Draft Genetic Test Review

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Breast Cancer
Analytic Validity

(95KB)

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ANALYTIC VALIDITY

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ANALYTIC VALIDITY

Question 9. How often is the test positive when a mutation is present (sensitivity)?
Question 10. How often is the test negative when a mutation is not present (specificity)?

Complicating factors in interpreting survey results These were limited challenges, and in each of the two years, two of the three samples did not contain a mutation. An additional aim of these external challenges was to assess the type of reporting and counseling information that the participating laboratories might include in their report. The results for interpretation are given in Table 2-5. In 2001, for each of the two challenges without a BRCA1/2 mutation, 10 of 17 laboratories (59%) responded that the risk of breast cancer could not be determined without testing an affected relative, while 7 (41%) stated that the risk of breast cancer is the same as that for the general population. The first interpretation is correct. Responses improved in 2002, with one exception (one laboratory correctly found no mutation in the sample, but indicated a lifetime risk of breast cancer of 50 to 85% - Table 2-5). In the challenge containing a BRCA2 mutation, 16 of 17 laboratories (94%) estimated the lifetime risk of breast cancer as 50 to 85 percent. The one remaining laboratory estimated the risk of breast cancer to be 80 to 95 percent. These post-analytic issues are as important as technical proficiency.

Gap in Knowledge: ACMG/CAP: Analytic performance estimates are preliminary. While these data are not complete or robust, there is no evidence of a problem in detecting a specific BRCA2 mutation with any of the existing laboratory methodologies. Expansion of these challenges to include different types of mutations and comparisons amongst methodologies will assist in validating the analytic performance of the U.S. laboratories providing clinical testing for a subset of BRCA1/2 mutations.

Gap in Knowledge: ACMG/CAP: Analytic performance estimates are available for only a small number of mutations. Only a small number of mutations (3) is included in external proficiency testing exercises (185delAG, 5382insC, 6174delT). Only one of these three mutations was challenged in the first two years. Other mutations, such as those identified in index cases, have not yet been subject to external proficiency testing.

Table 2-5. Responses for the post-analytic aspects of BRCA1/2 mutation challenges from laboratories participating in the ACMG/CAP Molecular Genetics Laboratory Surveys

Response	Participants N (%)
	2001	2002
Case 1 (no mutation)
Lifetime risk of breast cancer is reduced but cannot be determined without BRCA mutation testing of an affected relative	10 (59)	10 (91)
Lifetime risk of breast cancer is the same as that in the general population	7 (41)	1 (9)
Case 2 (no mutation)
Lifetime risk of breast cancer is reduced but cannot be determined without BRCA mutation testing of an affected relative	10 (59)	9 (82)
Lifetime risk of breast cancer is the same as that in the general population	7 (41)	1 (9)
Lifetime risk of breast cancer is approximately 50-85%	0	1 (9)
Case 3 (6174delT)
Lifetime risk of breast cancer is approximately 50-85%	16 (94)	11 (100)
Lifetime risk of breast cancer is approximately 80-95%	1 (6)	0

 

Appendix A. Data Used to Estimate Analytic Sensitivity and Specificity from external proficiency testing

European Molecular Genetics Quality Network Table 2-6 summarizes the familial cancer testing (BRCA1/2 mutations) external proficiency testing results obtained by European Molecular Genetics Quality Network (EMQN). Samples with known genotypes were distributed to participants from 1999 through 2002. The first column of the table contains the case number for the year. The second column contains number of participating laboratories, followed by the genotype of the sample. The number of laboratories reporting specific genotypes is then provided, along with a tabulation of their ‘correct' and ‘incorrect' responses. The table also contains the data used to compute the analytic sensitivity and specificity in a box, along with the yearly (and summary) totals.

Table 2-6. Computations for the EMQN Proficiency Testing Surveys

			Reported Alleles
Distribution	Labs	Genotype	Correct	Incorrect
1999
Case 1	13	C140T
	12	C140T	24	0
	1	Wild type	1	1 (fn)
Case 2	14	A5176G
	14	A5176G	28	0
	1	*		1 (fp)
Case 3	13	C4446T
	13	C4446T	26	0
Totals		80 alleles	79	2
Sensitivity		24+1+28+26/80

 

 

 

 

 

 

 

 

* One laboratory identified the correct mutation, but also reported a second base exchange that was not seen by the 2 reference labs or any of the other participants.

fn = false negative
fp = false positive

			Reported Alleles
Distribution	Labs	Genotype	Correct	Incorrect
2000
Case 1	24	185delAG
	24	185delAG	48	0
Case 2	24	1259delG
	22	1259delG	44	0
	1	Wild type	1	1 (fn)
	1	Wrong position	1	0 (wm)
Case 3	20	A10462G
	18	A10462G	36	0
	2	Wild type	2	2 (fn)
Totals		136 alleles	132	3
Sensitivity		48+44+1+1+36+2/136

 

 

 

 

 

 

 

 

 

fn = false negative, wm = wrong mutation

			Reported Alleles
Distribution	Labs	Genotype	Correct	Incorrect
2001
Case 1	41	3600del11
	39	3600del11	78	0
	1	Wild type	1	1 (fn)
	1	4600del11	1	1 (wm)
Case 2	38	G4603A
	34	G4603A	68	0
	2	Wild type	2	2 (fn)
	2	G4603T	2	2 (wm)
Case 3	40	G5075A
	38	G5075A	76	0
	2	Wild type	2	2 (fn)
Totals		238 alleles	230	8
Sensitivity		78+1+1+68+2+2+76+2/238

 

 

 

 

 

 

 

 

 

 

fn = false negative, wm = wrong mutation

			Reported Alleles
Distribution	Labs	Genotype	Correct	Incorrect
2002
Case 1	36	5677insA
	34	5677insA	68	0
	2	Wild type	2	2 (fn)
Case 2	36	300T>G
	36	300T>G	72	0
Case 3	36	3875del4
	34	3875del4	68	0
	2	Wild type	2	2 (fn)
Totals		216 alleles	212	4
Sensitivity		68+2+72+68+2/216

 

 

 

 

 

 

 

 

fn = false negative

American College of Medical Genetics/College of American Pathologists Table 2-7 summarizes the familial cancer testing (BRCA1/2 mutations) external proficiency testing results obtained by the American College of Medical Genetics and the College of American Pathologists (ACMG/CAP). Samples with known genotypes were distributed to participants in 2001 and 2002. The first column of the table contains the distribution label (e.g. MGL-07 indicates the 7^th DNA sample distributed as part of the Molecular Genetics Laboratory Survey). The second column contains the number of participating laboratories, followed by the genotype of the sample. The number of laboratories reporting specific genotypes is then provided, along with a tabulation of their ‘correct' and ‘incorrect' responses. The table also contains the data used to compute the analytic sensitivity and specificity in a box, along with the yearly (and summary) totals.

Table 2-7. Computations for the ACMG/CAP Proficiency Testing Surveys

			Reported Alleles
Distribution	Labs	Genotype	Correct	Incorrect
2001
MGL-07	12	normal
	12	normal	24	0
MGL-08	12	normal
	12	normal	24	0
MGL-09	17	6174delT
	17	6174delT	29*	0
Totals		77 alleles	77	0
Sensitivity		29/29
Specificity		(24 + 24)/(24 + 24)

 

 

 

 

 

 

 

* Five laboratories did not test the second allele when the mutation was identified in the first allele.

			Reported Alleles
Distribution	Labs	Genotype	Correct	Incorrect
2002
MGL3-04	11	normal
	11	normal	22	0
MGL3-05	11	normal
	11	normal	22	0
MGL3-06	11	6174delT
	11	6174delT	22	0
Totals		77 alleles	77	0
Sensitivity		22/22
Specificity		(22 + 22)/(22 + 22)

Appendix B. Analytic Methodologies Used for BRCA1/2 Mutation Analysis

Testing Methods by U.S. Laboratories Table 2-8 lists categories of methodologies that are used to detect BRCA1/2 mutations by laboratories participating in proficiency testing programs in the United States (ACMG/CAP MGL Survey), along with the proportions using each method. Because many laboratories utilize “home brew” assays, these categories are not homogeneous.

Table 2-8. Testing Methods Utilized by US Laboratories, According to ACMG/CAP External Surveys

Testing Method	2001 (%)	2002 (%)
Total Number of Laboratories	17	11
Allele Specific Oligonucleotide (ASO)	23.5	25.0
DNA sequencing, automated	11.8	8.3
DNA sequencing, manual	5.9	0
DNA sequencing, automated & Allele-specific PCR/ARMS	5.9	0
Allele-specific PCR/ARMS	5.9	25.0
Restriction endonuclease digestion and electrophoresis for size analysis	5.9	0
Restriction endonuclease digestion and electrophoresis for size analysis & DNA sequencing, automated	5.9	25.0
Restriction endonuclease digestion and electrophoresis for size analysis & Allele-specific PCR/ARMS	5.9	0
Restriction endonuclease digestion and electrophoresis for size analysis & Mutation Scanning Methods (SSCP, dHPLC, etc.)	5.9	8.3
Other & DNA sequencing, automated	5.9	0
Other & Restriction endonuclease digestion and electrophoresis for size analysis	5.9	0
Other & Oligonucleotide ligation assay & Restriction endonuclease digestion and electrophoresis for size analysis & DNA sequencing, automated & Allele specific PCR/ARMS & Mutation scanning	5.9	0
Other	5.9	8.3

Testing Methods in the European Community Laboratories participating in the European Molecular Genetics Quality Network (EMQN) external proficiency testing schemes from 2000 through 2002 used a variety of methods to screen for mutations. Of the 296 samples analyzed during these years, the following methodologies were used: denaturing high performance liquid chromatography(73), denaturing gradient gel electrophoresis (40), protein truncation test (39). For 144 additional samples, either no details about methodology were given, or individual exotic techniques were used.