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Modification （新鮮組織を使用）（Jun, 2007）–! K080252

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Class II Special Controls Guidance Document:
Cardiac Allograft Gene Expression Profiling Test Systems （October, 2009）
Original （血液を使用）

37（Aug , 2008）

Class II Special Controls Guidance Document:
Gene Expression Profiling Test System for Breast
Cancer Prognosis

Clinical Validation (continued)

38

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21-Gene expression profile assay on core needle biopsies predicts
responses to neoadjuvant endocrine therapy in breast cancer
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Akashi-Tanaka S, et al. Breast. 2009 Jun;18(3):171-4.

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：術前ホルモン療法!
1.! 臨床効果と有意の相関性が認められた!
2.! 乳房温存手術との関連性が認められた。!
3.! ルミナールタイプの乳がん患者における全身療法の適応決定において、有用性が示唆される。!

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56

MHLW Notification by Director, OMDE,Yakushokuki-hatsu No. 0404002, April 4, 2008
Attachment 2
Guidance on evaluation of diagnostic agents for genotyping using DNA microarrays
1. Introduction
With an advancement of pharmacogenomics, DNA microarrays are being developed forinvestigating the nucleotide sequence and the amount of multiple nucleic acid fragments in asample. A DNA microarray is used with a dedicated device for measurement and analysis, andunlike traditional IVD that directly takes physical or biochemical measurements, DNAmicroarrays provide medical information from the analysis of multiple measurement values in
multiple criteria using an algorithm. Therefore, the precision and reliability of measurement are
not the only evaluation items, but also the algorithm used in the analysis as well as the adequacyof the statistical analysis are evaluated. The DNA microarray is treated as a Class III IVD, and the
dedicated measurement and analytical devices are handled as Class I medical devices.
Genotyping diagnostic agents using DNA microarrays will be introduced to the market in thenear future to determine human genotypes from the analysis of genetic polymorphisms andmutations, as well as genotypes of pathogenic microbes. In most cases, the degree of hybridization
to the probe, which is targeted at a specific region of the genome, is measured, and the nucleotide
sequence is determined from this measurement. This method provides more information more
quickly and at lower cost compared to traditional sequencing techniques, but it is important todetermine the nucleotide sequence accurately. In this guidance for evaluation, check points havebeen summarized for confirming that the genotype was accurately determined by the DNA
microarray and its measurement/analytical device. The guidance also aims to help manufacturingfirms to prepare their approval application, and to accelerate the examination process conductedby the Pharmaceuticals and Medical Devices Agency (PMDA). In addition, this guidance will be areference for related products such as diagnostic agents for gene expression analysis using DNA
microarrays. In terms of adequacy in determining responsiveness to drugs based on humangenotype, data should be accumulated and each drug must be independently discussed by experts
at academic conferences, etc.
2. Scope
This guidance for evaluation covers diagnostic agents that utilize DNA microarrays todetermine the genotypes of humans and pathogenic microbes. The guidance aims to evaluate themeasurement system consisting of the DNA microarray and its measurement/analytical device.
3. Role of this guidance!

57

The guidance for evaluation of a genotyping diagnostic agent using DNA microarrays
specifies points that are currently considered important in the expected performance, etc... This
guidance is subjected to revision based on technical innovations and accumulation of findings in
the future, and is not intended to restrict the content of applications. When evaluating products, it
is important to fully understand the properties of each product, and to conduct the evaluation
flexibly with scientific rationality.
4. Points to consider during evaluation(1) Overview
The human or pathogenic genotype to be determined should be explained..
1) Clinical significanceThe clinical significance of determining the human or pathogenic microbial genotype fordiagnosis and treatment should be explained with citations. In this process, polymorphisms within
the Japanese population, or the distribution of pathogenic strains in Japan, should be considered.
2) Principle of measurement
Details of the principle behind genotyping using DNA microarrays and the dedicatedmeasurement/analytical device should be provided. An explanation of the difference in principle
from other traditional genotyping methods in humans and pathogens should be given, and it
should be shown that both methods give identical results. If there are other published articles orpatent literature, those publications can be cited, and any necessary measured data should be
attached. If instruments similar to the measurement/analytical device are used, then documents for
those instruments should be indicated as well. If foreign data is used, then the adequacy of the datain terms of difference in race or distribution of pathogenic strains should be explained.
3) Nucleotide sequence of primers and probes
The nucleotide sequence of the primers and probes used for gene amplification of the sample
should be provided, and the adequacy in selecting those sequences should be explained, including
the existence of pseudogenes or genes with similar sequences. If mismatch probes were used ingenotyping, the basis of selecting those sequences should be explained. Since there are manymutants in pathogens, methods for dealing with genomic diversity should be presented as well.
Details with attached measured data or citations should be provided as needed.
If a high number of probes are being used, PMDA consulting system should be utilized
regarding the submission of documents.
4) DNA microarray construction
The spatial position and method of fixing each probe in the DNA microarray should be
explained in detail.
5) Reference material in the DNA microarray
The selection of positive and negative controls, as well as their adequacy, should be
explained. In terms of negative controls, several DNA sequences similar to that of the detected!

58DNA should be used. The negative control will be the basis for calculating the background signal.
If the measured data is calibrated using background signals and internal standards with positive
controls, the principle and method should be explained in detail using the measured data.
6) Assay conditions
An overview of the reaction conditions of hybridization, washing, and drying (including theassay protocol and standard procedure) should be provided. Any possible nonspecific reactions
should be explained as well.
7) Software
If the final genotype is determined using software installed in the analytical device, based on
signal intensity obtained from fluorescence or current, the adequacy of the algorithm should be
explained. The method of validation concerning the performance of the software should be
provided as well.
(2) Specifications and stability of the DNA microarray1) Quality control method
The identity in the nucleotide sequence between the probe bound to the DNA microarray andthe designed sequence should be explained using measured data.
A standard test should be established to assure the sensitivity, precision and within-run
reproducibility of the detection of the target gene by the DNA microarray. A detailed method ofverifying these criteria from the results of this test should be explained using measured data.
2) Sensitivity, specificity and measurement range
A sample including a certain number of genomic copies should be diluted and measured todetermine the detection limit. If possible, the qualitative and quantitative detection limit should bedetermined as well. It is permissible to set an entire measurement range based on the investigationof typical genotypes. If nucleic acids manipulated through genetic engineering or pathogenic
genomes obtained from cell culturing are used as reference materials, the concentration and purityof the samples derived from clinical specimens should be concerned..
The nonspecific reactions and the stability and uniformity of background signals should be
examined, and the possibility of misjudgment should be explained. Attachment of information on
appropriate primer sequences, reaction conditions, repeated sequences and CG content isrecommended, for the assumed DNA sequencing..
The minimal amount of specimen and the corresponding minimal amount of DNA needed forgenotyping, should be presented. When a quantitaive measurement is done, the range in which
linearity is retained should be presented. The maximum amount of specimen should also be
determined when needed.
3) Calibration of measurement device
If the performance validation can be done regularly by using a calibration DNA microarray
which produces a constant signal in a stable manner for the calibration of the device., the!

59adequacy of the calibration microarray should be examined. If the calibration microarray is notavailable, measurements should be evaluated using positive and negative calibrating samples toconfirm the performance, and this should be noted together with the validation of the DNA
microarray. In addition, the adequacy of the calibrating samples should be examined. Note that the
information discussed so far will be necessary for evaluating the DNA microarray and its
measurement/analytical device as a whole.
4) Documents about stability
The storage conditions and expiration date of the DNA microarray should be set, and theadequacy of that information should be explained. If any reagents are prepared by theexperimenter, the method of preparation and quality control should be presented.
(3) Performance
1) Precision of genotyping
I. Human genotyping!

Tests using human specimens should be conducted, and data for both heterozygous andhomozygous specimens should be presented.

!

If there is a stored specimen with a known genotype, measured data for that specimen
should be presented. If measurements are taken from a human specimen with an unknown
genotype, the measured values should be compared with the results from a bidirectionalsequence analysis using a DNA sequencer.

!

Although it is preferable to measure all genotypes in question using human specimens, itis permissible, in cases of low-frequency mutations, to use specimens mixed with the
mutant DNA or genetically engineered DNA. However, the composition of the mixed
specimen should be as similar as possible to that of a human specimen.

II. Pathogen genotyping!

For instruments that determine the genotype of pathogens, detection data of all the
genotypes in question should be presented. However, in cases of low-frequency
genotypes, a pseudospecimen in which a genetically engineered standard preparation ismixed with a specimen derived from an uninfected individual could be used as a
substitute.

!

The precision of genotyping should be confirmed through comparison of the measured
data with the results from an already-approved IVD or other sequencing method. If there
are multiple genotypes existing for the pathogen in question, it should be shown thatspecific detection and quantification is possible.

!

Cases of complex infections should be examined, including quantitative measurements.
Since mutations occur frequently in pathogenic genomes, the effect of these mutations on
detection sensitivity and genotyping results should be presented, as well as methods for
dealing with the effects.

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!

The existence of mutations should be considered when setting the signal-to-cutoff ratio
and the algorithm.

2) Control material measured with the sample
Positive and negative controls should be set, and reasons for the selection should be
explained. Methods of controlling accuracy by means of these controls should be explained using
the measured data.
3) Reproducibility and robustness
Reproducibility of the signals and the genotyping results should be examined by repeatedmeasurements using reference materials. Measurements should be conducted at multiple facilities
to confirm reproducibility. Information on robustness and external quality control should bepresented when needed.
4) Countermeasures against contamination and data mix-up
If the amplification of nucleic acids, such as by PCR, was part of the sample preparation, thepossibility of misjudgment due to contamination and countermeasures to prevent it should be
explained using measured data when needed. In addition, tests to rule out sample carry-overshould be conducted to ensure the adequacy of the countermeasure against contamination.
Countermeasures to prevent errors in the correspondence of sample information and analysis
results are needed through data management systems using barcodes or other measures.
5) Sample preparation
The quality of the sample can have a great effect on the accuracy of genotyping. In order toobtain high-quality DNA and RNA samples, an appropriate method for collecting, storing andtransporting samples should be set according to the types of samples to be collected and thepathogens in question, and the adequacy of each method should be explained. Countermeasures toprevent degradation should especially be taken for RNA samples.
With consideration of the type of sample (blood, saliva, etc.), methods to extract DNA or
RNA from the sample and to assess the quality of the sample should be presented, along with any
reference values (amount, purity, level of degradation, etc.).
The stability of the prepared sample should be explained. Substances that disturb reactions(such as triglycerides, hemoglobin, bilirubin in serum, lipids, administered drugs, anticoagulants
used for sample collection, etc.) should be evaluated beforehand.
For pathogenic genomes, there is a possibility of contamination by patient DNA and RNA orthat of the normal flora. Therefore, the possibility of such contamination disturbing the reaction
should be explained, and a standard for quality assessment of the specimen and sample should be
provided when needed.
(4) Risk analysis
Factors leading to human or mechanical errors and nonspecific reactions during operation
should be analyzed, and methods for dealing with these factors should be presented when needed,!

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by attaching a caution note, for example. The possible risk of misjudgment in diagnosis andtreatment should be evaluated using literature references. Methods for independently confirming
genotyping results using different techniques should be actively presented.

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