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Clinical Cancer Research Vol. 10, 2379-2385, April 2004
© 2004 American Association for Cancer Research

Molecular Oncology, Markers, Clinical Correlates

Real-Time Gap Ligase Chain Reaction

A Rapid Semiquantitative Assay for Detecting p53 Mutation at Low Levels in Surgical Margins and Lymph Nodes from Resected Lung and Head and Neck Tumors

Susan V. Harden1, David C. Thomas3, Nicole Benoit1, Khalid Minhas1, William H. Westra2, Joseph A. Califano1, Wayne Koch1 and David Sidransky1

1 Department of Otolaryngology-Head and Neck Surgery, and 2 Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, Maryland, and 3 National Cancer Institute-HIV Drug Resistance Program, Frederick, Maryland


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: We have developed a real-time semiquantitative gap ligase chain reaction for detecting p53 point mutations at low level in a background of excess of wild-type DNA.

Experimental Design: This method was validated by direct comparison to a previously validated but cumbersome phage plaque hybridization assay. Forty-one surgical margins and lymph nodes from 10 cases of head and neck squamous cell carcinoma and lung carcinoma were tested for p53 mutant clones.

Results: Both methods detected p53 mutants in margins from 8 of the 10 cases, whereas standard pathology detected cancer cells in only 3 cases. Positive margins included tissue samples with a tumor/normal DNA ratio of up to 1:1000.

Conclusions: This novel molecular approach can be performed in <5 h facilitating intraoperative use for real-time surgical resection.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mutation of the p53 tumor suppressor gene is a common event in many solid malignancies, including head and neck, lung, and bladder cancers (1, 2, 3) . Sequence analysis of primary tumors will generally only detect mutations that are present in >20% of the tested DNA. Thus, detection of mutant genes among an excess of wild-type genes requires a more sensitive assay. This low-level identification of mutated cells is of significant interest for early cancer detection, the assessment of minimal residual disease after surgery, staging of disease based on analysis of lymph nodes and/or serum, and as a technique for monitoring patients for cancer recurrence (4, 5, 6, 7, 8, 9) .

Over a decade ago, a technique for low-level detection of mutated p53 was shown to have clinical validity: the phage plaque oligonucleotide hybridization assay (3) . PCR is used to amplify p53 exons from a DNA sample and the PCR products are then cloned into a bacteriophage vector and additionally amplified in Escherichia coli. Resulting phage plaques are then transferred to nylon membranes, hybridized to mutation specific radiolabeled oligonucleotide probes, and finally exposed to X-ray film. This assay has the capacity to detect a single mutant cancer cell among 1,000–10,000 normal cells, and positive plaques can be picked and sequence confirmed. Using this assay for analysis of surgical margins and lymph nodes from patients with head and neck squamous cell carcinoma, we previously showed that those with molecularly positive margins displayed a substantially increased risk of local recurrence (6) . Other groups have also confirmed these findings using a similar approach (10 , 11) .

However, a major limitation of this assay is the time and technical challenges involved to optimize the assay for each individual case and the cumbersome 3-day turnaround time. Prior reports have described numerous alternative methods for detecting point mutations at low-level, including variations on allele-specific PCR as well as ligation based assays, denaturing gradient gel electrophoresis and restriction enzyme-selected amplification (12, 13, 14, 15, 16, 17, 18, 19) . Most of these assays can detect mutations with high sensitivity (to 1:1000), but specificity, quantitation, and robustness when applied to a variety of different mutations have hindered their more routine use.

We therefore developed a novel assay with similar sensitivity and high specificity, in a compressed time scale, in anticipation of intraoperative assessment of surgical resections. This assay is based on a modified ligase chain reaction (20 , 21) to substantially increase sensitivity (22) and uses fluorescent labeled probes to allow quantitative real-time analysis. In this study, we compared quantitative gap ligase chain reaction (QGLCR) to the standard plaque hybridization assay for the detection of rare neoplastic cells in surgical margins and lymph nodes of patients with lung and head and neck cancer.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients and Sample Collection.
Primary carcinomas of the head and neck and lung were surgically resected at The Johns Hopkins Hospital. After appropriate approval of the Institutional Review Board and patient consent, tumor, surgical margins, and resected lymph nodes were collected during surgical resection at the Johns Hopkins Medical Institutions. Those tissues not used for diagnostic histological analysis were fresh frozen for molecular analysis. Primary tumors with <70% tumor cells were microdissected to remove areas of normal tissue. The p53 gene was analyzed in primary tumors by direct sequencing as described previously (3 , 7) . After resection of the primary tumor, margins were examined by frozen section to confirm adequacy of resection, and then additional normal-appearing tissue was removed from the edges of the surgical defect and fresh frozen for molecular analysis. For this validation study, 10 cases were selected to encompass a range of common p53 mutations in the primary tumor, identified in previous studies (Table 1)Citation . All tissue samples were cut into 12-µm sections and placed in a mixture of 1% SDS and proteinase K (0.5 mg/ml) at 48°C overnight. In addition, 5-µm sections were taken every 15 slices and stained with H&E for examination under light microscopy by a pathologist in a blinded manner. DNA was then extracted from all samples with phenol/chloroform and precipitated with ethanol. A total of 41 lymph node or tumor margin samples was prepared and tested.


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  		Table 1 Clinical details and assay results for all 10 cases

 
Plaque Hybridization Assay.
The exon containing the known p53 mutation for each DNA sample was amplified by PCR using primers containing EcoRI digestion sites (5/6F 5'-GTAGGAATTCACTTGTGCCCTGACTTTC-3', 5/6R 5'-CATCGAATTCTTAACCCCTCCTCCCAGAG-3'; 7/9F 5'-GTAGGAATTCTTGGGCCTGTGTTATCTCC-3', 7/9R 5'-CATCGAATTCAGAAAACGGCATTTTGAGTG-3'). Reactions were carried out in a volume of 50 µl using 500 ng of template, 2 µM of each primer (Invitrogen, Carlsbad, CA), 5 units of TaqDNA polymerase (Invitrogen), 1.5 mM each dATP, dCTP, dGTP, dTTP, 16.6 mM ammonium sulfate, 67 mM Trizma, 6.7 mM magnesium chloride, 10 mM mercaptoethanol, and 0.1% DMSO. The exons were amplified for 35 cycles (95°C for 30 s, 57°C for 1 min and 72°C for 1 min).

The PCR products were then cloned into Lambda ZapII bacteriophage vector and amplified additionally in XL1Blue E. coli cells (Stratagene, La Jolla, CA). The PCR products were initially purified using a Qiagen PCR purification kit (Qiagen, Valencia, MA), digested overnight at 37°C with EcoRI (New England Biolabs, Beverly, MA) and repurified using a Qiagen Gel extraction kit before overnight ligation with the vector at 14°C using T4 ligase (Invitrogen) and packaging into Gigapack III Gold Packaging Extract as per manufacturers instructions (Stratagene). Serial dilutions of phage were plated with 200 µl XL1Blue E. coli cells (A600 nm 0.5) and 3 ml of L-topose onto 100-mm LB agar plates and incubated overnight at 37°C. The resulting clone colonies were transferred to nylon membranes (NEN, Boston, MA) and hybridized with oligonucleotide probes end labeled with {gamma}P32 ATP (3 , 6 , 7) . In each case, the probe was specific for the known mutation in the primary tumor (sequences available on request). After hybridization the membranes were washed stringently at 53°C–60°C to detect mutant-specific binding of the probes. The membranes were then exposed to X-ray film (Kodak) and hybridizing plaques counted. In addition, wild-type end-labeled probes were also hybridized to the membranes as a measure of total plaques containing insert present. The percentage of tumor cells in each specimen was estimated by dividing the number of labeled mutant plaques by the total number of plaques present containing p53 insert (hybridizing to a wild-type probe). For each set of margins or lymph nodes examined, positive (primary tumor) and negative (leukocyte) controls were simultaneously analyzed. All positive assays were repeated.

Real-Time QGLCR.
Tumor, lymph node, margin, and normal DNA samples for each case were amplified by PCR using primers for the specific exon containing the mutation in each case. Reaction conditions were as described in the previous section but using 100–200 ng templates. (Exon 5, F 5'-CACTTGTGCCCTGACTTTCA-3', R 5'-AACCAGCCCTGTCGTCTCT-3'; exon 6, F 5'-AGAGACGACAGGGCTGGTT-3', R 5'-CTTAACCCCTCCTCCCAGAG-3'; exon 7, F 5'-CCTCATCTTGGGCCTGTGT-3', R 5'-CCGGAAATGTGATGAGAGGT-3'; and exon 8, F 5'-TTTCCTTACTGCCTCTTGCTTC-3', R 5'-GCTTCTTGTCCTGCTTGCTT-3'.)

The PCR products were purified using a Qiagen gel extraction kit (Qiagen). The concentration of all purified products was stringently determined using the TD-360 MiniFluorometer (Turner Designs, Sunnyvale, CA). All samples from a single patient were measured concurrently on two separate occasions with multiple readings taken each time. Accuracy of the fluorometer was also validated with standards of known DNA concentration and by comparison to agarose gel analysis with a Low DNA Mass Ladder (Invitrogen) and real-time quantitative PCR (data not shown). Stock solutions of each PCR product were made in 10 mM Tris (pH 8) to give 1010 amplicon copies/5 µl calculated from the size of the amplicon for each exon and assuming 1 bp of double-stranded DNA = 660pg/pmol and 1 pmol = 6.02 x 1011copies.

Ligase detection reactions involve the use of two adjacent oligonucleotide primers, which hybridize to a single strand of target DNA, which will be ligated only if there is an exact match to the target sequence. In the presence of a mismatch, ligation will not occur. Point mutations are best detected by designing oligonucleotides so that the mutation site is at the 3'-end of the upstream 5'-primer. This linear reaction can increase exponentially by the addition of two further complementary oligonucleotides that hybridize to the cDNA strand able to ligate only in the presence of an exact match. Using a thermostable ligase in a cycling reaction, ligated products from both reactions are subsequently used as templates, creating exponential amplification, dubbed a LCR. Although highly sensitive, background ligation can still occur at low level with this basic technique. An additional modification to further improve specificity is the use of a single nucleotide gap at the site of the point mutation between the adjacent oligo primers (22) . This gap is filled using a thermostable DNA polymerase in the presence of the appropriate mutant nucleotide in the reaction mix adding substantial additional specificity to the reaction. On the basis of these principles, we developed a modified gap-LCR reaction that could be monitored in real-time using fluorescent-labeled oligos to allow quantitation, QGLCR. In our assay, one oligonucleotide is labeled with a reporter dye at the 5'-end and the adjacent oligonucleotide to which it will ligate is labeled at the 3'-end with a quencher dye. Excitation of the reporter dye results in fluorescence energy transfer to the quencher dye and subsequent fluorescence at the characteristic spectrum of the quencher dye. The onset of rise in fluorescence by cycle number is proportional to the initial amount of target mutation. A schematic representation of QGLCR is shown in Fig. 1Citation .



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  		Fig. 1. Schematic diagram of the quantitative gap ligase chain reaction (QGLCR). F, FAM; T, TAMRA; p, phosphorylation.

 
The assay was modified and developed over a 2-year period, initially using K-ras- and p53-mutated cell lines, radiolabelled primers, and high percentage polyacrylamide gels before converting to real time using fluorescent labeled primers. The assay was then optimized for each mutation, using varying concentrations of tumor DNA initially diluted in water and subsequently diluted in normal DNA. Different lengths and concentrations of primers were tested at varying temperatures and with varying amounts of the other reagents. Blocking oligonucleotides, extra gaps, differential positioning of the gap, and additional mismatches in the primers were also tested. In general, fluorescent primer lengths of 15 or 16 bp were found to be optimal for producing the best signal, creating a ligated product of 30–32 bp. Some mutations worked best with an additional mismatch nucleotide. All primer sequences are available on request. A two-step cycling reaction was found to work better than an initial three-step reaction, and annealing temperature was found to be critical for maximizing specificity for each particular mutation.

For the QGLCR assay, 109 copies of PCR product template from each sample was used. In addition, serial 10-fold dilutions of tumor mixed into normal template (down to 1:10,000) were constructed for each case and 109 copies from each dilution used to generate standard comparison curves against which to compare the unknown margin and lymph node samples (Fig. 2)Citation . Each reaction also contained in a 25-µl reaction volume: 2.5 µl of Platinum Taq 10x buffer; 0.625 µl of Taq ligase 10x buffer; 1 mM NADß, 100 µM mutant-complementary insertion nucleotide; 1 unit of Platinum Taq polymerase (Invitrogen); 8 units TaqDNA ligase (NEB); 400 nM two nonlabeled reverse strand mutation specific oligomers; 400 nM one FAM 5'-labeled forward strand mutation specific oligomer and 600 nM one TAMRA 3'-labeled forward strand mutation-specific oligomer. All oligos 3' of the ligation site were phosphorylated at their 5'-end. Reactions were run for 40 cycles (94°C for 2 min initiation, then 50°C 30 s, 94°C 30 s) on a 96-well plate using an Applied Biosystems 7700 Sequence detector (Applied Biosystems, Foster City, CA). All samples were run in duplicate and each plate was run twice. All samples from each patient were run concurrently on the same plate. Each plate also included multiple water blanks, containing everything except DNA, as an additional negative control. Threshold values for the margin and lymph node amplification curves were quantitated relative to the thresholds for the corresponding serial dilutions of tumor in normal for each case.



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  		Fig. 2. Standard curve for quantitative gap ligase chain reaction, generated from dilutions of tumor DNA in normal DNA (case 7). X axis, starting quantity of mutant DNA (copy number)/well; y axis, quantitative gap ligase chain reaction cycle number.

 

   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Forty-one lymph nodes and surgical margin samples were analyzed for the presence of carcinoma by histology and two quantitative assays for p53 mutation. The QGLCR assay is diagrammed schematically in Fig. 1Citation and described in detail in "Materials and Methods." Standard curve generation through correlation coefficient is further depicted in Fig. 2Citation . The results for all three methods and clinical outcome are summarized in Table 1Citation . Of the 41 lymph node and surgical margin samples, 6 (15%) were identified by the pathologist on routine histological examination as positive for carcinoma from 3 cases. No histological evidence of malignancy was seen in the remaining 35 samples.

Using the plaque hybridization assay (Fig. 3, A and B)Citation , p53 mutant DNA was detected in the lymph nodes or tumor margins of 8 of 10 (80%) cases, a total of 15 of 41 (37%) samples, including all 6 margins found positive by light microscopy. The estimated percentage of mutant clones to total clones in the lymph nodes and margins ranged from 26% (in a lymph node also histologically positive) to 0.7% (Table 1)Citation .



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  		Fig. 3. Identification of p53 gene mutation in a tumor margin (M2) from case 7: A, many clones hybridized to a wild-type p53 oligomer. B, a substantial number of clones also hybridized to a oligomer probe specific for the mutant p53 identified in the primary tumor.

 
The QGLCR assay (Fig. 4, A and B)Citation detected mutant p53 DNA in the identical 15 of 41 samples. The percentage of mutant p53 detected in each sample compared relative to the equivalent tumor sample ranged from 88.7% (in the above noted histologically positive lymph node) to 0.02% (Table 1)Citation .



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  		Fig. 4. Quantitative gap ligase chain reaction amplification curves for case 1 with negative lymph nodes (A) and case 7 with two positive margins, M2 and M3 (B): T, tumor (orange); N, normal (pink); W, water; M, margin (blue, green, red); L, lymph node (blue, green); 1–4 (yellow) serial 10-fold dilutions of T in N (T/10, T/100, T/1,000, T/10,000), respectively. X-axis, cycle number; Y-axis, change in TAMRA fluorescence from start of reaction on a log scale.

 
To compare the two assays for p53 mutation more directly, the plaque assay results were also converted into a ratio for each sample calculated relative to the corresponding tumor sample result. This correlated more closely with the QGLCR analysis of sample relative to corresponding tumor and ranged from 96.3% down to 2%. In general, the results from both assays compared very well with strong or weakly positive samples being detected at similar levels by both tests. For 4 of 5 cases with multiple molecularly positive margins or lymph nodes, both assays quantitated the positive samples relative to each other in the identical order with 1 of 5 (case 6) showing a discrepancy (all three samples from this case identified as weakly positive by both tests).


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have demonstrated that the molecular detection of tumor-specific p53 mutations can be performed using a rapid easy novel assay, QGLCR, based on a modified fluorescent LCR with the same sensitivity and specificity as a previously reported plaque hybridization assay. Advantages of this novel assay include decreased assay time that allows adaptation to real-time clinical analysis and use of fluorescent rather than radioactive-based detection.

The most important advantage of QGLCR is the speed with which the assay can be carried out. With modifications to our protocol, this technique may be performed in <5 h. In brief, this may be accomplished by performing a rapid DNA extraction (1 h), a rapid PCR and gel purification (1.5 h), fluorometer-based dilutions (30 min), and a rapid QGLCR (1.5 h) to provide assay results within a timeframe suitable for major operative cases that entail extensive or complex neck dissection reconstruction. Additional modifications in DNA extraction and quantitation as well as PCR product purification are likely to decrease the total assay run time.

This assay is currently a two-step process with an initial PCR step to amplify the exon of interest, a step also used in the plaque hybridization assay. However, this may alter the ratio of mutant to wild-type DNA within each sample to some extent due to nonlinearities between amplicon number over the number of PCR cycles used, and so, current efforts are aimed at removing this step by substituting genomic DNA for QGLCR, which would require larger amounts of initial DNA but have the advantage of making it a one-step sealed process. In a manner analogous to that of the plaque hybridization assay, variability in the sensitivity of the assay is noted in a mutation specific manner. For some mutations (Fig. 4)Citation , late amplification curves are seen for normal DNA at levels 1,000–100,000-fold less than for the same input amount of tumor DNA but greater than the absent or very late curves for water and reagents alone. We suspect that this is because for certain point mutations; there is some late targeting of wild-type DNA by the mutant primers at this very low level. This is characteristic of the difficulties involved when mutant sequence only differs by 1 bp from wild-type. Despite these limitations, however, dilutions of 1:1000 tumor:normal DNA are routinely detected and differentiated from background wild-type DNA late signals, with some specific mutations demonstrating a level of detection of 1:10,000 or greater.

The QGLCR also differs from the plaque assay in that samples are assessed relative to tumor DNA, which is not 100% pure, despite conscientious microdissection. In contrast, plaque assay calculates the percentage of mutant plaques relative to wild-type plaques for the sample and the tumor in each case. Therefore, to allow a more direct comparison of values, calculating the ratio of these percentages (sample/tumor) obtained from the plaque assay gives a measure of sample relative to the corresponding tumor as for the QGLCR assay. Although it is impossible to show an exact match between two completely different assays (each affected by different variables—differential probe binding and so on), the results are remarkably similar, and there is general agreement within each case as to the samples that harbor the greatest amount of mutated cells.

Both techniques are capable of detecting p53 mutations that occur commonly in head and neck squamous cell carcinoma and other tumor types but not in all tumors. Clearly molecular analyses using alternative tumor markers are required for these p53 wild-type (23 , 24) cases and may include rapid, sensitive assays for analysis of other tumor markers. One promising approach, include the quantitative promoter methylation assay, has the potential to be carried out intraoperatively. The results of a major prospective 470 patient trial in head and neck cancer patients using the plaque hybridization assay to assess the impact on tumor recurrence and overall survival are due soon. If molecular analysis in this trial precisely identifies the site of recurrence (for margins), real-time intraoperative analysis represents a promising approach to actually guide surgical therapy.


   FOOTNOTES
 
Grant support: Genetic Alterations as Diagnostic and Prognostic Biomarkers for Oral Cancer Grant 5RO1 DE13152, Molecular Progression Model of HNSCC Grant RO1 DEO12588-04, and OncoMethylome Sciences, SA.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: Under a licensing agreement between OncoMethylome Sciences, SA and the Johns Hopkins University, D. Sidransky is entitled to a share of royalty received by the university on sales of products described in this article. D. Sidransky owns OncoMethylome Sciences, SA stock, which is subject to certain restrictions under University policy. D. Sidransky is a paid consultant to OncoMethylome Sciences, SA and is a paid member of the company’s Scientific Advisory Board. The term of this arrangement is being managed by the Johns Hopkins University in accordance with its conflict of interest policies.

Requests for reprints: David Sidransky, Director, Head and Neck Cancer Research, The Johns Hopkins University SOM, 818 Ross Research Building, 720 Rutland Avenue, Baltimore, MD 21205-2196. Phone: (410) 502-5153; Fax: (410) 614-1411; E-mail: dsidrans{at}jhmi.edu

Received 10/14/03; revised 12/29/03; accepted 1/ 8/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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