
==== Front
Oncologist
Oncologist
oncolo
The Oncologist
1083-7159
1549-490X
Oxford University Press US

38776551
10.1093/oncolo/oyae098
oyae098
Brief Communication
AcademicSubjects/MED00010
Oncolo/5
A comprehensive analysis of POLE/POLD1 genomic alterations in colorectal cancer
https://orcid.org/0000-0002-3779-185X
Mosalem Osama Department of Medicine, Division of Hematology and Oncology, Mayo Clinic, Jacksonville, FL, USA

https://orcid.org/0000-0002-5843-2506
Coston Tucker W Department of Medicine, Division of Medical Oncology, Duke Cancer Center, Raleigh, NC, USA

https://orcid.org/0000-0001-5413-087X
Imperial Robin Department of Medicine, Division of Hematology and Oncology, Mayo Clinic, Jacksonville, FL, USA

https://orcid.org/0000-0002-9012-0502
Mauer Elizabeth Tempus AI, Chicago, IL, USA

Thompson Christopher Tempus AI, Chicago, IL, USA

Yilma Binyam Tempus AI, Chicago, IL, USA

https://orcid.org/0000-0003-4653-4537
Bekaii-Saab Tanios S Department of Medicine, Division of Hematology and Oncology, Mayo Clinic, Phoenix, AZ, USA

Stoppler Melissa Conrad Tempus AI, Chicago, IL, USA

https://orcid.org/0000-0002-8674-8102
Starr Jason S Department of Medicine, Division of Hematology and Oncology, Mayo Clinic, Jacksonville, FL, USA

Corresponding author: Jason S. Starr, Department of Medicine, Division of Hematology and Oncology, Mayo Clinic, 4500 San Pablo Road South, Jacksonville, FL 32224, United States (starr.jason@mayo.edu).
9 2024
22 5 2024
22 5 2024
29 9 e1224e1227
10 1 2024
17 4 2024
© The Author(s) 2024. Published by Oxford University Press.
2024
https://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.

Abstract

Introduction

Pathogenic mutations in POLE/POLD1 lead to decreased fidelity of DNA replication, resulting in a high tumor mutational burden (TMB-H), defined as TMB ≥ 10 mut/Mb, independent of deficient mismatch repair (dMMR) and microsatellite instability high (MSI-H) status.

Methods

De-identified records of patients with colorectal cancer (CRC) profiled with the Tempus xT assay (DNA-seq of 595-648 genes at 500×) were identified from the Tempus Database.

Results

Among 9136 CRC samples profiled, the frequency of POLE/POLD1 genomic alterations was 2.4% (n = 217). Copy number loss was the most common genomic alteration (64%, n = 138) of POLE/POLD1, followed by copy number amplifications (18%, n = 40) and short variant mutations (18%, n = 39). The POLE/POLD1 mutated group presented with a higher frequency of TMB-H phenotype relative to wild type (WT; 22% vs. 9%, P < .001), with a median TMB of 127 mut/Mb in the TMB-H POLE/POLD1 subset. The TMB showed a dramatic contrast between POLE/POLD1 short variant mutations as compared to the group with copy number alterations, with a TMB of 159 mut/Mb vs 15 mut/Mb, respectively. Thus, the short variant mutations represented the so-called ultra-hypermutated phenotype. The POLE/POLD1 mutated group, as compared to WT, exhibited a higher rate of coexisting mutations, including APC, ALK, ATM, BRCA2, and RET mutations.

Conclusion

Patients with POLE/POLD1 mutations exhibited significant differences across immunological markers (ie, TMB, MMR, and MSI-H) and molecular co-alterations. Those with short variant mutations represented 18% of the POLE/POLD1 cohort and 0.4% of the total cohort examined. This group of patients had a median TMB of 159 mut/Mb (range 34-488), representing the ultra-hypermutated phenotype. This group of patients is important to identify given the potential for exceptional response to immune checkpoint inhibitors.

This article reports the characteristic of POLE/POLD1 mutations in a large, real-world cohort of patients with colorectal cancer.

colorectal cancer
genetics
DNA repair
tumor biomarkers
genomics
Tempus AI
==== Body
pmcIntroduction

The POLE and POLD1 genes encode for the catalytic and proofreading DNA polymerase subunits ε and δ, respectively.1 Pathogenic missense mutations within the exonuclease domain of the POLE/POLD1 genes lead to the loss of the proofreading function and reduce the fidelity of DNA repair with the subsequent accumulation of mutations.2 This leads to the so-called ultra-hypermutated phenotype, typically with a tumor mutational burden (TMB-H) greater than 100 mutations/megabase (mut/Mb). Studies have shown that this ultra-hypermutated phenotype, namely in colorectal cancer (CRC), predicts a significant benefit from immune checkpoint inhibitors (ICIs).3-5 Our study aims to expand the existing knowledge about the genomic landscape of POLE/POLD1 mutations in CRC.3,6 Here, we report the characteristic of POLE/POLD1 mutations in a large, real-world cohort of CRC patients.

Methods

We used de-identified records of patients with CRC profiled using the Tempus xT next-generation sequencing assay (DNA-seq of 595-648 genes at 500×). Immunological markers analyzed included TMB, microsatellite instability status (MSI), and mismatch repair deficiency (dMMR). The assay determined MSI-high (MSI-H) by assessing 239 loci, while dMMR was determined by immunohistochemistry (MLH1, MSH2, MSH6, and PMS2). Our study encompassed patients who exhibited molecular aberrations in either POLE or POLD1, including pathogenic/likely pathogenic mutations and copy number alterations (including loss or amplification [copy number ≥ 8]). These patients were compared to wild-type (WT) POLE/POLD1.

Results

Among 9136 CRC samples profiled, the POLE/POLD1 genomic alterations (copy number variant or mutation) frequency was 2.4% (n = 217; 203 POLE, 14 POLD1). The breakdown of types of genomic alterations of POLE/POLD1 were as follows: copy number loss (64%, n = 138), copy number amplification (18%, n = 40), and short variant mutations (18%, n = 39). Of the total cohort, 61% of the patients had tumor-normal (blood sample) matched samples. The mutations in this cohort were analyzed and deemed somatic in nature. For the remaining 39% of patients, only tumor was analyzed without parallel blood testing for germline mutations. It is possible, although unlikely, that these patients had germline mutations of POLE/POLD1. No significant differences were seen in demographics among patients with POLE/POLD1 WT vs the mutated group (Supplementary Table S1). The median TMB between the POLE/POLD1 mutated vs the WT cohort was non-significant (5 vs 4 mut/Mb). The POLE/POLD1 mutated subgroup presented with a higher frequency of “TMB-H” phenotype (≥ 10 mut/Mb) as compared to WT (22% vs 9.9%, P < .001). In addition, the median TMB in the POLE/POLD1 “TMB-H” group was markedly higher than the WT TMB-H subgroup, averaging 127 mut/Mb vs 29 mut/Mb, respectively. POLE/POLD1 mutated tumors were mostly MSS (98%, n = 213), with only 2% being MSI-H. Of note, dMMR was present in 5.3% of the mutated cohort.

Among the POLE/POLD1 mutant cohort, copy number loss was the most common genetic alteration (59% POLE, 5% POLD1), followed by short variant mutation (including single nucleotide variants, insertions, or deletions) and copy number amplification. Seventy-eight percent (n = 132) of the POLE/POLD1 TMB-low (< 10 mut/Mb) subgroup had copy number loss as their predominant genetic alteration. This contrasts with the TMB-H POLE mutated phenotype, where short variant mutations were exclusively observed in this subgroup (83%, n = 39), with Pro286Arg, Val411Leu, Ser297Phe, and Ala456Pro being the most common mutation sites; all mutations are located within the exonuclease domain of the gene (Supplementary Figures S1 and S2). All the short variant mutations were associated with the ultra-hypermutated phenotype. The group with short variant mutations had a significantly higher TMB than those with copy number variation (CNV), with a median TMB of 159 mut/Mb (range 34-488) vs 15 mut/Mb (range 11-27) (Figure 1). We next analyzed the co-mutational profile. Overall, POLE/POLD1 mutant tumors exhibited a higher co-mutational frequency than WT (Figure 2). The most frequently noted genetic co-mutations in the POLE/POLD1 mutant group were APC, ALK, LRP1B, RET, FGF4, and NTRK3 (P < .001), while KRAS, BRAF, and ERBB2 mutations were seen equally in mutant and WT cohorts (KRAS 41% vs 44%, BRAF 8% vs 11%, ERBB2 0.3% vs 0.3%, respectively). Comparing TMB-H vs TMB-L POLE/POLD1 mutant cohorts, a higher load of co-mutations was seen in the TMB-H vs TMB-L, mainly APC, LRP1B, KMT2C, PIK3CA, BRCA1/2, and PTEN mutations (P < .05). Meanwhile, p53, RET, and FGF6 mutations were seen more frequently in the TMB-low subgroup (P < .05).

Figure 1. Box plot highlights differences in tumor mutational burden (TMB) among POLE/POLD1 short variant mutations vs copy number variants (CNV). The TMB was significantly higher in the short variant mutation group as compared those with CNVs, with a median TMB of 159 mut/Mb (range 34-488) vs 15 mut/Mb (range 11-27), respectively.

Figure 2. A heatmap illustrating co-occurring genomic alterations in patients with POLE/POLD1 mutant vs wild type (WT). A higher rate of co-mutations was seen in the POLE/POLD1 mutant subgroup, specifically APC, ALK, LRP1B, RET, FGF4, and NTRK3 (P < .001), while KRAS, BRAF, and ERBB2 mutations were equally observed in both mutant and WT cohorts.

Discussion

Our findings provide further insights into the prevalence, genomic characteristics (ie, TMB, MSI, and MMR), and POLE/POLD1 heterogeneity in the colorectal cancer patient population. In our large cohort, 2.4% of the patients with CRC harbored POLE/POLD1 genomic alterations. The majority of the genomic alterations involved copy number variants (ie, copy number loss [n = 138, 64%] or amplification [n = 40, 18%]). This group was mainly associated with the TMB-L phenotype (ie, TMB < 10 mut/Mb). When this group was noted to have TMB-H status, the median TMB was noted to be 15 mut/Mb (range 11-27 mut/Mb). To further emphasize this point, Hwang et al previously reported on 47 cases with POLE mutated cancers, and the median TMB in this cohort was 12.5 mut/Mb, with only 2 patients in this study having the ultra-hypermutated phenotype.7 This is in stark contrast to the short variant mutations which exhibited a median TMB of 159 mut/Mb. Thus, it is the group of patients with short variant mutations, which represented 18% of the POLE/POLD1 genomic alterations and 0.4% of the total examined cohort, who exhibited the ultra-mutated phenotype. It is critically important to identify these patients as they have been reported to have significant benefit from ICIs.3,8 Among those with short variant mutations, most of the amino acid changes (Pro286Arg, Val411Leu, Ser297Phe, and Ala456Pro) occurred within the “hot spot” sites, which were previously described to be associated with pathogenic mutations and TMB ≥ 100 mut/Mb.9

The main limitation of our study is the lack of clinical annotation for the genomic findings. It would have been particularly important to know whether there is a differential response to immunotherapy in this group of patients. For example, do patients with CNVs of the gene where the TMB may be in the 10-20 (mut/Mb) range respond as favorably as those with short variant mutations and TMB in the 100s (mut/Mb). Further exploration of these types of questions are warranted.

Supplementary material

Supplementary material is available at The Oncologist online.

oyae098_suppl_Supplementary_Table

oyae098_suppl_Supplementary_Figures

Acknowledgments

The authors thank Stephen Park, MPH for figure production.

Author contributions

Osama Mosalem, Tucker W. Coston, Jason S. Starr contributed to the project design, data analysis, and drafted the manuscript. Elizabeth Mauer, Christopher Thompson, Binyam Yilma, and Melissa Conrad Stoppler assisted in the data collection and data analysis. Robin Imperial, Osama Mosalem, Tanios S. Bekaii-Saab, Jason S. Starr reviewed the manuscript and helped in data interpretation. All authors read and approved the final manuscript.

Funding

This work was supported by Tempus AI.

Conflicts of interest

Elizabeth Mauer is an employee of Tempus AI. Christopher Thompson is an employee of Tempus AI. Binyam Yilma is a full-time employee of Tempus AI and receives restricted stock units of Tempus AI. Melissa Conrad Stoppler is an employee of Tempus AI, Inc., with stock ownership. Tanios S. Bekaii-Saab has a consulting or advisory role with Abbvie; Amgen (Inst); Arcus Biosciences (Inst); AstraZeneca; Bayer (Inst); BeiGene; Boehringer Ingelheim; Celularity; Daiichi Sankyo/UCB Japan; Deciphera; Eisai; Eisai; Exact Sciences; Foundation Medicine; Foundation Medicine; Illumina; Immuneering; Incyte (Inst); Ipsen (Inst); Janssen; Kanaph Therapeutics; Lilly (Inst); Natera; Pfizer (Inst); Roche/Genentech (Inst); Seagen (Inst); SOBI; Stemline Therapeutics; Treos Bio. Jason Starr has a consulting or advisory role with Advanced Accelerator Applications; Ipsen; Natera; Pfizer; Taiho Oncology; Terser. Research Funding—RayzeBio (Inst), Amgen (Inst), Arcus Bioscience (Inst), Perspective Therapeutics (Inst). Aminex Therapeutics (Inst), Cardiff (Inst).

Data availability

The data underlying this article are available in the article and in its online supplementary material.
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