Analytical Performance of Next-Generation Sequencing and RT-PCR on Formalin-Fixed Paraffin-Embedded Tumor Tissues for PIK3CA Testing in HR+/HER2− Breast Cancer
Abstract
:1. Introduction
2. Materials and Methods
2.1. Study Design
2.2. Patients and Tissue Specimens
2.3. Nucleic Acids Purification
2.4. Next-Generation Sequencing (NGS) Analysis
2.5. RT-PCR Analysis
2.6. Statistical Analyses
3. Results
3.1. Clinicopathological Features
3.2. Concordance between Primary Tumor and Metastasis Samples Using NGS
3.3. Concordance between Primary Tumor and Metastasis Samples Using RT-PCR
3.4. Comparison of NGS and RT-PCR
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Patients (n = 16) | |
---|---|
Age at diagnosis, range (median) | 32–73 (52.5) |
Histology, n (%) | |
NST | 15 (93.7) |
Lobular | 1 (6.3) |
ER, n (%) | |
Positive | 16 (100) |
Negative | 0 |
PgR, n (%) | |
Positive | 8 (50.0) |
Negative | 8 (50.0) |
HER2, n (%) | |
Low | 7 (43.8) |
Zero | 9 (56.2) |
Metastatic site, n (%) | |
Bone (iliac crest) | 2 (12.5) |
Bone (pelvis) | 1 (6.3) |
Lymph node | 2 (12.5) |
Liver | 8 (50.0) |
Pleura | 2 (12.5) |
Thoracic wall | 1 (6.3) |
Therapy with CDK4/6, n (%) | |
Yes | 6 (37.5) |
No | 10 (62.5) |
#Case | Age at Original Diagnosis | Time between Primary Tumor and Metastasis (Months) | Sample Type | ER (%) | PgR (%) | HER2 (%) |
---|---|---|---|---|---|---|
PIK_004 | 26 | 69 | Primary | 95 | 95 | 1+ |
Metastasis | 95 | 0 | 1+ | |||
PIK_007 | 46 | 102 | Primary | 95 | 70 | 1+ |
Metastasis | 90 | 2 | 2+ not amplified | |||
PIK_008 | 45 | 54 | Primary | 95 | 0 | 1+ |
Metastasis | 95 | 0 | 2+ not amplified | |||
PIK_010 | 53 | 126 | Primary | 95 | 0 | 1+ |
Metastasis | 95 | 0 | 2+ not amplified | |||
PIK_012 | 43 | 38 | Primary | 95 | 20 | 2+ not amplified |
Metastasis | 95 | 10 | 1+ | |||
PIK_013 | 44 | 117 | Primary | 95 | 95 | 0 |
Metastasis | 80 | 90 | 0 | |||
PIK_014 | 41 | 116 | Primary | 95 | 95 | 1+ |
Metastasis | 95 | 0 | 0 | |||
PIK_016 | 40 | 127 | Primary | 95 | 95 | 0 |
Metastasis | 60 | 0 | 0 |
Metastases | Primary Tumors | |||||||
---|---|---|---|---|---|---|---|---|
#Case | [DNA] (ng/µL) | Tumor Cell Content (%) | Mutation | VAF (%) | [DNA] (ng/µL) | Tumor Cell Content (%) | Mutation | VAF (%) |
PIK_001 | 0.26 | 20 | p.E545K/c.1633G>A | 3 | - | - | - | - |
PIK_002 | 0.84 | 60 | p.H1047R/c.3140A>G | 29 | - | - | - | - |
PIK_003 | 0.37 | 60 | p.H1047R/c.3140A>G | 67 | - | - | - | - |
PIK_004 | 0.24 | 70 | p.H1047R/c.3140A>G | 38 | 2.99 | 60 | p.H1047R/c.3140A>G | 42 |
PIK_005 | 0.44 | 80 | p.N1068Kfs*5/c.3203_3204insA | 20 | - | - | - | - |
PIK_006 | 0.42 | 70 | p.E545K/c.1633G>A | 35 | - | - | - | - |
PIK_007 | 0.36 | 30 | p.H1047R/c.3140A>G | 10 | 0.54 | 20 | p.H1047R/c.3140A>G | 41 |
PIK_008 | 4.06 | 85 | p.H1047R/c.3140A>G | 56 | 0.41 | 35 | p.H1047R/c.3140A>G | 22 |
PIK_009 | 0.43 | 70 | p.H1047L/c.3140A>T | 24 | - | - | - | - |
PIK_010 | 0.24 | 70 | p.E545K/c.1633G>A | 25 | 0.66 | 60 | p.E545K/c.1633G>A | 49 |
PIK_011 | 4.52 | 60 | p.H1047R/c.3140A>G | 44 | - | - | - | - |
PIK_012 | 0.48 | 60 | p.E542K/c.1624G>A | 18 | 0.31 | 80 | p.E542K/c.1624G>A | 4 |
PIK_013 | 113.00 | 80 | p.E453K/c.1357G>A | 13 | 3.06 | 75 | p.E453K/c.1357G>A | 37 |
PIK_014 | 0.42 | 50 | p.N1044K/c.3132T>A | 54 | 1.83 | 75 | p.N1044K/c.3132T>A | 37 |
PIK_015 | 0.12 | 90 | p.Q546K/c.1636C>A | 37 | - | - | - | - |
PIK_016 | 0.07 | 80 | p.Q546R/c.1637A>G | 33 | 0.97 | 40 | p.Q546R/c.1637A>G | 45 |
Metastases | Primary Tumors | |||||
---|---|---|---|---|---|---|
#Case | [DNA] (ng/µL) | Tumor Cell Content (%) | Mutation | [DNA] (ng/µL) | Tumor Cell Content (%) | Mutation |
PIK_001 | 0.26 | 20 | E545x | - | - | - |
PIK_002 | 0.84 | 60 | H1047x | - | - | - |
PIK_003 | 0.37 | 60 | H1047x | - | - | - |
PIK_004 | 0.26 | 35 | Failed * | 9.11 | 60 | H1047x |
PIK_005 | 0.44 | 20 | WT # | - | - | - |
PIK_006 | - | - | - | - | - | - |
PIK_007 | 0.18 | 40 | Failed * | 0.54 | 20 | H1047x |
PIK_008 | 4.06 | 85 | H1047x | 0.41 | 90 | H1047x |
PIK_009 | 0.43 | 70 | H1047x | - | - | - |
PIK_010 | 0.24 | 70 | E545x | 2.49 | 30 | E545x |
PIK_011 | - | - | - | - | - | - |
PIK_012 | 0.48 | 60 | E542x | 0.47 | 90 | WT |
PIK_013 | 28.00 | 70 | WT # | 12.00 | 70 | WT # |
PIK_014 | - | - | - | 7.31 | 35 | WT # |
PIK_015 | 0.12 | 90 | Failed * | - | - | - |
PIK_016 | 0.14 | 80 | WT | 0.97 | 40 | Q546x |
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Venetis, K.; Pepe, F.; Munzone, E.; Sajjadi, E.; Russo, G.; Pisapia, P.; Ivanova, M.; Bonizzi, G.; Vacirca, D.; Rappa, A.; et al. Analytical Performance of Next-Generation Sequencing and RT-PCR on Formalin-Fixed Paraffin-Embedded Tumor Tissues for PIK3CA Testing in HR+/HER2− Breast Cancer. Cells 2022, 11, 3545. https://doi.org/10.3390/cells11223545
Venetis K, Pepe F, Munzone E, Sajjadi E, Russo G, Pisapia P, Ivanova M, Bonizzi G, Vacirca D, Rappa A, et al. Analytical Performance of Next-Generation Sequencing and RT-PCR on Formalin-Fixed Paraffin-Embedded Tumor Tissues for PIK3CA Testing in HR+/HER2− Breast Cancer. Cells. 2022; 11(22):3545. https://doi.org/10.3390/cells11223545
Chicago/Turabian StyleVenetis, Konstantinos, Francesco Pepe, Elisabetta Munzone, Elham Sajjadi, Gianluca Russo, Pasquale Pisapia, Mariia Ivanova, Giuseppina Bonizzi, Davide Vacirca, Alessandra Rappa, and et al. 2022. "Analytical Performance of Next-Generation Sequencing and RT-PCR on Formalin-Fixed Paraffin-Embedded Tumor Tissues for PIK3CA Testing in HR+/HER2− Breast Cancer" Cells 11, no. 22: 3545. https://doi.org/10.3390/cells11223545