| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Technical Briefs |
1 Department of Pathology, National University of Singapore;2 Molecular Diagnosis Centre, Department of Laboratory Medicine, and3 Department of Hemato-Oncology, National University Hospital, Singapore
aaddress correspondence to this author at: Department of Pathology, National University of Singapore, 5 Lower Kent Ridge Road, Singapore 119074; fax 65-67751757, e-mail patkoaye{at}nus.edu.sg
Microsatellite instability (MSI) (1) is the consequence of a failure in the DNA replication proofreading mechanism. In two-thirds of colorectal cancer (CRC) patients with high-frequency MSI (H-MSI), the cause is an epigenetic hypermethylation of one of the mismatch repair (MMR) genes; in the remaining 30% [the hereditary non-polyposis colorectal cancer (HNPCC) subgroup], the cause is inherited mutations in these genes. Distinction between H-MSI and microsatellite-stable (MSS) CRC may also have prognostic and therapeutic implications (2)(3).
The MSI test should be complemented with morphologic (4) and clinicopathologic evaluation, immunohistochemical (IHC) staining (5) (as manifested by absence of hMLH1 and/or hMSH2 antibody reactivity), and when indicated, screening and confirmation of MMR gene mutations. Direct sequencing, another viable diagnostic approach, is limited by the complex nature of the genes, the broad mutational spectrum, and the cost (6).
In the present study, we reviewed the MSI testing performed in our routine molecular diagnostic laboratory and correlated the findings with mutation analysis and IHC studies; we also propose the most cost-effective manner of diagnosing CRC and report the fidelity of tissue microarrays (TMAs) for high-throughput analysis of large tissue archives.
Approximately 20% (29 tumors from 27 patients) of the 131 MSI cases we analyzed underwent surgery in our hospital; thus, samples for IHC studies and sequencing were available. CRC tissue and noncancerous colonic mucosa were manually microdissected, and DNA was extracted with the DNeasyTM Tissue Kit (Qiagen GmbH). The microsatellite repeat sequences analyzed were Bat-25 (4q12/c-kit), Bat-26 (2p16.3/hMSH2), D2S123 (2p16/hMSH2), D5S346 (5q21/APC), and D17S250 (17q11.2/BRCA1). The method (7) was modified from that of Berg et al. (8) and based on international recommendations (9)(10). Individual PCR tubes were set up for each of the five markers, each tube containing 
50 ng (5 µL) of DNA extract from the healthy or tumor tissue; a PCR control [with ß-globin primers (8)] and a water blank control were included. PCR was performed on an ABI PE9600 thermocycler, and the amplified products were detected by use of the ABI PRISM® 310 Genetic Analyzer and GeneScanTM software [Applied Biosystems Incorporation (ABI)] (7).
TMAs were constructed with double punches 1 mm in diameter from formalin-fixed, paraffin-embedded surgical resection specimens (CRC and healthy colon tissue) of the 25 patients, as described previously (11). For IHC analysis, monoclonal antibodies against h-MLH1 and h-MLH2 (Ab-1 and Ab-2; Oncogene Research Products) at antibody dilutions of 1:10 and 1:50, respectively, were applied to 4-µm-thick TMA sections, according to the manufacturer’s instructions. Cases with unequivocal nuclear staining were considered positive. If no staining was noted in the internal control cells (the normal fibro-inflammatory and vascular component), the result was labeled indeterminate.
For the hMLH1 and hMSH2 sequencing, genomic DNA was extracted from peripheral mononuclear cells by use of the PuregeneTM DNA Isolation Kit (Gentra Systems). Each of the 19 exons of hMLH1 and 16 exons of hMSH2 was amplified by PCR, with 50–200 ng of genomic DNA used as template. PCR products were visualized on 1% agarose gels and purified with the QIAquick PCR Purification Kit (Qiagen). Purified PCR products were sequenced on the ABI PRISM 3100 Genetic Analyzer with the Big Dye Terminator, Ver. 3.0, reagent set (ABI), either with the forward or reverse PCR primer or with an internal primer, when appropriate, and analyzed with Sequencing Analysis, Ver. 3.0, software. See Fig. 1
for representative sample results.
|
The mean age of the patients involved (17 males and 10 females) was 43 years [mean overall age of CRC patients in Singapore is 60 years (12)]. There were 22 (81%) of Chinese, 4 (15%) of Malay, and 1 (4%) of Indian descent, compared with 89%, 7%, and 4% for CRC patients in Singapore in general (12). The tumors were right-sided in almost 50% of our cases, whereas the right-to-left ratio in Singapore is 1:10 (12). Clinically, the patients were grouped as follows: satisfying all Amsterdam criteria (n = 2); three or more family members with CRC or HNPCC-like cancer (n = 5); two family members with CRC or HNPCC-like cancer (n = 11); index patient in the pedigree with multiple primary CRC or HNPCC-like cancer (n = 3); and young CRC diagnosed before age 40 without family history (n = 6). The most common features identified in the series were poor differentiation (n = 12), pushing infiltrative margin (n = 11), Crohn-like reaction (n = 10), and cribiform architecture (n = 8). These features were also prominent among the H-MSI cases. The cases that we found who had more of these features were not necessarily those with H-MSI status.
Selective results are shown in Table 1
(for the full results, see Table 1
in the Data Supplement that accompanies the online version of this Technical Brief at http://www.clinchem.org/content/vol50/issue6/). Eleven tumors (38%) were H-MSI, 2 tumors (7%) showed one unstable microsatellite (the low-frequency MSI group), and 16 tumors (55%) were MSS CRC. The most frequent microsatellite markers exhibiting instability were D2S123 and D17S250 (in eight cases each), followed by D5S346 (in seven cases) and the mononucleotide repeats (in six cases each).
|
IHC analysis of full sections from all H-MSI cases produced full concordance with the TMA IHC results. Full concordance was also achieved with the duplicate punches for every case. Analysis of the IHC staining on TMA sections (done "blinded" by the histopathologist) showed 1 case (case 15) with loss of hMSH2 reactivity and 1 case (case 1) with loss of hMLH1 reactivity (7.4% of all patients and 18% of those with H-MSI status). One case (case 9) was indeterminate. Interestingly, lack of antibody reactivity was not 100% accurate when used as a surrogate test to predict the presence of MMR gene mutations. The sequencing analysis showed hMLH1 gene mutations in two cases: a 887T>G mutation in case 24, which changed a leucine-encoding codon to a termination codon; and a 350C>T (splice-site) mutation in case 1. However, concurrent loss of hMLH1 reactivity was found only in case 1; case 24 had seemingly paradoxical conserved reactivity for the hMLH1 antibody and showed no HNPCC-related histologic features. A single hMSH2 mutation (2446C>T; glutamine > termination) was identified in case 15, which was H-MSI and showed no hMSH2 antibody reactivity. Case 9, with an indeterminate result for hMSH2 IHC staining, showed no hMSH2 mutation.
The clinicopathologic profiles of our patients were in keeping with the known profiles of H-MSI patients. This was also reflected in the high percentage of H-MSI in our series (37.9%), which is more than double that described in the general population (1) and in the ethnic group distribution (12). Our results raise several issues of clinical relevance. For example, they confirm the difficulty in predicting H-MSI status on histologic grounds (4)(13) alone (even one of the cases with hMLH1 mutations was a left-sided carcinoma with no characteristic histologic features). In addition, both cases with a known mutation in our series were in the H-MSI subgroup. Moreover, in our series, the dinucleotide markers D2S123 and D17S250 were the more frequent and relevant ones for instability. Had we not performed tests for these markers routinely, four cases in our series (cases 9, 10, 12, and 22) would not have been classified as H-MSI, whereas not testing for the mononucleotide repeats [postulated as the microsatellite marker with a higher informativity (8)] would not have changed the overall H-MSI status of our cases. The reason is difficult to ascertain in a relatively small sample group, but it indeed has a clear molecular diagnostic implication.
We observed that antibody reactivity may not be a reliable indicator of MMR gene abnormality. Case 24 is a cogent reminder that mutations may be present with at least partial antibody reactivity (14). This is also relevant for TMA-based screening to detect MMR gene mutations. This study shows how a duplicate tissue array of 1-mm-diameter punches offers full correlation with the same analysis in full sections, as we have described for other neoplasms (11).
We propose a diagnostic flowchart for cost-efficient analysis of samples from HNPCC-like patients. Because the chances of having a MSS tumor with hMLH1 or hMSH2 mutations are uncertain (15), MSI testing should be the first-line laboratory investigation. We propose that the MSI-documented cases be followed by IHC analysis, which would allow the targeting of specific genes for follow-up mutation analysis, minimizing costs and eliminating unwarranted testing. Although the cost of first-line direct sequencing of the three most common genes for all cases in our series would have been US $81 000, which would be followed in the negative cases with full MSI/IHC analysis (another US $12 150), a protocol that started with the MSI test as a first-line investigation with subsequent IHC staining, to be followed by sequencing only in those cases that require it [three-gene (hMLH1, hMSH2, and hMSH6) or one-gene sequencing depending on the IHC result], would substantially reduce the cost of diagnostic investigations, to US $43 850, or by 50–60%.
In summary, our study highlights the importance of the microsatellite dinucleotide repeats D2S123 and D17S250 in MSI testing; the necessity of using MSI testing as the first-line investigation, followed by selective IHC analysis to recommend MMR gene sequencing in the appropriate patients; and the general cost-effectiveness of this approach. Furthermore, our study establishes the adequacy of TMAs for high-throughput screening of paraffin-embedded tissue collections of known genes.
Acknowledgments
This work was funded by grants from the Health Services Development Program, Ministry of Health, Singapore (HSDP01N02) and the National Health Group of Singapore (NHG-RP01121). The study was carried out with the ethics approval of our Institutional Review Board (IRB Reference Code 03.104).
References
The following articles in journals at HighWire Press have cited this article:
|
|
 |
|
  M. Salto-Tellez A Case for Integrated Morphomolecular Diagnostic Pathologists Clin. Chem., July 1, 2007; 53(7): 1188 - 1190. [Full Text] [PDF]
|
|
|
|
 |
|
 K. Ito, Q. Liu, M. Salto-Tellez, T. Yano, K. Tada, H. Ida, C. Huang, N. Shah, M. Inoue, A. Rajnakova, et al. RUNX3, A Novel Tumor Suppressor, Is Frequently Inactivated in Gastric Cancer by Protein Mislocalization Cancer Res., September 1, 2005; 65(17): 7743 - 7750. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
