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Molecular Oncology, Markers, Clinical Correlates |
Maxillofacial Unit/Oncology, King’s College Hospital, London SE5 8RX, United Kingdom
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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6% of all cancers. Despite very
significant improvements in surgery and radiotherapy, comparison with
historical controls shows only a modest improvement in overall
survival, although there have been significant improvements for tumors
at some sites [for examples, see Franceschi et al.
(1)
and references therein]. There is also evidence that
the overall pattern of disease is changing so that today, fewer
patients die as a result of locoregional disease but more succumb
because of development of distant metastases or a second primary tumor
(2, 3, 4, 5, 6, 7, 8)
. These studies suggest that occult tumor cells must
remain in the body but are not detected by the current diagnostic
procedures. This residual cancer may be present at the surgical
margins, in lymph nodes, or be disseminated throughout the body, where
it is most readily detected in the hematopoietic cell compartment.
Treatment may also fail if a second tumor develops, because the primary
lesion develops within a field of cancerization where the mucosa
harbors the genetic aberrations required for tumorigenesis. There is a wealth of evidence identifying tumor-positive surgical margins as an important source of treatment failure, and in view of this, most surgeons aim for a minimum of 1-cm clearance when resecting these cancers. However, the anatomy of the oropharynx means that this is often not achievable, and studies reveal that 12–32% of cases have positive surgical margins (9, 10, 11, 12) , although the true frequency may be much higher. For example, Brennan et al. (13) have shown, using a sensitive molecular diagnostic based on finding the same p53 gene mutation in the primary tumor and the surgical margins, that malignant cells can be detected for 52% of patients with histopathologically negative margins. The same study reported that 38% of these cases with positive margins after molecular analysis developed local recurrence, whereas no patient with negative margins failed treatment at the local site. This methodology can also be used to provide a sensitive approach to screen lymph nodes, which are negative for tumor by light microscopy, for the presence of micrometastases.
A proportion of recurrent tumors share the same p53 mutation (14, 15, 16, 17) , and p53 mutations in mucosa adjacent to head and neck tumors (13 , 18, 19, 20, 21) have been taken as evidence of likelihood of the development of a malignant lesion. There is also data that suggests that second primary tumors develop more frequently when p53 mutations are present in single (18) or multiple biopsies of tumor-distant mucosa (22) . This later study used a FASAY2 (23) to detect mutations that abrogate the function of p53, an approach that has the advantage of detecting polyclonal aberrations against a background of wild-type.
In addition to minimal residual cancer and precancerous change within the upper aerodigestive tract, patients may also fail treatment because of the presence of malignant cells in the hematopoietic cell compartment (24, 25, 26) . At present, the prognostic significance of disseminated tumor cells has not been established conclusively, although tumor cells identified in mononuclear cells obtained from the bone marrow of 32–64% of cases with SCC of the head and neck were associated with poor outcome (24 , 25) . Subsequent studies have revealed that when density gradients are used to prepare leukocyte subsets, although tumor cells sediment predominantly with the mononuclear fraction, they may also be detected in the granulocyte layer (26) . Thus, to improve the sensitivity of tumor cell detection, protocols for detection of disseminated tumor cells for patients with tumors of squamous epithelium must screen all leukocytes, using an approach that incorporates appropriate controls and morphological assessment of immunostained cells, to minimize the risk of false-positive results (26) .
To shed light on the vexing question as to why a proportion of patients develop local and distant recurrence despite seemingly adequate treatment, we have used the p53 phage plaque assay (13) to detect occult tumor cells in surgical margins, immunocytochemistry to detect disseminated tumor cells (26) , and FASAY (23) to search for molecular evidence of field cancerization by detecting p53 mutations in tumor-distant mucosa. Our preliminary findings confirm that detection of minimal residual cancer can provide additional prognostic information and support the need for long-term prospective studies to assess the contribution of these remaining malignant cells to treatment failure for this group of patients.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The indications for postoperative radiotherapy to the local site were close resection margins (all cases with involved resection margins were excluded from the study), a noncohesive tumor margin, or perineural spread. The neck was irradiated when two or more tumor-positive lymph nodes were identified and when extracapsular spread was present. In addition, elective irradiation was used when the risk of microscopic tumor involvement was considered to be at least 20% in clinically negative necks, where no surgery was performed. Details of the radiation dose and timing are not shown for these cases because it was felt that this was outside the purview of this report.
All clinical samples were coded and analyzed blind. Local recurrence was confirmed by histopathologically demonstrable SCC at a site adjacent to the index lesion. All patients have been followed for a minimum period of 36 months. Ethical Committee Approval for the minimal residual cancer program was granted at King’s College Hospital.
Surgical Margins and Lymph Nodes.
After excision of the primary tumor, four mucosal and two deep margins,
each approximately 1 cm x 8 mm x 8 mm, were harvested. The
mucosal biopsies were taken at opposite ends of the defect for
T1 and T2 lesions, but for
larger T3 and T4 tumors and
lesions which involved bone, the shape of the defect meant that the
mucosal margins were often taken at random. Two deep margins were taken
from the floor of the resection unless the tumor was excised in
continuity with the neck dissection and there were no other sites that
could be sampled. Each margin was divided into two; the inner portion
was processed for conventional light microscopy, and the outer half
frozen for molecular analysis. Sections of paraffin-embedded lymph
nodes >1 cm in diameter, considered to be tumor-free after light
microscopy, were also analyzed.
Biopsies of Tumor-distant Mucosa.
Three to five biopsies of clinically normal mucosa were taken from
patients undergoing preoperative diagnostic endoscopy or at the time of
primary surgery. Each biopsy was divided in two; one half was sent for
histology, and the other was used for molecular analysis. All samples
were taken at least 5 cm from the tumor, and the distance between
biopsies was at least 2 cm. Samples to be analyzed using the modified
FASAY (22)
were placed directly in 500 µl of RNA lysis
buffer [500 mM LiCl, 1% LiDS, 100 mM Tris (pH
8), 10 mM EDTA, and 5 mM DTT], immediately
frozen on dry ice, and stored at -80°C until use. All biopsies were
examined by light microscopy for evidence of hyperplasia or dysplasia.
FASAY for Identification of a Signature p53 Gene
Mutation in the Primary Tumor.
The presence or absence of a p53 mutation was determined by screening
p53 cDNA by FASAY, according to the method modified by
Tada et al. (27)
, with the yeast reporter
strain yIG397 (23)
. Ten-µm frozen sections were prepared
from 50–100 mg of tumor tissue and lysed with LiDS buffer as described
above. The sample was homogenized in 500 µl of LiDS lysis/binding
buffer by passing it repeatedly through a 22-gauge needle and
freeze/thawing twice on dry ice. After centrifugation, mRNA was
prepared using Dynabeads (Dynal, Oslo, Norway) according to the
manufacturer’s instructions. RNA was eluted in 20 µl of water
containing 50 units of human placental RNase inhibitor (Roche, Lewes,
United Kingdom). p53 cDNA was synthesized from 5 µl of
mRNA at 46°C for 1 h in a final volume of 20 µl containing 100
units of Superscript II reverse transcriptase (Life Technologies, Inc.,
Paisley, Scotland) and 0.1 µg of RT1 primer (CGG GAG GTA CAA), as
described by Flaman et al. (23)
. The
p53 cDNA was amplified in 20 µl of reaction mixture
containing 2 µl of the reverse transcriptase reaction product, 1.25
units of pfu DNA polymerase (Stratagene, Amsterdam, Holland), 10 pmol
of primers, 10% DMSO, and 50 mM deoxynucleotide
triphosphates. Primers P3 and P4 were used for the standard FASAY
(22)
. PCRs were run in the Thermal Cycler Model 2400
(Perkin-Elmer, Warrington, United Kingdom) at 96°C for 1 min; then 35
cycles of 95°C for 40 s, 65°C for 70 s, and 78°C for
90 s; followed by 78°C for 2 min. Satisfactory amplification was
confirmed by examining the PCR product on a 1% agarose gel. Each crude
PCR product was used for yeast transformation. More than 200 colonies
were examined for each sample analyzed, and yeast plasmids harboring a
p53 mutation and wild-type p53 were included in each run. Plasmid DNA
was rescued from yeasts yielding red colonies by alkaline lysis (Wizard
Minipreps; Promega, Southampton, United Kingdom) and electroporated
into Escherichia coli, strain DH5
. At least six plasmids
were rescued from all samples yielding >10% red colonies, and
mutations were identified on both strands by automated sequencing.
Genomic DNA was prepared for cases found to have intronic
p53 gene deletions, and the mutation was determined by
sequencing a PCR fragment encompassing the deletion.
Modified FASAY for Identification of Polyclonal Mutations in
Tumor-distant Mucosa.
To increase the sensitivity of the assay and discriminate clonal
mutations from PCR-induced mutations, the modified split form of this
assay was used to examine three to five samples of normal mucosa with
primers P3, P17, P4, and P16 (22)
for each case. cDNA
obtained from normal mucosa yields 3–15% red colonies, which
represents the background for the FASAY (22
, 27)
. Plasmid
DNA was rescued from at least six colonies for all patient samples,
yielding >10% red colonies for sequencing.
p53 Phage Plaque Assay for Detection of Residual
Disease at the Surgical Margins and Micrometastases in Lymph Nodes.
Twenty 5-µm sections were examined for the paraffin-embedded portion
of each surgical margin and lymph nodes >1 cm for cases 1–3. The
sections were stained with H&E and examined by light microscopy. Only
cases pronounced tumor free by the histopathologist were forwarded for
the molecular analysis. DNA was prepared from the frozen portion of the
surgical margins and paraffin-embedded lymph nodes using DNA Stat 50
(Biogenesis, Poole, United Kingdom) according to the manufacturer’s
instructions. Exons harboring the p53 mutation detected in
the primary tumor were amplified by PCR; products were cloned into a
bacteriophage vector and amplified further in E. coli
(13)
. Each sample was plated in duplicate to prepare
filters for hybridization with mutant and wild-type-specific
oligonucleotide probes. At least 2000 clones were transferred to nylon
membranes for each margin analyzed. Filters were hybridized with probes
end-labeled with [
32P]ATP. After
hybridization, the membranes were washed to identify plaques after
exposing the membranes to X-ray film. The assay included positive and
negative controls for each set of margins and lymph nodes examined. The
positive control was the amplified p53 gene product derived
from the patient’s primary carcinoma; the negative control cloned
p53 products derived from patients with a different p53
mutation in the primary tumor. All positive assays resulting in 1–10
plaques/plate were repeated from the genomic DNA step. Because the
ploidy status of the tumors examined was unknown, we assumed that each
cancer cell contained one mutant p53 allele and estimated
the percentage of clonal (mutated) tumor cells in each sample by
counting the number of plaques labeled with the mutant-specific
oligonucleotide probe and dividing this number by the total number
counted.
Bone Marrow and Central Venous Blood.
Central venous blood (30 ml) and bone marrow aspirates (5–10 ml) were
collected preoperatively, and buffy coat cells were prepared, mixing
the sample with 0.1 volume of dextran (average
Mr 500,000; 6% w/v) to sediment the
erythrocytes. Bone marrow aspirates were diluted 1:1 with PBS prior to
preparation of leukocyte-rich plasma, and the leukocytes were washed in
10x volume PBS with 2% FCS to reduce the viscosity of the sample
(26)
.
Detection of Disseminated Tumor Cells.
Leukocytes (6 x 107) were enriched for
tumor for each patient using M450 Dynabeads (Dynal, Oslo, Norway)
coated with anti-CD45 (recognizing the common leukocyte antigen; Ref.
26
). After negative immunomagnetic selection, the
remaining cells (approximately 2 x 107)
were sedimented onto slides coated with Cell-Tak tissue adhesive
(Becton Dickinson, Oxford, United Kingdom) for immunocytochemistry with
the pancytokeratin antibody AE1/AE3, diluted 1:50 (Dako, High Wycombe,
United Kingdom), using an alkaline phosphatase/monoclonal mouse
anti-alkaline phosphatase technique (26)
. Briefly, slides
were fixed in acetone for 10 min. After washing in Tris-buffered saline
(TBS), tissues were incubated for 10 min in fetal bovine serum (Life
Technologies, Inc., Paisley, Scotland) diluted 1:5 in TBS to block
nonspecific binding before incubation with 100 µl of AE1/AE3 in a
humid chamber. After washing in TBS, slides were incubated for 30 min
with 100 ml of rabbit antimouse immunoglobulin antibody (Dako; 1:25)
diluted in 20% human serum and washed again, and 100 µl of alkaline
phosphatase/monoclonal mouse anti-alkaline phosphatase (Dako; 1:30)
were added for 30 min. After additional washing, the reaction product
was developed with New Fuchsin substrate solution (Dako) containing
levamisole (1 mM) to block endogenous alkaline phosphatase
activity for 10 min. Slides were washed with water and counterstained
with hematoxylin. Isotype controls were stained with mouse anti-FITC
(Sigma, Poole, United Kingdom), of the same subclass (IgG1), using the
same concentration of immunoglobulin. Positive controls were SCC cell
lines. Immunoreactive cells were scored as tumor using the criteria
defined previously (26)
if they lacked recognizable
hematopoietic characteristics and were typically large, often with an
irregular shape and a high nuclear:cytoplasmic ratio when compared with
the surrounding leukocytes. Cases found to have a single immunoreactive
cell, which could not be categorically identified as tumor, were scored
as tumor negative.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Thirteen of 18 (72%) tumors examined
were found to harbor a p53 gene mutation. Four to six
surgical margins from 12 patients, assessed as tumor negative after
conventional light microscopic examination, were probed with
mutant-specific and wild-type oligonucleotides (for an example, see
Fig. 1
). The amplified p53
region from two or more surgical margins hybridized to the
tumor-specific probe for six cases (nos. 1, 2, 3, 4, 5, and 16),
demonstrating residual tumor cells harboring p53 mutations at the
operative site. The estimated percentage of occult tumor cells in the
surgical margins ranged from 0.05 to 10%. All patients received
standard adjuvant treatment, including postoperative radiotherapy.
During the period of study, six patients (nos. 1, 2, 3, 4, 5, 6, and
16) with tumor-positive surgical margins by molecular analysis had
biopsy-proven recurrences of carcinoma at the site of the primary tumor
or in the neck. In addition, one case (12)
, which could
not be assessed by the phage plaque assay because of the nature of the
p53 mutation (insertion codon 152 CCG-CCCG), and a case with
wild-type p53 (14)
developed a recurrence at the local
site.
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. For each
sample giving a value >10%, at least six plasmids were rescued from
red colonies and sequenced. Nonclonal mutations were detected for 17
cases, but this is the expected result when the mutant content
approaches the background. One case (no. 6, primary tumor in the buccal
sulcus and recurrence in the fauces) was found to have 16–43% red
colonies when multiple biopsies of tumor-distant mucosa taken from
different sites were examined (Fig. 2)
.
Two of the three biopsies examined harbored clonal mutations at codon
205 (TAT-GAT); nonclonal mutations were found for the other sample
tested. The mutation detected in the primary tumor was at codon 175
(CGC-ACG), whereas the local recurrence in the fauces harbored codon
205 (TAT-GAT) and lower levels of 175 (CGC-ACG). Local recurrence also
developed in one case (14)
for which the margins could not
be analyzed using the molecular approach, because the primary tumor
harbored wild-type p53.
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Evidence of systemic disease, manifested by the finding of tumor cells
in the bone marrow preoperatively, was found for 6 of 18 cases (nos. 3,
4, 5, 7, 16, and 18; for examples, see Fig. 3
). Two of these cases developed multiple
lung lesions (cases 3 and 5) and a third case developed a solitary lung
nodule (case 4), which were ultimately the causes of death for these
patients. The presence of SCC was confirmed by bronchoscopy for cases 3
and 4. All cases with evidence of widespread tumor dissemination had at
least one tumor-positive lymph node after conventional light
microscopic examination, and two of these nodal metastases showed
extracapsular tumor spread. There was no relationship between the
number of nodal metastases and the frequency of disseminated tumor
cells. Tumor cells were also detected in the central venous blood
preoperatively for two cases (3
, 7)
, both with
extracapsular nodal deposits of tumor detected by conventional light
microscopy.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The present study used the p53 phage plaque assay in a prospective study to detect residual cancer at surgical margins considered to be tumor free after conventional light microscopy. We found a p53 mutation rate that is considerably higher (13 of 18; 72%) than the findings reported for a previous series [40–60%; reviewed by Partridge and Warnakulasuriya (29) ]. The high mutation detection rate could reflect the small number of cases examined or a true difference in sensitivity; because the FASAY tests a larger region of the p53 cDNA than is normally examined (amino acids 53–364), the technique can detect mutations in the presence of large amounts of normal tissue, and the simple red/white read-out means that mutations are not overlooked easily.
Our findings confirm the pilot studies conducted by Brennan et al. (13) , showing that molecular detection of malignant cells in apparently tumor-free surgical margins can identify patients likely to fail treatment at the local site. Application of the modified FASAY identified one patient with clinically and histologically normal mucosa harboring a p53 mutation, confirming that genetic aberrations can precede histological changes. The analysis also provides information about the likely clonal relationship of the index and second tumor, because a different p53 mutation (codon 205, TAT-GAT) was detected in several biopsies of tumor-distant mucosa and was present, together with very low levels of the mutation detected in the index tumor (codon 175, CGC-ACG), in the second tumor. These data strongly suggest that the recurrence arose as a result of a polyclonal process of field cancerization in the oral cavity and oropharynx.3 However, screening tumor-distant mucosa for p53 gene mutations only revealed molecular evidence of field cancerization for a single case. A further disadvantage of the FASAY is that this approach cannot differentiate between nonclonal PCR-induced mutations and true mutations in a very small number of cells. Thus, in view of the large number of yeast plasmids that must be rescued, purified, and sequenced to detect clonal aberrations, our data suggest that it is not be feasible to use this assay to screen patients for molecular evidence of field cancerization in future studies.
In the present study, radiotherapy was given according to standard protocols, and the additional information obtained from the molecular assay was not taken into account when the decision regarding the need for adjuvant therapy was made. Four cases who received postoperative radiotherapy developed local recurrence, despite aggressive adjuvant therapy. This lack of response may reflect an aggressive tumor phenotype or a failure of p53-dependent apoptosis, because the product of the p53 gene modulates a significant proportion of the cytotoxic effects of radiotherapy (30) . At this stage, analysis of further cases is required to clarify whether p53 status influences the response to radiotherapy. Nevertheless, the possibility that this group of patients may show a poor response to this treatment modality emphasizes the need for adequate surgery (31) or effective repeat surgery when positive surgical margins are found (11 , 32, 33, 34, 35) .
It is well recognized that conventional light microscopy can miss the early stages of metastatic disease in lymph nodes (13 , 36) , and the finding of occult micrometastases in the present study confirms the value of the molecular approach. Clinically, this information is very important because two patients would have been designated as having a more advanced stage of carcinoma if the results of molecular analysis had been taken into account. The current TNM tumor classification system (37) is based on size rather than the number of tumor-positive lymph nodes. Patients found to have more than two tumor-positive nodes are treated routinely with postoperative radiotherapy. Thus, if this information had been taken into account when planning adjuvant treatment for cases 1 and 4, found to have additional tumor-positive nodes with the molecular assay, postoperative radiotherapy to both the primary site and the neck might have prevented development of recurrence for these cases.
The present strategies for supraomohyoid (levels I, II, and III) neck dissection and postoperative radiotherapy are largely based on the patterns of cervical lymph node metastases described by Shah et al. (38) . However, recent evidence suggests that this pivotal study may underestimate the true frequency of micrometastases, because molecular analysis identified more tumor-positive lymph nodes and can stage patients more precisely. Today, the majority of patients have at least a functional neck dissection carried out as a staging procedure or to provide access to vessels for microanastomosis. Thus, the prognostic significance of these nodal micrometastases will never be known because they are removed routinely. In view of this, although the present study serves to highlight the fact that nodal micrometastases may be present, we did not compare the frequency of nodal micrometastases detected by light microscopy and molecular methods for many patients, because this would be labor intensive and of questionable value in the absence of prognostic data. Nevertheless, we need to bear in mind that the change in treatment protocol from radical to selective forms of neck dissection may leave micrometastases in the body that may not necessarily give rise to recurrences in the neck but which may provide a niche for the evolution of tumor cells with a phenotype conducive to development of distant metastases, which are ultimately responsible for the patient’s demise.
The finding of disseminated tumor cells in the bone marrow and central venous blood for cases 3, 4, and 5 developing lung lesions shows that a proportion of the disseminated tumor cells are true micrometastases, capable of forming a focus of tumor at a new site. Whether these disseminated tumor cells permeate the hematopoietic cell compartment indirectly via the lymphatics or as a consequence of direct perivascular extension is unclear at present. In this preliminary series, all patients found to have disseminated tumor cells in the bone marrow had lymph node metastases, but there are also reports of tumor dissemination in tumor-free necks (24 , 25) . We also observed a higher frequency of tumor cells in the hematopoietic cell compartment when extracapsular spread, a recognized risk factor for development of distant metastases, was present. There are reports in the literature suggesting that the presence of three or more tumor-positive lymph nodes correlates with development of distant metastases (5) , suggesting that tumor cells with a migratory phenotype are more likely to be disseminated throughout the body and detected in both the hematopoietic and lymphatic systems. The presence of disseminated tumor cells may be a more reliable marker for risk of development of distant metastases than lymph node status alone, and this relationship will be investigated in a definitive study. However, it will also be important to establish whether the number of disseminated cells falls postoperatively. The study of other cancer types suggests that some tumor cells that remain after surgery are recognized and destroyed by the immune system within 3 months, whereas in other cases, disseminated cells persist after this time and represent a true minimal residual disease situation (39) .
These preliminary findings support the need for substantive prospective studies to compare the sensitivity of light microscopy and new molecular approaches to detect residual disease and establish the prognostic significance of detection of minimal residual cancer. If these confirm that the presence of disseminated tumor cells and molecular analysis of surgical margins provides additional prognostic information for all or subgroups of patients, the results obtained with this new generation of diagnostics can be combined with conventional prognostic factors to provide a basis for identifying patients likely to benefit from early systemic adjuvant treatment.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 To whom requests for reprints should be
addressed, at Maxillofacial Unit/Oncology, King’s College Hospital,
Denmark Hill, London SE5 8RX, United Kingdom. Fax: 020-7346-3754. 
2 The abbreviations used are: FASAY, functional
analysis of separated alleles in yeast; SCC, squamous cell carcinoma;
TNM, Tumor-Node-Metastasis.
3 M. Partridge, S. Pateromichelakis, E. Phillips,
G. G. Emilion, and J. D. Langdon. Analysis of the clonal origin of
multiple premalignant and malignant oral lesions-implications for
prognosis and therapy, submitted for publication.
Received 1/15/00; revised 3/23/00; accepted 3/23/00.
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