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Urinary screen- ing tests for fetal Down syndrome: II. Hypergly- cosylated hCG. Prenat Diagn 1999;19:351–9. 2. Kovalevskaya G, Birken S, Kakuma T, Ozaki N,.
Letters to the Editor lier commercial assay, which also used B152 as the capture antibody, stated that diluting serum samples produced the expected results and that results for serum and plasma were identical (5 ). This assay has been used in several clinical studies. It remains to be seen whether earlier promising results on the clinical use of hCG-h can be verified and possibly improved by eliminating the variable effect of complement in serum.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article. Authors’ Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the Disclosures of Potential Conflict of Interest form. Potential conflicts of interest: Employment or Leadership: None declared. Consultant or Advisory Role: None declared. Stock Ownership: None declared. Honoraria: None declared. Research Funding: None declared. Expert Testimony: U.-H. Stenman, testimony in Princess Diana hearing.

References 1. Cole LA, Shahabi S, Oz UA, Rinne KM, Omrani A, Bahado-Singh RO, Mahoney MJ. Urinary screening tests for fetal Down syndrome: II. Hyperglycosylated hCG. Prenat Diagn 1999;19:351–9. 2. Kovalevskaya G, Birken S, Kakuma T, Ozaki N, Sauer M, Lindheim S, et al. Differential expression of human chorionic gonadotropin (hCG) glycosylation isoforms in failing and continuing pregnancies: preliminary characterization of the hyperglycosylated hCG epitope. J Endocrinol 2002;172:497–506. 3. Birken S, Yershova O, Myers RV, Bernard MP, Moyle W. Analysis of human choriogonadotropin core 2 O-glycan isoforms. Mol Cell Endocrinol 2003;204:21–30. 4. Cole LA, Butler S. Detection of hCG in trophoblastic disease. The USA hCG reference service experience. J Reprod Med 2002;47:433– 44. 5. Pandian R, Lu J, Ossolinska-Plewnia J. Fully automated chemiluminometric assay for hyperglycosylated human chorionic gonadotropin (invasive trophoblast antigen). Clin Chem 2003;49:808 –10. 6. Bormer OP. Interference of complement with the binding of carcinoembryonic antigen to solid-

phase monoclonal antibodies. J Immunol Methods 1989;121:85–93. 7. Valmu L, Alfthan H, Hotakainen K, Birken S, Stenman UH. Site-specific glycan analysis of human chorionic gonadotropin beta-subunit from malignancies and pregnancy by liquid chromatography– electrospray mass spectrometry. Glycobiology 2006;16:1207–18. 8. Alfthan H, Haglund C, Dabek J, Stenman U-H. Concentrations of human chorionic gonadotropin, its ␤-subunit and the core fragment of the ␤-subunit in serum and urine of men and nonpregnant women. Clin Chem 1992;38:1981–7.

Ulf-Håkan Stenman2* Steven Birken3 Anna Lempia¨inen2 Kristina Hotakainen2 Henrik Alfthan4 2

Department of Clinical Chemistry Helsinki University and HUSLAB Helsinki, Finland 3 Department of Obstetrics and Gynecology College of Physicians and Surgeons Columbia University New York, NY 4 Department of Clinical Chemistry, HUSLAB Helsinki University Central Hospital Helsinki, Finland * Address correspondence to this author at: Department of Clinical Chemistry Biomedicum, PB 700 Helsinki University Central Hospital FIN-00029 Helsinki, Finland E-mail [email protected] Previously published online at DOI: 10.1373/clinchem.2010.159939

The Detection of Double Mutations in KRAS Depends on the Mutation-Detection Assay Used To the Editor: Epidermal growth factor receptor is frequently overexpressed in colorectal cancer, and targeted therapies have been developed to block this receptor. Monoclonal antibodies such as cetuximab and panitumumab have demonstrated

effectiveness in increasing the survival rates for some metastatic patients. Patients with mutations in codon 12 or 13 of the KRAS (v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog) gene are not responsive to this therapy, however, and tumors from patients are systematically screened for somatic KRAS mutations before treatment. Several techniques have been developed to screen for KRAS mutations, and the differences in these techniques with respect to diagnostic sensitivity, diagnostic specificity, and efficiency for mutation detection have been described (1– 4 ). The main potential weakness of direct sequencing is its low diagnostic sensitivity (approximately 20%). Other techniques, such as TaqMan® PCR– based assays and SNaPshot® (Applied Biosystems), have diagnostic sensitivities between 1% and 10%. In this study, we analyzed 1970 colorectal carcinoma tumors for KRAS mutations by direct sequencing. We detected 10 cases with double mutations at codons 12 and 13 via direct sequencing and compared the ability of singlenucleotide extension (SNE) and hydrolysis probe real-time PCR to detect these rare mutations. The nucleotide pairs implicated in the nucleotide changes are 34 and 35, 35 and 36, 35 and 37, 35 and 38, 37 and 38, and 38 and 39. Sequencing analysis did not allow us to determine whether the 2 mutations were located on the same allele. With hydrolysis probe realtime PCR, we detected both mutations in only 1 case (c.35G⬎T and c.38G⬎A); 9 of the tumors were classified as wild type for codons 12 and 13. The lack of detection of either one of the double mutations implies that the 2 mutations are located on the same allele. Yet, the detection of mutations c.35G⬎T and c.38G⬎A suggests that these mutations are located on separate alleles, although the c.35G⬎T muClinical Chemistry 57:7 (2011) 1077

Letters to the Editor

Table 1. Detection of double mutations in KRAS exon 2 in a case of colorectal adenocarcinoma tumor, according to the method of analysis. Method of analysis

a

Site

Hydrolysis probe real-time PCR

SNE

Direct sequencing

c.38

WTa

G⬎A (both strands)

G⬎A

c.39

ND

ND

C⬎T

Authors’ Disclosures or Potential Conflicts of Interest: No authors declared any potential conflicts of interest.

WT, wild type; ND, not done.

tation is detected at only a low level. For 5 of the 9 tumors analyzable by SNE, each of the 2 mutations was detected only with either the forward or the reverse primers. This observation supports the hypothesis that the 2 mutations are colocalized on the same allele. For 3 cases, only 1 of the 2 mutations was detected with the forward primers. For these tumors, the second mutation involved nucleotide 36 or 39, neither of which was screened by SNE. The detection of the mutation in only the forward strand confirmed the fact that these 2 mutations are located on the same allele. For 1 case (c.38G⬎A, c.39C⬎T), the c.38G⬎A mutation was detected by both the forward and reverse primers, suggesting that the second mutation, c.39C⬎T, which was not screened by SNE, was localized on a separate allele. This finding indicated a discrepancy between the SNE result and the hydrolysis probe real-time PCR result (Table 1). The detection of double mutations at codons 12 and 13 by direct sequencing corresponded to 1.3% of the KRAS mutations at codons 12 and 13. Although the rate of double mutations is low, it is important to detect such mutations; otherwise, these patients would be considered eligible for treatment with antibodies to epidermal growth factor receptor, which potentially would have no clinical benefit but could have adverse effects. The hydrolysis probe real1078 Clinical Chemistry 57:7 (2011)

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.

time PCR analysis reported codons 12 and 13 of KRAS as wild type for 9 of the 10 cases screened. The SNE technique was more successful because one of the 2 mutations was detected for all cases that could be amplified from the forward and/or reverse sequences. Although these mutations are rare events, they have been reported in the literature (5 ). Metastatic colorectal carcinomas occur at a high frequency, and thus numerous cases are routinely tested for KRAS mutations. Given that patients need quick and efficient treatment, the testing procedure must be performed in the shortest time with the best available technique in terms of rapidity, diagnostic specificity, and diagnostic sensitivity. Different in-house methods (1–3 ) and commercially available kits [Signature® KRAS/ BRAF Mutations Kit (Asuragen), Therascreen KRAS PCR Kit (Qiagen)] have been developed, some of which are based on the hybridization of mutation-specific probes. The detection of 1.3% KRAS double mutations located on the same allele must be kept in mind when choosing the KRAS mutation– detection method for routine clinical setting. It is essential not only that the test used be diagnostically sensitive but also that it be able to detect all possible mutations (even if rare) so that the patient can be offered the most appropriate treatment.

Acknowledgments: We are grateful to Pippa McKelvie-Sebileau for help with the English manuscript.

References 1. Di Fiore F, Blanchard F, Charbonnier F, Le Pessot F, Lamy A, Galais MP, et al. Clinical relevance of KRAS mutation detection in metastatic colorectal cancer treated by cetuximab plus chemotherapy. Br J Cancer 2007;96:1166 –9. 2. Zinsky R, Bolukbas S, Bartsch H, Schirren J, Fisseler-Eckhoff A. Analysis of KRAS mutations of exon 2 codons 12 and 13 by SNaPshot analysis in comparison to common DNA sequencing. Gastroenterol Res Pract 2010;2010: 789363. 3. Lievre A, Bachet JB, Boige V, Cayre A, Le Corre D, Buc E, et al. KRAS mutations as an independent prognostic factor in patients with advanced colorectal cancer treated with cetuximab. J Clin Oncol 2008;26:374 –9. 4. Mariani P, Lae M, Degeorges A, Cacheux W, Lappartient E, Margogne A, et al. Concordant analysis of KRAS status in primary colon carcinoma and matched metastasis. Anticancer Res 2010;30:4229 –35. 5. He Y, Van’t Veer LJ, Mikolajewska-Hanclich I, van Velthuysen ML, Zeestraten EC, Nagtegaal ID, et al. PIK3CA mutations predict local recurrences in rectal cancer patients. Clin Cancer Res 2009;15:6956 – 62.

Isabelle Hostein1* Aude Lamy2 Nicolas Faur1 Charlotte Primois1 Se´verine Verdon1 Jean-Christophe Sabourin2,3 Isabelle Soubeyran1 1

Department of Pathology Institut Bergonie´ Bordeaux, France 2 Laboratory of Molecular Genetics and 3 Department of Pathology Rouen University Hospital Rouen, France

Letters to the Editor

* Address correspondence to this author at: Department of Pathology Institut Bergonie´ 229 cours de l’Argonne 33076 Bordeaux Cedex, France Fax ⫹33-0-5-56-33-04-38 E-mail [email protected]

Previously published online at DOI: 10.1373/clinchem.2010.161190

Multiplex Ligation-Dependent Probe Amplification Analysis Is Useful for Diagnosing Congenital Adrenal Hyperplasia but Requires a Deep Knowledge of CYP21A2 Genetics To the Editor: We read with great interest the recent report in Clinical Chemistry by Cantu¨rk et al. (1 ). These authors affirmed that the CYP21A1P1 (cytochrome P450, family 21, subfamily A, polypeptide 1 pseudogene) genotype interferes with quantitative multiplex ligation-dependent probe amplification (MLPA) analysis of the CYP21A2 (cytochrome P450, family 21, subfamily A, polypeptide 2) gene. They also reported that the p.I172N and p.Q318X mutations were absent in 3.6% and 8.5%, respectively, of the CYP21A1P alleles (200 unrelated individuals examined). Because CYP21A2 MLPAspecific probes contain the wild-type sequences for the Del8bp, p.I172N, Cluster E6, and p.Q318X mutations, Cantu¨rk et al. stated that “without a parallel CYP21A1P [sequencing] analysis, one would falsely infer a

1

Human genes: CYP21A1P, cytochrome P450, family 21, subfamily A, polypeptide 1 pseudogene; CYP21A2, cytochrome P450, family 21, subfamily A, polypeptide 2.

heterozygous duplication of CYP21A2 with a risk of 3.6%” (p.I172N) with an MLPA method, or 8.5% for p.Q318X. From our experience, we believe that such assertions are incorrect. Indeed, it is not possible to establish the CYP21A2 copy number from the signal of only 1 specific gene probe (the exon 4 or exon 8 CYP21A2 probe in this case). When MLPA analysis is performed, it is necessary to consider the value of all specific gene probes (2 ). If CYP21A2 duplication were actually present, all 5 specific probes would show a ratio ⬎1.3. That occurs when all CYP21A2 alleles are wild type for the following mutations: exon 3 8-bp deletion, p.I172N, clusterE6, and p.Q318X. On the contrary, if a duplicated CYP21A2 allele is present and carries the p.Q318X mutation, only the exon 8 probe would show a typical ratio (0.7–1.3) (2 ). Furthermore, if the p.I172N or p.Q318X mutation is absent in CYP21A1P, the CYP21A2specific probes for exon 4 or exon 8 would also bind the wild-type CYP21A1P pseudogene sequence, giving a ratio ⬎1.3. For this reason and given the data of Cantu¨rk et al., we think it more correct to affirm that exon 4 and exon 8 CYP21A2 probes may give a ratio ⬎1.3 in 3.6% and 8.5% of the cases, respectively. Therefore, when that occurs, it is incorrect to immediately assume gene duplication, but it is necessary to consider the value of all specific CYP21A2 probes for a definitive interpretation of the MLPA analysis. In this case, pseudogene sequencing can also be performed to strengthen the diagnosis. Quantitative CYP21A2 diagnosis could provide an incorrect result when the CYP21A1P pseudogene lacks all of the following mutations: Del8bp, p.I172N, ClusterE6, and p.Q318X. In fact, in this case all specific CYP21A2 gene probes might also recognize

the pseudogene sequence showing a ratio ⬎1.3. To the best of our knowledge, that has never been reported. Our group first used MLPA analysis for the diagnosis of congenital adrenal hyperplasia (2 ). Other groups with considerable experience have used this technique successfully (3, 4 ), and it is currently used for routine analysis of congenital adrenal hyperplasia as a valid alternative to the Southern blot. As previously reported (2 ), we confirm that MLPA analysis is a very informative tool for the molecular diagnosis of congenital adrenal hyperplasia. Considering also the findings of Cantu¨rk et al., however, we stress that the use of this methodology requires a deep knowledge of CYP21A2 genetics. The CYP21A2 gene, which encodes a 21hydroxylase, has a complex structure and is considered one of the most polymorphic of human genes.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article. Authors’ Disclosures or Potential Conflicts of Interest: No authors declared any potential conflicts of interest.

References 1. Cantu¨rk C, Baade U, Salazar R, Storm N, Pörtner R, Höppner W. Sequence analysis of CYP21A1P in a German population to aid in the molecular biological diagnosis of congenital adrenal hyperplasia. Clin Chem 2011;57: 511–7. 2. Concolino P, Mello E, Toscano V, Ameglio F, Zuppi C, Capoluongo E. Multiplex ligationdependent probe amplification (MLPA) assay for the detection of CYP21A2 gene deletions/ duplications in congenital adrenal hyperplasia: first technical report. Clin Chim Acta 2009;402: 164 –70. 3. Coeli FB, Soardi FC, Bernardi RD, de Arau´jo M,

Clinical Chemistry 57:7 (2011) 1079