The Japanese Society of Pathology Practical Guidelines on the handling of pathological tissue samples for cancer genomic medicine

Abbreviations CDx companion diagnostics CGM cancer genome medicine CLIA Clinical Laboratory Improvement Amendment Cq quantification cycle Ct threshold cycle FFPE formalin-fixed paraffin-embedded H&E hematoxylin and eosin IHC immunohistochemistry ISH in situ hybridization IVD in vitro diagnostics NBF neutral buffered formalin NGS next-generation sequencing QC quality control INTRODUCTION

In Japan, cancer genomic testing (also termed cancer gene panel testing or genomic profile testing) using solid tumor tissue samples has increased rapidly since the national health insurance system coverage started in 2019.1, 2 In routine pathological practice, most tissue samples obtained by biopsy or surgery are processed as formalin-fixed paraffin-embedded (FFPE) materials. The FFPE process enables long-term storage of tissues and cells at room temperature, facilitating multiple molecular tests for diagnostic, prognostic, and predictive markers in addition to morphological diagnosis. Moreover, the development of various breakthrough technologies, such as multiplex or comprehensive genomic analyses, have accelerated the use of FFPE samples in clinical practice and research settings. As with fresh samples used in research, FFPE samples must be prepared to minimize the degeneration of biomolecules such as nucleic acids and proteins; however, this has long been considered difficult in most hospitals due to the heavy workload of routine practice. Meanwhile, given the introduction of advanced technologies such as next-generation sequencing (NGS) in clinical settings, the quality of FFPE samples processed by pathological laboratories should be kept high for use in cancer genomic testing.

Several variable preanalytic factors affect the FFPE sample quality.3 Therefore, it is essential for the clinicians who collect and submit specimens, pathology technologists who prepare them, and their supervisors to be aware of these factors in their medical institutions (Table 1). As a result, several guidelines established for the diagnostics utilized for predicting drug efficacy (companion diagnostics [CDx]) in cancer patients provide recommendations for the fixation process. Over 2000 FFPE samples from gastrointestinal cancer patients were analyzed in the nationwide cancer genome screening project, SCRUM-Japan/GI-SCREEN, which revealed inter-laboratory differences in sample quality (Figure 1).4 In another clinical research project, TOP-GEAR, FFPE samples from over 200 patients with various types of cancers were analyzed, and the results of genomic testing based on FFPE samples were clinically validated in hospitals that comply with Clinical Laboratory Improvement Amendment (CLIA) requirements.5

Table 1. Main variable factors affecting the preanalytical phase of FFPE sample processing Processes Persons in charge of the process Preanalytical variable factors Pre-fixation process Clinicians (persons who collect specimens) Fixation process

Pathologists

Pathology technologists

Composition of the fixative (i.e., concentration, buffered action, and pH of solution)

Time and temperature of formalin fixation

Ratio of the volume of fixatives to tissue volume during formalin fixation

Treatment methods to penetrate tissues (i.e., immersion, injection, or acceleration by microwaves)

Post-fixation process

Pathologists

Pathology technologists

Conditions for decalcification (i.e., avoidance of acid decalcification)

Tissue processor type and the frequency of replacement of the reagents used in the machine

Conditions for dehydration and clearing (i.e., the types of reagents, temperature, and time used)

Conditions for paraffin immersion (i.e., the type of paraffin, temperature, and time used)

(Post-FFPE process) Pathology technologists Abbreviation: FFPE, formalin-fixed paraffin-embedded. image

Inter-institutional differences in the results of genomic testing using formalin-fixed paraffin-embedded (FFPE) samples prepared in routine practice. In total, 2573 gastrointestinal cancer FFPE samples (biopsy and surgical specimens) prepared during routine practice were submitted by 19 institutions participating in the SCRUM-Japan/GI-SCREEN nationwide, large-scale genome screening research project. Among them, data were analyzed for 16 institutions that submitted ≥40 samples for genomic testing. The FFPE samples were sent to a Thermo Fisher Scientific Clinical Laboratory Improvement Amendments-certified laboratory, where they were subjected to quality control (QC) assay and next-generation sequencing according to the laboratory's standard operating procedures. Manual microdissection was performed when required, and the ΔCt values were determined after DNA and RNA extraction. Targeted sequencing (Oncomine Cancer Research Panel; OCP, 143 genes and Oncomine Solid Tumour DNA/Fusion Transcript Kit; CE-IVD, 26 genes) using the Ion PGM™ System (Thermo Fisher Scientific) was performed on the samples that passed the QC. (a) The samples were classified into three categories based on the ΔCt values (high [HQ], intermediate [IQ], and low quality [LQ]); those for which ΔCt values could not be obtained owing to very LQ were classified as PCR-failures (AmpFail). Overall (indicated as “All”), 48.7%, 35.4%, and 15.9% of the samples from the 19 institutes were classified as high, intermediate, and LQ/PCR-failure samples for DNA integrity, respectively. There were marked differences in quality among the samples from the 16 institutions that submitted ≥40 samples. (b) Samples (HQ, IQ, and LQ) for which ΔCt values were obtained were analyzed using a comprehensive gene panel (143-gene panel) and a small panel (26-gene panel). The success rates of these analyses highly correlated with the result from the QC assay shown in (a). The institutions with high proportions of LQ samples and PCR-failures also exhibited high failure rates for both panels, and marked differences in quality were observed among the samples from these 16 institutions. Overall (“All” in b), the success rate was 68.1% for the comprehensive gene panel and 14.7% for the small panel, while the failure rate for both panels was 17.2%. (c) The sequencing success rates for the three quality categories are shown, with the HQ samples exhibiting success rates of 90.2% for the 143-gene panel and 97.4% for the 26-gene panel

In 2017, the Ministry of Health, Labour, and Welfare (MHLW) initiated a consortium to promote genomic cancer medicine that outlined the framework for the national cancer genomic medicine platform in Japan and presented the blueprint for genomic medicine using NGS (Table 2). Medical institutions and hospitals have been requested to meet the FFPE sample quality standards for NGS testing when performing cancer genome profiling tests approved for Japan's national insurance system.2 Stricter sample quality controls are required for genomic testing in analyzing multiple genes than the current requirements for singleplex gene testing.

Table 2. Use of NGS in the national platform of CGM in Japan Type of genomic testing Institution Type of medical care Multiplex CDx system (approved as IVD) Common hospitals or commercial laboratories Regulatory approval and insurance coverage Gene Panel testing of clinically relevant genes including genes for which the level of therapeutic evidence is not high (approved as IVD) CGM hospitals and medical institutions Regulatory approval and insurance coverage (after advanced medical care is performed, if needed) Whole-genome sequencing, and immuno-oncology testing (non-IVD) Performed at medical institutions that meet certain requirements Combined treatment using advanced medical care that is not covered by insurance Abbreviations: CGM, cancer genome medicine; IVD, in vitro diagnostics; NGS, next-generation sequencing.

Hence, the Japanese Society of Pathology (JSP) released Japanese-language guidelines on the handling of pathological tissue samples for genomic medicine in March 2018, following the release of the tentative edition in September 2017. This article (practical guidelines) is based on the content of the Japanese-language guidelines and intends to cover the FFPE sample handling requirements used in cancer genomic testing systems approved as in vitro diagnostics (IVD) and medical devices by the MHLW and covered by the national health insurance system. Therefore, the guidelines deal with requirements for highly comprehensive genomic analyses such as whole-exome and whole-transcriptome sequencing secondarily. Due to technological innovation, advances in knowledge, and improvements in the genomic medicine system, the scope of these practical guidelines is expected to change continuously.

EFFECTIVE HANDLING OF FFPE TISSUE SAMPLES

The practical guidelines described here aim at enabling the introduction of cancer genomic medicine in the future. Parts 1 and 2 outline the proper handling of FFPE samples for cancer genomic testing in the entire preanalytical phase and the initial process of the analytical phase, which are both performed in the pathology laboratories of medical institutions. Effective FFPE sample processing is necessary for accurate diagnosis; simultaneously, it is essential to follow the recommended molecular testing methods such as immunohistochemistry, particularly for CDx. Therefore, extreme shortening of the fixation process is difficult, as this can lead to insufficient fixation of protein molecules, although some degeneration and modification of nucleic acids and proteins is unavoidable even with adequate fixation. As the FFPE sample preparation requires extensive processing and is significantly more time-consuming than other samples such as blood, even a slight modification of operating procedures can significantly burden pathology laboratories in medical institutions that have limited technical resources and infrastructure. Therefore, it is recommended that the practical guidelines are followed to the greatest extent possible.

The recommendations in the practical guidelines for genomic medicine are based on extensive empirical data, information from the scientific literature, and the previously released JSP guidelines for genome research.6 The recommendation categories are classified into three groups as described in the explanatory notes below. However, recommendations for general clinical practice according to evidence-based medicine are not presented.

(C) Items recommended as the best practice in a routine clinical setting (C indicates “Clinical recommendation”)(R)Items recommended when FFPE samples are used for the comprehensive genomic analysis (including whole-exome and whole-transcriptome sequencing) in the interventional study or as genomic testing not covered by the national health insurance system in Japan (R means “Research recommendation”). (N)Items that should be avoided (N indicates “Not recommended”) Part 1: Recommendations for the preanalytical phase (a)

Pre-fixation process

Handling of tissue immediately after resection or collection 1.1. Surgical specimens should be stored at 4°C until formalin fixation and preferably fixed within 1 h, or not later than 3 h after resection (C).6, 7 1.2. Small endoscopically resected specimens (e.g., digestive tract specimens obtained from endoscopic mucosal resection) should be placed in formalin fixative immediately after sample collection (C). 1.3. Tissue specimens obtained by biopsy should be immediately placed in formalin fixative (C). 1.4. Specimens for preparing cell blocks should be immersed in formalin fixative as soon as possible after the necessary pretreatment (C). 1.5. Keeping surgical specimens at room temperature for 30 min or longer after resection should be avoided as much as possible (N).

Note for 1.1: It has been reported that the time from tissue resection to fixation affects the results, if it exceeds 2 h for the in situ hybridization (ISH) (HER2) and 1 h for the immunohistochemistry (IHC; hormone receptors).8 The ASCO/CAP guidelines for breast cancer recommend tissue fixation within 1 h.7

Note for 1.1: As the penetration rate of formalin fixative is approximately 1 mm/h, it is recommended that appropriate cuts should be made in the specimen before fixation, particularly for surgical specimens, so that they are thin enough to enable complete tissue fixation by the time of gross cutting.9

Note for 1.1–1.3: If the specimens are intended for clinical research, it is necessary to immerse them in fixing solution immediately after resection or collection.6

Note for 1.1 and 1.5: Due to the complex pre-fixation process, surgical specimens tend to be of lower quality and yield less nucleic acid than biopsy specimens (Figure 2).10

Note for 1.4: Among cytological specimens, some fluid specimens obtained from the body cavity are processed as cell blocks. Commonly used preparing method of cell block include cell collection method via centrifugation and cell solidification method via gelation, in each of which there are several different procedures, and none of which is currently standardized. Although the applicability of these processing methods to genomic medicine is unknown, they have been used in many institutions in Japan, and can be used in molecular testing for CDx.11

(b)

Fixation process

Composition of formalin fixative 1.6. For buffering formalin fixative, a neutral buffered solution should preferably be used; avoid acidic or unbuffered solutions (C). 1.7. A 10% solution (3.7% formaldehyde) should preferably be used as a formalin fixative (C). Optimal fixation time 1.8. Following the recommendations in several CDx guidelines (Table 3), tissue specimens (surgically or endoscopically resected specimens and biopsy specimens) should be fixed for 6−48 h (C) (Figures 3 and 4 and6). 1.9. Sample quality deteriorates due to inadequate fixation; therefore, insufficient fixation and over fixation should be avoided (N). Optimal volume of fixative for formalin fixation 1.10.Ten times the sample volume per tissue should be used for formalin fixation (C). 1.11.Formalin fixation can be performed at room temperature (C).

Note for 1.6 and 1.7: In several CDx assays using IHC, a 10% neutral buffered formalin solution is recommended7, 12-19 (Table 3). Detection of protein expression by IHC is affected by the composition and concentration of the formalin fixative.20 Moreover, results of a comparative analysis of DNA data quality using ΔCt and DNA integrity number (DIN) values supported the use of a 10% neutral buffered solution.6

Note for 1.8: The known effects of formalin fixation on the quality of nucleic acids include chemical modifications of nucleic acid bases in addition to nucleic acid fragmentation. Notably, the deamination of cytosine that changes cytosine into uracil produces thymine (a C > T substitution) during the PCR amplification process.21, 22 Extent of deamination significantly increases with increasing fixation time over 72 h; therefore, samples should preferably not be fixed for more than 48 h (C) (Figures 3-6).

Note for 1.8: The fixation of minute tissue specimens obtained by biopsy using techniques such as endobronchial ultrasonography (EBUS) or cytological specimens for cell block preparation can be completed in a shorter duration (e.g., 6−24 h).16

Note for 1.8: It is difficult to prepare an NGS library (particularly when using comprehensive gene panels) from FFPE samples that were fixed for prolonged duration, particularly when using samples that were fixed for 7 days or longer (N) (Figures 3 and 4 and6).

Note for 1.8: If there are remaining specimens that can be used for future genomic testing after performing routine pathological diagnosis, or when a re-biopsy is planned immediately for cancer genomic testing, the use of superior, non-formalin fixatives can be considered for preserving nucleic acids (R).6

(b)

Fixation process

Composition of formalin fixative 1.6. For buffering formalin fixative, a neutral buffered solution should preferably be used; avoid acidic or unbuffered solutions (C). 1.7. A 10% solution (3.7% formaldehyde) should preferably be used as a formalin fixative (C). Optimal fixation time 1.8. Following the recommendations in several CDx guidelines (Table 3), tissue specimens (surgically or endoscopically resected specimens and biopsy specimens) should be fixed for 6−48 h (C) (Figures 3 and 4 and6). 1.9. Sample quality deteriorates due to inadequate fixation; therefore, insufficient fixation and over fixation should be avoided (N). Optimal volume of fixative for formalin fixation 1.10.Ten times the sample volume per tissue should be used for formalin fixation (C). 1.11. Formalin fixation can be performed at room temperature (C).

Note for 1.6 and 1.7: In several CDx assays using IHC, a 10% neutral buffered formalin solution is recommended7, 12-19 (Table 3). Detection of protein expression by IHC is affected by the composition and concentration of the formalin fixative.20 Moreover, results of a comparative analysis of DNA data quality using ΔCt and DNA integrity number (DIN) values supported the use of a 10% neutral buffered solution.6

Note for 1.8: The known effects of formalin fixation on the quality of nucleic acids include chemical modifications of nucleic acid bases in addition to nucleic acid fragmentation. Notably, the deamination of cytosine that changes cytosine into uracil produces thymine (a C > T substitution) during the PCR amplification process.21, 22 Extent of deamination significantly increases with increasing fixation time over 72 h; therefore, samples should preferably not be fixed for more than 48 h (C) (Figures 3-6).

Note for 1.8: The fixation of minute tissue specimens obtained by biopsy using techniques such as endobronchial ultrasonography (EBUS) or cytological specimens for cell block preparation can be completed in a shorter duration (e.g., 6−24 h).16

Note for 1.8: It is difficult to prepare an NGS library (particularly when using comprehensive gene panels) from FFPE samples that were fixed for prolonged duration, particularly when using samples that were fixed for 7 days or longer (N) (Figures 3 and 4 and6).

Note for 1.8: If there are remaining specimens that can be used for future genomic testing after performing routine pathological diagnosis, or when a re-biopsy is planned immediately for cancer genomic testing, the use of superior, non-formalin fixatives can be considered for preserving nucleic acids (R).6

(c)

Post-fixation processes

Decalcification 1.12. When using specimens containing hard tissues, an EDTA decalcification should be performed (C); acid decalcification should be avoided (N).6 Paraffin-embedding 1.13. Conventional tissue processors (closed automated instruments) can be used while referring to general procedures (C). However, the influence of the reagents, processing protocol, and processor maintenance (e.g., the frequency of replacement) remains unknown. There are insufficient data on rapid-type processors (continuous rapid automated instruments). Storage of FFPE blocks 1.14. FFPE blocks can be stored at room temperature (C). However, the blocks should be stored in a cool, dark place (C) and not exposed to high humidity (N) (C). FFPE blocks for genomic testing may be stored under refrigeration (4°C) (R).23 Storage of unstained FFPE slides 1.15. For the storage of unstained FFPE slides, preventive measures such as low-temperature storage or coating with a thin-layer paraffin should be undertaken to avoid deterioration of nucleic acid quality. However, in principle, long-term storage of unstained FFPE slides for genomic testing should be avoided (N), and unstained FFPE slides should preferably be freshly prepared from the FFPE block, if possible (C). image

Yield and quality of DNA obtained from formalin-fixed paraffin-embedded (FFPE) samples prepared in routine practice. The yield and quality of DNA obtained from FFPE tissue samples of 233 patients with solid tumors analyzed in the first term of the TOP-GEAR project were examined. DNA was extracted from five 10-µm sections using the QIAamp DNA FFPE Tissue Kit (Qiagen). (a) The cross-sectional tissue area was measured and multiplied by 50 µm to calculate the tissue volume. The surgical and biopsy specimens were assessed and compared (including samples not because they were from other institutions). The DNA yield per volume varied widely among the samples, with the biopsy specimens exhibiting a higher yield than the surgical specimens. (b) After DNA quality assessment using Q-values (quantity of DNA measured using qPCR/quantity of double-stranded DNA measured using the fluorescence method), the quality was compared between the surgical and biopsy specimens. The Q-values varied widely among the samples, with the biopsy specimens exhibited better quality than the surgical specimens

image

Effects of prolonged formalin fixation on DNA and next-generation sequencing (NGS) library quality. The effects of formalin fixation time (1, 2, 3, 7, and 14 days) were examined at one institution using colorectal cancer specimens. DNA quality was determined by performing a real-time PCR assay following DNA extraction using commercial formalin-fixed paraffin-embedded (FFPE) tissue DNA extraction kits (Qiagen). The libraries for amplicon sequencing (TruSeq Amplicon Cancer Panel; Illumina) were prepared using the MiSeq system (Illumina) and samples that passed the quality control (QC) assay. (a) The sample quality was determined by measuring the ΔCt values using the FFPE QC assay (Illumina) recommended for this gene panel. Samples fixed for 7 or 14 days did not pass the QC assay [A]. The other assay results were similar [B–F] (red line indicates cut-off value). In the GI-SCREEN study described above, the samples were classified into the three quality categories based on the results of assays performed using primer sets for specific housekeeping genes that differed in amplicon size [D]. As the FFPE blocks used in this analysis were prepared 3 years earlier, the ΔCt value of 2-day-fixed samples was classified as “intermediate.” For assays using primer sets for the RNase P gene that yield a different amplicon size, using an indicator utilizing calibration curves [F] instead of the usual ΔCt values [E] is recommended. (b) The libraries were prepared using 150 ng DNA, and library QC checks were performed on the Bioanalyzer 2100 (Agilent). Samples with 7- and 14-day fixation that did not pass the ΔCt value-based QC assay failed to produce library peaks. All samples with 1, 2, or 3-day fixation produced library peaks and yielded sequencing results, although the peaks in the Case 2 sample with a 3-day fixation were minute

image Effects of prolonged formalin fixation on base substitution. Amplicon sequencing using the TruSeq Amplicon Cancer Panel (Illumina) was performed using colorectal cancer specimens from two cases. The samples were fixed for 1, 2, or 3 days. Refer to Figure 3 for the sample preparation details. When the DNA quality was determined using ΔCt values, samples fixed for 7 or 14 days did not meet the quality standard for use with this panel, and the libraries could not be prepared. Therefore, only samples fixed for 1, 2, or 3 days were used in this analysis. An analysis of the sequencing data revealed no large deviations in the total number or read depth among these three samples. (a) An analysis of the numbers of each type of base change in the sequenced regions of this gene panel (35 kb) revealed that the number of base changes, including the C > T, A > G, and A > T substitutions, increased significantly on Day 3 of formalin fixation. (b) In the additional analyses focusing on an allele frequency of ≤0.1%, the number of base changes in (b) was similar to the total number of changes in (a), suggesting that the change was likely to be an artifact generated by fixation Table 3. Recommendations for the fixation process in the CDx-guidance Type of cancer Biomarkers Target molecule Method Formalin fixatives Fixation time Breast cancer HER2 Protein IHC 10% NBF

6–72 h

<6 h should be avoided

HER2 DNA ISH ER/PgR Protein IHC Non-small cell lung cancer EGFR DNA Real-time PCR 10% NBF 6–48 h ALK Protein IHC ALK DNA FISH ROS1 RNA RT real-time PCR 10% NBF

For surgical specimens, 18–36 h

For biopsy specimens, 4–24 h

PD-L1 Protein IHC 10% NBF 6–48 h Gastric cancer HER2 Protein IHC 10% NBF 6–48 h HER2 DNA ISH Colorectal cancer

RAS

(KRAS/NRAS)

DNA PCR-rSSO 10% NBF 6–48 h Malignant melanoma BRAF DNA Real-time PCR – – Note: –, not described. Abbreviations: CDx, companion diagnostics; IHC, immunohistochemistry; ISH, in situ hybridization; 10% NBF, 10% neutral buffered formalin. image Effects of prolonged formalin fixation on total read number. Amplicon sequencing using two types of small gene panels, the GeneRead Actionable Insight Tumor Panel (12 genes) [AIT12] (Qiagen) and the TruSight Tumor 15 Kit (15 genes) [TST15] (Illumina) was performed on four tumor samples (two colon, one lung, and one gastrointestinal stromal tumor) that had undergone 1-, 3-, or 7-day fixation. For the TST15 panel, the DNA quality was determined using a real-time PCR assay (quality control [QC] results are shown in Figure 3a, Panel B). For amplicon sequencing using the AIT12 and TST15 panels, libraries were prepared using 40 and 20 ng dsDNA, respectively. After performing a library QC, the samples were analyzed using the GeneReader (Qiagen) and MiSeq systems (Illumina). All the samples were successfully analyzed with both small gene panels. Therefore, it can be inferred that the small gene panels are useful for the analysis of formalin-fixed paraffin-embedded (FFPE) samples containing low-quality nucleic acids. However, the total number of reads decreased as formalin fixation time increased [A, B]; this effect varied depending on the gene panel used for analysis image

Effects of prolonged formalin fixation on microarray testing. The effects of fixation time (1, 2, 3, 7, and 14 days) on microarray analysis (GeneChip Human Genome U133 + 2.0 arrays; Affymetrix) performed for breast cancer-recurrence risk prediction testing were examined using breast cancer samples from 11 cases. The cDNA was synthesized using 100 ng RNA extracted from each sample. After the yield was determined, the microarray testing was performed, and the fluorescence intensity was measured. (a) Examining the cDNA yield after the reverse transcription reaction and two quality control (QC) parameters for microarray testing (SF and % p values) revealed a significant change after 3-day fixation (*p < 0.05; **p < 0.01) [A–C]. The SF value reflects the mean fluorescence intensity corresponding to the expression level obtained from all probes. It is expressed as a coefficient and used to normalize the mean fluorescence intensity to a specific level. A high value indicates the array is dark due to inappropriate measurement resulting from sample quality or procedural complications. The % p values indicate the proportion of expressed probes compared to all probes. A value below a certain proportion indicates that the measurement is likely to be inappropriate, and that the microarray data are less reliable. (b) Using 1-day fixation as the control, a correlation analysis was performed to examine the effects of formalin fixation time on the overall expression level of the microarray probes. The results indicated that the correlation coefficients decreased with increased fixation time. In particular, the correlation coefficients for gene probes with a low expression level decreased significantly

Note for 1.15: The use of a thin-layer paraffin coating should be carefully considered because it may affect the process of manual microdissection or nucleic acid extraction.

Part 2: Recommendations for the analytical phase (a)

Selection and thin sectioning of FFPE blocks and marking of H&E-stained samples

Selection of FFPE blocks 2.1. In principle, tissue samples for genomic testing are selected by a pathologist from FFPE blocks with sufficient tumor volume, based on observation of the H&E-stained slides, and the pathological diagnosis report.24 The use of blocks containing blood, necrotic tissue, or numerous non-tumor cells, such as inflammatory cells, should be avoided as much as possible (C, R). 2.2. If there are multiple FFPE samples from the same patient that were prepared at different time points, the most recently prepared samples should be selected first (C, R) (Figures 7-11). Thin sectioning of FFPE blocks and preparation of unstained samples 2.3. Thin sectioning of FFPE blocks should be performed with extreme caution to avoid cross-contamination. As a precaution, the microtome blade should be changed for each sample. Additionally, care (such as wearing gloves) should be taken to prevent nucleic acid degradation (C). Reconfirmation with H&E-stained slides and marking 2.4. For genomic testing, H&E-stained and unstained slides are freshly prepared from the FFPE block selected by the pathologist. Next, in principle, the pathologist should mark the test area for nucleic acid extraction on the H&E-stained slides and assess the tissue volume (total number of nucleated cells), the tumor volume (total number of tumor cells), and tumor content (percentage of tumor cells with respect to total nucl

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