcripts is similar to that of the ERBB2 target gene and because there are no known TBP retropseudogenes. (Retropseudogenes lead to coamplification of contaminating genomic DNA and thus interfere with RT-PCR despite the use of primers in separate exons). We therefore rejected the ß-actin, GAPDH (glyceraldehyde-3-phosphate dehydrogenase), and HPRT (hypoxanthine phosphoribosyltransferase) genes as endogenous controls because of the existence of corresponding retropseudogenes (19)(20); we also rejected the human 18S rRNA gene, which is intron-less, has no poly(A) tail, and has a very high abundance of transcripts; and the ß2-microglobulin gene, expression of which may be altered in some tumors (21).
For each experimental sample, the amounts of the targets and endogenous reference were determined from the calibration curve. The target amount was then divided by the endogenous reference amount to obtain a normalized target value.
The relative gene target expression was also normalized to a healthy breast tissue sample (calibrator), or 1X sample. Each of the normalized target values was divided by the calibrator-normalized target value to generate the final relative expression.
Final results, expressed as N-fold differences in
ERBB2 gene expression relative to the TBP gene
and the calibrator, termed "NERBB2",
were determined as follows:
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Primers, probes, and PCR consumables.
Primers and probes for
the TBP and ERBB2 genes were chosen with the
assistance of the computer programs Oligo 4.0 (National Biosciences)
and Primer Express (Perkin-Elmer Applied Biosystems). We conducted
BLASTN searches against dbEST and nr (the nonredundant set of GenBank,
EMBL, and DDBJ database sequences) to confirm the total gene
specificity of the nucleotide sequences chosen for the primers and
probes and the absence of DNA polymorphisms. The primer pairs for
ERBB2 were selected to be unique when compared with the
sequences of the closely related ERBB1 (EGFR),
ERBB3, and ERBB4 genes. The nucleotide sequences
of the oligonucleotide hybridization probes and primers are shown in
Table 2
. The primers and probes are designated by the nucleotide
position (relative to TBP GenBank accession no. X54993 and
ERBB2 GenBank accession no. M11730) corresponding to the 5'
position, followed by the letter U for upper (sense strand) or L for
lower (antisense strand). To avoid amplification of contaminating
genomic DNA, one of the two primers or the probe was placed at the
junction between two exons or in a different exon. For example, the
upper primer of TBP (706U) was placed in exon 5, the probe
(745U) was placed at the junction between exon 5 and exon 6, and the
lower primer (794L) was placed in exon 6.
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Primers were purchased from Scandinavian Gene Synthesis AB and probes from Perkin-Elmer Applied Biosystems.
RNA extraction.
Total RNA was extracted from breast specimens
by the acid-phenol guanidinium method (22). The quality of
the RNA samples was determined by electrophoresis through denaturing
agarose gels and staining with ethidium bromide, and the 18S and 28S
RNA bands were visualized under ultraviolet light.
Calibration curve.
The calibration curve was constructed with
fourfold serial dilutions of total RNA from healthy human breast tissue
(cat. no. 64037-1; Clontech,) in mouse total RNA (cat. no. 64042-1;
Clontech) with 1000, 250, 62.5, 15.6, and 3.9 ng of human normal breast
total RNA. The series of diluted human total RNAs were aliquoted and
stored at -80 °C until use.
cDNA synthesis.
Reverse transcription of RNA was performed in
a final volume of 20 µL containing 1x RT-PCR buffer (500 mmol/L each
dNTP, 3 mmol/L MgCl2, 75 mmol/L KCl, 50 mmol/L
Tris-HCl, pH 8.3), 10 U of RNasinTM ribonuclease
inhibitor (Promega), 10 mmol/L dithiothreitol, 50 U of Superscript II
RNase H- reverse transcriptase (Life
Technologies), 1.5 mmol/L random hexamers (Pharmacia), and 1 µg of
total RNA (calibration curve points and patient samples). The samples
were incubated at 20 °C for 10 min and 42 °C for 30 min, and
reverse transcriptase was inactivated by heating at 99 °C for 5 min
and cooling at 5 °C for 5 min.
PCR amplification.
All PCR reactions were performed on an ABI
Prism 7700 Sequence Detection System (Perkin-Elmer Applied Biosystems).
For each PCR run, a master mixture was prepared on ice with 1x TaqMan
buffer; 5 mmol/L MgCl2; 200 mmol/L dATP, dCTP,
and dGTP, and 400 mmol/L dUTP; 300 nmol/L each primer; 150 nmol/L
probe; and 1.25 U of AmpliTaq Gold DNA polymerase (Perkin-Elmer Applied
Biosystems). Ten microliters of each appropriately diluted reverse
transcription sample (calibration curve points and patient samples) was
added to 40 µL of the PCR master mixture. The thermal cycling
conditions comprised an initial denaturation step at 95 °C for 10
min and 50 cycles at 95 °C for 15 s and 65 °C for 1 min.
Experiments were performed with duplicates for each data point. Each PCR run included the five points of the calibration curve (fourfold serially diluted human normal breast cDNAs), a no-template control, the calibrator cDNA, and 41 unknown patient cDNAs. The target gene mRNA copy value of the 41 patients was obtained in ~2 h with this assay format. All patient samples with a CV of the number of ERBB2 mRNA copies >10% were retested.
statistical analysis
Relapse-free survival was determined as the interval between
diagnosis and detection of the first relapse (local and/or regional
recurrences and/or metastases).
Clinical, histological, and biological factors were compared using the
2 test. Differences between the two
populations were judged significant at confidence levels greater than
95% (P <0.05). Survival distributions were estimated by
the Kaplan-Meier method (23), and the significance of
differences between survival rates was ascertained using the log-rank
test (24).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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0.99). The
efficiency of the reaction (E), calculated by the formula:
E = 101/|m| - 1, where m is
the slope of calibration curve, was 90–100%.
erbb2 mRNA IN HEALTHY BREAST TISSUES
To determine the cutoff for altered ERBB2 gene
expression at the RNA level in breast cancer tissue, the
NERBB2 value (ratio of ERBB2
mRNA to TBP mRNA, calculated as described in Materials
and Methods) was determined for 20 normal breast tissue RNAs.
Because this value consistently fell between 0.5 and 1.7 (mean
0.95, 0.37), values of 3 (mean + 5 SD) or more were considered to
represent overexpression of the ERBB2 gene in tumor RNA
samples.
erbb2 mRNA IN TUMOR BREAST TISSUES
Among the 134 breast tumor RNA samples tested, 23
(17.2%) showedERBB2
overexpression. Major differences in ERBB2 mRNA were
observed (NERBB2 from 3.2 to 135), with 13
(9.7%) tumors giving an expression level 3- to 10-fold, 6 (4.5%)
tumors giving an expression level 10- to 30-fold, and 4 (3.0%) tumors
giving an expression level >30 higher than that of healthy breast
tissue. The strongest expression was 135-fold the basal level in tumor
ERB98, which showed the greatest ERBB2 gene amplification
(15-fold) in our previously tested breast tumor DNA series
(16). Fig. 1
and Table 3
represent data on tumors in which the ERBB2 gene was
expressed 135-fold the basal value (sample ERB98), 11-fold the basal
value (sample ERB37), and normally (sample ERB55). ERBB2
expression was also investigated in five breast tumor lines, and only
SK-BR-3 showed overexpression (NERBB2 =
13). Lastly, among the 10 patients from whom both primary breast tumors
and matched healthy breast tissue were investigated, two tumors (ERB114
and ERB67) showed clearly higher ERBB2 expression in the
tumor (NERBB2 = 11.2 and 4.7,
respectively) than in the healthy tissue
(NERBB2 = 0.9 and 1.2, respectively).
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The ERBB2 primer pair used in this study was placed in the 5'-terminal region of the ERBB2 gene (extracellular domain of p185c-erbB2), and total concordance was obtained with a second ERBB2 primer pair in the 3' region (intracytoplasmic domain of p185c-erbB2; data not shown).
erbb2 mRNA STATUS AND CLINICAL AND
PATHOLOGICAL FACTORS
We sought links between the ERBB2 mRNA status and
standard clinical, pathological, and biological factors in breast
cancer (Table 4
). The statistically significant associations were between
ERBB2 gene overexpression and Scarff-Bloom-Richardson (SBR)
histopathological grade III (P <0.05) and negative
progesterone receptor status (P <0.001). A trend toward a
link between ERBB2 gene overexpression and estrogen receptor
negativity was also observed (P = 0.09).
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Moreover, patients with tumors overexpressing ERBB2 did not
relapse more frequently (Table 4
) and did not have significantly
shorter relapse-free survival after surgery (log-rank test) compared
with patients with tumors not overexpressing ERBB2.
NERBB2 ANDERBB2 AMPLIFICATION
Among the 134 tumors studied for ERBB2 expression at
the RNA level, 94 had previously been tested for ERBB2
amplification, using the same TaqMan technology (16). A near
complete correlation between the results of real-time RT-PCR
(ERBB2 gene expression status) and real-time PCR
(ERBB2 gene amplification status) was obtained for
tumors with high and moderate amounts of ERBB2
messengers (NERBB2 >5). There were only
three cases in which real-time RT-PCR showed ERBB2 gene
overexpression, whereas real-time PCR did not, but these three tumors
had a low level of ERBB2 gene overexpression
(NERBB2 = 3.7, 4.5, and 4.8,
respectively). We observed ERBB2 gene overexpression in all
tumors in which real-time PCR showed ERBB2 gene
amplification.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Gene expression status has also been studied at the RNA level by means of Northern blotting, but this method is not sensitive enough to detect low-level gene overexpression or accurate enough to quantify the full range of expression values. Northern blotting is also time-consuming, uses radioactive reagents, and requires large amounts of RNA, which means that it cannot be used routinely in clinical laboratories. An amplification step is required to determine the mRNA copy number of a given target gene from minimal quantities of tumor RNA (small early-stage tumors or cytopuncture specimens).
In this study, we validated an RT-PCR method recently developed for the quantification of gene expression (13). The method, based on real-time analysis of PCR amplification and TaqMan methodology, has several advantages over other RT-PCR-based quantitative assays such as competitive quantitative RT-PCR (14). The first advantage is that the real-time PCR method is performed in a closed-tube system, avoiding the risk of contamination by amplified products. The second advantage is the simplicity and rapidity of sample analysis because no post-PCR manipulations are required. The third advantage is that the system has a linear dynamic range of at least four orders of magnitude, meaning that samples do not need to contain equal starting amounts of RNA. This technique should, therefore, be suitable for analyzing small early-stage tumors, cytopuncture specimens, or formalin-fixed, paraffin-embedded tissues. The fourth, and perhaps most important, advantage is that real-time RT-PCR makes RNA quantification much more precise and reproducible because it is based on Ct values rather than end-point measurement of the amount of accumulated PCR product. Indeed, the ABI Prism 7700 Sequence Detection System enables Ct to be calculated when PCR amplification is still in the exponential phase and when none of the reaction components are rate-limiting. The within-run CV of the number of ERBB2 mRNA copies for calibrator human RNA (five replicates) was always below 10%, and the between-assay CV in five different analytical runs was always below 20% (data not shown). Moreover, unlike competitive quantitative RT-PCR, real-time RT-PCR does not require an internal control. (The design and storage of internal controls and the validation of their amplification efficiency is painstaking).
The only potential disadvantage of real-time RT-PCR, like all other RT-PCR-based methods and solid matrix blotting techniques (Northern blots and dot blots), is that it cannot avoid dilution artifacts inherent in the extraction of RNA from tumor cells contained in heterogeneous tissue specimens. However, real-time RT-PCR has a fast turnaround time and requires small amounts of total RNA (5 ng/reaction in routine use), which means that it can be applied to several biopsy punches of very heterogeneous tumor samples.
Overall, the results of this study are in agreement with those reported in the literature: (a) The frequency (17%) of ERBB2 alterations in our breast tumor series was similar to that reported by other teams (11)(32). (b) The maximal ERBB2 overexpression was 135-fold, in keeping with previous data obtained with other quantitative PCR-based assays (33). (c) The ERBB2 results obtained for the six breast tumor cell lines with real-time RT-PCR (i.e., SK-BR-3 overexpressed 13-fold) were in good agreement with previous data (34). (d) The results of this study also confirm that ERBB2 overexpression rarely occurs in breast cancer in the absence of ERBB2 gene amplification (11). (e) We found an association between ERBB2 gene overexpression and SBR histopathological grade III and steroid receptor negativity but not a poor prognosis, confirming that ERBB2 is a marker of tumor aggressiveness rather than a major prognostic factor in human breast cancer [reviewed in Ref. (35)]. These latter findings must, however, be confirmed in a prospective and larger series of breast cancer patients. The relationship between ERBB2 gene overexpression and the response and/or resistance to different therapies could not be studied in this retrospective series because the therapies used after surgery were highly variable.
We observed no prognostic importance of very low ERBB2 expression in our series (only 3 relapses among the 10 (30%) patients with tumors showing an NERBB2 value <0.5), as suggested by Koscielny (36). Finally, we observed total concordance of our results when we used a primer pair placed in the 5'-terminal region of the ERBB2 gene (extracellular domain of p185c-erbB2) or in the 3' region (intracytoplasmic domain of p185c-erbB2), suggesting that the truncated ERBB2 transcript (5', 2.1 kb) that produces a secreted form of ERBB2 receptor containing only the extracellular ligand-binding domain (37) is not overexpressed alone without the full-length 4.6-kb ERBB2 transcript.
In conclusion, we need today a reliable and reproducible technique for assessing ERBB2 status with an established quality-control procedure to compare the ERBB2 values between laboratories. Should we report gene amplification, protein expression, or mRNA levels? ERBB2 status mainly has been studied with two methods, FISH and immunohistochemistry, that are not accurate enough to quantify (with SD determination) the full range of alterations. Here we describe a rapid, highly sensitive, high-throughput RT-PCR assay to determine ERBB2 status. This method should be very useful for routine use in support of new ERBB2-based therapies for breast cancer.
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Acknowledgments |
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Footnotes |
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References |
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