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Molecular Oncology, Markers, Clinical Correlates |
Novel Therapeutic Approaches Section, Oncologia Sperimentale D [A. D. L., A. C., N. N.], Oncologia Medica B [S. P., C. G., A. R.], Anatomia Patologica [A. D.], Chirurgia Oncologica C [F. C., V. P.], and Endocrinologia Oncologica [A. D. M.], ITN-Fondazione Pascale, 80131 Naples, Italy
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2 test). The expression of EGFR, cytokeratin 19,
and carcinoembryonic antigen mRNA in blood samples from patients with
metastatic colon carcinoma was compared. EGFR, cytokeratin 19, and
carcinoembryonic antigen transcripts were found in 8 of 11 (73%), 3 of
11 (27%), and 5 of 11 (45%) patients, respectively. Furthermore, two
of seven (29%) Dukes’ B and five of nine (55%) Dukes’ C colon
carcinoma patients were found to express EGFR mRNA in the peripheral
blood. All patients that expressed EGFR transcripts in the peripheral
blood were found to express the EGFR protein in the corresponding
primary carcinoma, as assessed by immunohistochemistry. These data
suggest that the EGFR assay that we developed is a highly specific and
sensitive technique to detect circulating tumor cells in patients
affected by different carcinoma types. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The metastatic process is a complex cascade of events: tumor cells in the primary site must erode the basement membrane; penetrate a blood vessel; and spread to distant sites (1) . In this regard, detection of carcinoma cells in the blood could be important to identify carcinoma patients at high risk of relapse. In fact, immunochemical detection of micrometastases in the bone marrow of breast, colon, and gastric carcinoma patients has been shown to correlate with early disease relapse (2, 3, 4) . More recently, the RT-PCR3 has been proposed as a rapid screening assay to detect systemic tumor cell dissemination in peripheral blood and/or bone marrow. RT-PCR shows a 10–100-fold higher sensitivity than routine immunocytochemistry methods. Several markers have been used to detect circulating cancer cells. In particular, CKs and CEA have been proposed as markers to detect circulating tumor cells in gastrointestinal, breast, and/or lung carcinoma patients (5, 6, 7, 8) . Few reports have suggested that a correlation between expression of these markers in the peripheral blood and/or in the bone marrow and patient outcome might exist (6 , 9, 10, 11, 12) .
The EGFR is expressed in all cell types with the exception of hematopoietic cells. More importantly, the EGFR has been found to be overexpressed in 50–70% of human primary colon, lung, and breast carcinomas as well as in other tumor types (13) . Therefore, the EGFR might represent a suitable marker for detection of circulating tumor cells in patients affected by different carcinoma types. In this context, Mapara et al. (14) developed an EGFR RT-PCR assay to detect tumor cells in leukapheresis products obtained from breast cancer patients. However, this assay used a single-step RT-PCR, and it was less sensitive than immunocytochemical detection of tumor cells. More recently, a nested PCR assay for detection of EGFR mRNA has been developed (15) . This assay also showed a low sensitivity because EGFR transcripts were found in only 22% of peripheral blood samples from metastatic breast cancer patients.
The aim of this study was to develop a novel, nested RT-PCR-Southern blot assay for the detection of EGFR mRNA in peripheral blood. For this purpose, we analyzed peripheral blood samples from healthy volunteers and from breast, colon, and lung carcinoma patients. Finally, we compared the sensitivity and specificity of this technique with those of other previously reported RT-PCR assays.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and 4)
. The only exclusion criterion was
previous treatment. Thirty-eight healthy volunteers were also analyzed
as controls. After obtaining informed consent from patients, peripheral
blood samples (5 ml) were obtained by aspiration into EDTA-containing
syringes.
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RNA Preparation.
Five-ml aliquots of peripheral blood were processed within 1 h of
being obtained from the patient. The blood samples were mixed with 1 ml
of 5% dextran-saline solution and left to set for 30 min at room
temperature to yield erythrocyte sediment. Supernatant was collected
and centrifuged at 500 x g for 10 min at 4°C. The
cells were then suspended in 1 ml of nucleic acid extraction buffer and
frozen at -70°C until RNA extraction was performed. Total cellular
RNA was obtained by the acid guanidine isothyocianate-phenol-chloroform
extraction procedure, as described previously (18)
.
PCR Primers and RT-PCR.
EGFR primers for the nested PCR were designed from the sequences
of the EGFR human gene and cDNA. In the first-round PCR, we used
the EGFR primers published by Mapara et al.
(14)
: primer A, 5'-TCTCAGCAACATGTCGATGG-3', which
corresponds to sequence 702–721 of primer B, EGFR cDNA; and primer B,
5'-TCGCACTTCTTACACTTGCG-3' (amino acids 1156–1175). These primers
amplify a 473-bp fragment of the EGFR cDNA. The nested PCR was
performed by using primer A and primer C (5'-TCACATCCATCTGGTACGTG-3';
amino acids 1005–1024), and the PCR product was 322 bp in length. The
RT reaction was performed using 2-µg aliquots of total cellular RNA.
First-strand cDNA synthesis was carried out in a reaction volume of 20
µl using the RNA synthesis kit of Perkin-Elmer (Branchburg, NJ) as
suggested by the manufacturer. An 8-µl aliquot of this reaction was
subsequently used for EGFR PCR amplification in a 25-µl reaction
buffer containing 10 mM Tris-HCl, 1.5 mM
MgCl2, 50 mM KCl, the four deoxynucleoside
triphosphates (0.25 mM each), 10 pm of each primer (A and
B), and 0.5 unit of Taq Gold polymerase (Perkin-Elmer). Samples for PCR
amplification were prepared in a laminar flow hood to avoid
contamination. PCR was performed for 30 cycles consisting of 5 cycles
of 30 s at 94°C, 45 s at 60°C, and 45 s at 72°C
and 25 cycles of 30 s at 94°C, 45 s at 55°C, and 45 s at 72°C in a GeneAmp PCR System 9700 (Perkin-Elmer). The samples
were heated for 10 min at 94°C before the first cycle, and the
extension was lengthened to 10 min during the last cycle. One µl of
the first-round PCR product was used for the nested PCR. The PCR
conditions for the nested PCR reaction were similar to those for the
first-round PCR, with the following exceptions: (a) primers
A and C were used for amplification; and (b) the total PCR
number of cycles was 35 (5 + 30 cycles). Fifteen µl of the PCR
product were electrophoresed through a 1.5% agarose gel. The gel was
stained with ethidium bromide to allow visualization of the DNA, which
was then denatured and transferred to a nylon membrane. Finally, the
membrane was hybridized with an EGFR cDNA probe. The cDNA product was
visualized using streptavidin-alkaline phosphatase-coupled enhanced
chemiluminescence (New England Biolabs, Beverly, MA).
The GAPDH PCR reaction was performed as described previously (19) . CEA and CK19 nested PCR-Southern blot assays were performed as described previously (5 , 7) , with the following exceptions: (a) the PCR reaction was carried out in a 25-µl volume; (b) a hot start technique with TaqGold was used; and (c) different amounts of cDNA were used, as detailed in "Results."
Analysis of each sample was repeated at least two times. Two identical, consecutive results completed testing. A positive control and several negative controls were included in each experiment.
Immunohistochemistry.
EGFR expression in formalin-fixed, paraffin-embedded tissues was
assessed as described previously (20)
by using the C216
anti-EGFR monoclonal antibody (DBA, Milan, Italy).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; data not shown).
We have previously found that a high in vitro sensitivity
does not always correspond to high sensitivity in the patients
(19)
. Therefore, we further developed the assay by
contemporary analysis of peripheral blood samples obtained from a small
group of colon carcinoma patients and healthy volunteers (Table 1)
. In particular, we increased the
amount of cDNA step by step in the first PCR reaction to increase the
sensitivity of the assay. We detected EGFR mRNA in 5 of 11 (45%) colon
carcinoma patients and in none of the healthy donors by using 500 ng of
cDNA in the first PCR reaction. When we increased the amount of cDNA up
to 800 ng in the first-round PCR, EGFR transcripts were observed in 8
of 11 (73%) colon carcinomas and in 1 of 10 (10%) healthy volunteers.
The latter PCR conditions were used in the following experiments.
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.
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. All of the samples showed a positive
GAPDH RT-PCR signal, indicating the presence of intact RNA and
successful first-strand cDNA preparation (data not shown). We detected
EGFR transcripts in 44 of 75 (59%) carcinoma patients and in 4 of 38
(10.5%) healthy donors (Fig. 3
; Table 2
). This difference was statistically significant (P <
0.001, 2 test). The highest sensitivity was observed in
colon carcinoma (73%), whereas only 48% of the breast carcinoma
patients were found to be positive. However, this difference was not
statistically significant as assessed by the 2 test.
Southern blot analysis of the PCR product confirmed the specificity of
the reaction, and it also increased the number of positive patients. In
fact, an EGFR band was evident after ethidium bromide staining of the
gel in approximately two-thirds of the positive samples (Fig. 3A).
An additional 35% of samples were found to be positive
after Southern blot analysis (Fig. 3B).
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. Three of 11 (27%)
colon carcinoma patients and 1 of 10 (10%) healthy donors were
positive for CK19 expression when 200 ng of cDNA were used (Table 3)
.
As well as we did for EGFR, we tried to improve the sensitivity of the
technique by increasing the amount of cDNA in the first PCR reaction.
However, this also resulted in a decrease of the specificity of both
the CEA and CK19 assays (Table 3)
.
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. Finally, we analyzed by
immunohistochemistry the expression of EGFR in the primary tumors of 10
patients with either Dukes’ B or Dukes’ C colon carcinoma (Table 5)
. All patients who expressed EGFR
transcripts in the peripheral blood were found to express the EGFR
protein in the corresponding primary carcinoma. The expression of the
EGFR transcripts in the primary tumors was also confirmed by RT-PCR
analysis of the RNA extracted from the same formalin-fixed,
paraffin-embedded tissues that were used for immunohistochemical
analysis (data not shown). We assessed EGFR expression in tumors from
two Dukes’ D patients (patients 104 and 110; Table 5
). In fact, both
patients underwent surgery because distant metastases were not detected
by routine staging procedures. However, liver metastases were found in
both cases during surgery. The tumors from the two patients were both
positive for EGFR expression, and the peripheral blood samples from
these patients were both found to express EGFR transcripts (Table 5)
.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The higher in vivo sensitivity of our assay might also justify the fact that we found EGFR transcripts in 4 of 38 (10.5%) healthy donors, whereas Leitzel et al. (15) found no expression of EGFR mRNA in 23 control individuals. None of the volunteers who were screened in this study had clinical evidence of tumor disease at the time of blood withdrawal. It has long been established that the EGFR is not usually expressed in hematopoietic cells (13) . It is conceivable that our technique is able to detect illegitimate transcription of the EGFR gene in leukocytes (21) . However, the specificity of our EGFR assay was confirmed by the observation that no false positives were found in a small group of colon carcinoma patients in whom the expression of the EGFR protein in the tumor was assessed. In fact, all patients who were positive for EGFR mRNA expression in the blood showed detectable levels of EGFR protein in the primary tumor, whereas none of the patients with tumors negative for EGFR expression was found to express EGFR transcripts in the peripheral blood. In this context, no correlation was found between the intensity of EGFR staining in the tumor and the quantitative results of the RT-PCR assay on peripheral blood. The ability of the RT-PCR assay to detect EGFR transcripts is clearly correlated to both the level of expression of the EGFR and the number of tumor cells that are circulating. These two parameters are not necessarily correlated. The high sensitivity of our assay also allowed us to detect EGFR mRNA in approximately 45% (7 of 16) of colon carcinoma patients with resectable disease, whereas Leitzel et al. (15) failed to demonstrate the presence of EGFR transcripts in locally recurrent breast cancer patients and in adjuvant breast cancer patients. Clinical follow-up of patients with localized, resectable colon carcinoma has been started at our institution to assess whether patients at high risk of relapse might be recognized by using this assay.
Our data suggest that the background level of expression of EGFR in
leukocytes is lower when compared to other markers, such as CEA and
CK19. In fact, a lower number of false positives for EGFR expression
were seen in a group of healthy donors, when compared with CEA and
CK-19 (Table 3)
. In particular, CK19 showed a very low specificity.
Several reports have suggested that illegitimate transcription of CK19
mRNA occurs in human leukocytes (22
, 23)
. The use of a
nested PCR technique often results in detection of CK19 transcripts in
healthy volunteers (24)
. Expression of CEA mRNA has been
described recently in 76–80% of peripheral blood samples from
patients with metastatic carcinomas by using a nested PCR or a PCR
dot-blot technique, respectively (6
, 9)
. A significant
correlation has been found between expression of CEA mRNA and tumor
recurrence in breast and gastrointestinal carcinoma patients by using a
nested PCR approach (9)
. Several reports have also
demonstrated that a high percentage of false positive results occurs
when using a nested PCR analysis for CEA mRNA expression (23
, 25)
. In our hands, the nested PCR CEA assay showed a higher
specificity when compared with CK19. However, when we tried to increase
its sensitivity by increasing the amount of cDNA in the PCR reaction, a
high number of false positive results were observed.
These results suggest that the EGFR might represent a suitable marker for detection of circulating tumor cells. In this regard, this study is the first to demonstrate that circulating tumor cells can be detected in the peripheral blood of patients with lung or colon carcinoma. However, the EGFR is expressed in several human carcinoma types other than breast, lung, and colon tumors in which this assay could be used (13) . Several pieces of experimental evidence suggest that the EGFR is involved in tumor formation and progression. Overexpression of EGFR has been correlated with prognosis and metastatic spreading in several carcinoma types, such as breast, head and neck, and lung malignancies (13) . In this context, EGFR expression might be more informative on patient’s prognosis than CK or CEA expression because of its involvement in the pathogenesis of human carcinomas. Finally, novel therapeutic approaches based on EGFR blocking antibodies, EGFR tyrosine kinase inhibitors, or antisense oligonucleotides directed against either EGFR or its ligands have been developed (26 , 27) . The EGFR assay could be used to monitor the response of the tumor to these novel agents by monitoring the levels of EGFR-positive cells shed by the carcinoma during therapy.
In conclusion, we believe that the EGFR assay that we have developed is a highly specific and sensitive technique to detect circulating carcinoma cells. Studies on a larger cohort of patients and in different carcinoma types are required to confirm these findings. More importantly, clinical follow-up of patients with resectable disease will allow us to assess whether high-risk patients can be selected by using this approach.
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FOOTNOTES |
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1 Supported by a grant from the Associazione
Italiana per la Ricerca sul Cancro (to N. N.). 
2 To whom requests for reprints should be
addressed, at Novel Therapeutic Approaches Section, Oncologia
Sperimentale D, ITN-Fondazi-one Pascale, 80131 Naples, Italy.
Phone: 390815903826; Fax: 390815461688.
3 The abbreviations used are: RT-PCR, reverse
transcription-PCR; EGFR, epidermal growth factor receptor; CK,
cytokeratin; CEA, carcinoembryonic antigen.
Received 8/12/99; revised 1/ 4/00; accepted 1/ 6/00.
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REFERENCES |
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