Arndt Borkhardt

Layal Yasin

Haematologica

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Haematologica | 109 September 20242772EDITORIALDo it once, but do it rightCorrespondence: A. Borkhardtarndt.borkhardt@med.uni-duesseldorf.deReceived: March 5, 2024.Accepted:  April 16, 2024.Early view:  April 24, 2024.https://doi.org/10.3324/haematol.2024.285246©2024 Ferrata Storti FoundationPublished under a CC BY-NC license Layal Yasin and Arndt BorkhardtDepartment of Pediatric Oncology, Haematology and Clinical Immunology, Medical Faculty, Heinrich-Heine-University, Düsseldorf, GermanyDespite the widespread implementation of molecular tu-mor profiling for diagnostics, classification and, finally, treatment selection, there is still a debate as to whether tumor-normal sequencing is preferred or deemed un-necessarily costly compared to sequencing of only tumor material. In this issue of Haematologica, Newman et al.1 have contributed a nice addition to this discussion, and gave a clear and unequivocal answer, at least for children and young adults with hematologic cancers. Through a retrospective analysis of nearly 1,200 cases (0-35 years of age) with B-/T-cell acute lymphoblastic lymphoma (ALL), myelodysplastic syndromes (MDS) / juvenile myel-omonocytic leukemia (JMML) or acute myeloid leukemia (AML) who underwent tumor-only (N=1,034) or tumor-normal (N=156) gene panel testing, the authors confirmed 16 cases who had a proven cancer-predisposition syndrome (CPS). Ten cases were revealed from the much larger group of tumor-only panel sequencing and 6 cases came from the tumor-normal sequencing group (Figure 1). The first thing that stands out is the high drop-out rate in the tumor-only group. Even if an underlying germline predisposition was suspected in 111 cases, only 29 of them underwent con-firmatory gene testing. This low number might not come as a surprise for those involved in the daily clinical man-agement of patients. Once tumor chemotherapy has start-ed, discussions with children and parents about therapy response and prognosis, as well as acute and long-term side effects occupy much of the attention of both doc-tors and the family. Genetic germline testing might pose an additional psychological burden, leading to perpetual postponement. Another remarkable result of the study is the fact that all 10 cases identified and confirmed in the tumor-only group would have had a clear indication for germline testing even without the suspicious tumor-only sequencing results. Specifically, a low-hypodiploid leukemia karyotype in a child with B-ALL points towards Li-Frau-meni Syndrome and requires testing of the TP53 gene. Furthermore, a child with overgrowth syndrome who was cured from neuroblastoma but presented thereafter with a secondary malignancy (T-lymphoblastic lymphoma), and all children with JMML or MDS, would require either panel or whole exome analysis anyway. It is not surprising to clinicians that germline testing found an underlying CPS in all of those 10 cases. In contrast, the 6 cases with CPS who had been detected in the much smaller tumor-normal sequencing group may have escaped their attention when only a phenotype-driven approach is applied. There are some subtle clinical and laboratory findings that may be indicative for a pathogenic germline ETV6, IKZF1 or RUNX1 variant, but these are far less obvious or very unspecific.2 (See Table 1 from Newman et al., for details.) Finally, there are several inherent bioinformatic challenges when the results of tumor-only sequencing lead to the as-sumption of an underlying CPS. The main challenge bioin-formaticians face is the ability to find innovative strategies for accurate differentiation between germline and somatic hits. One approach is to use the expected variant allele frequency (VAF) of germline mutations, which typically falls between 40% and 60%, or equals 100%, to differenti-ate them from somatic mutations. However, copy number variants (CNV), such as the loss of the wild-type allele in the tumor sample or mutation amplification, complicate this differentiation process. Germline variants may become undetectable or could be mistakenly classified as somatic.3-5 Another approach used by bioinformaticians involves char-acterizing and filtering germline variants using population databases. Yet the problem here is that these databases do not reflect the genetic diversity of the population, par-ticularly for under-represented ethnicities. This can lead to misclassification of variants and miscalculation of the overall tumor mutational burden (TMB).6,7 Taken together, the lack of matched normal tissue complicates variant filtering. This can lead to inaccuracies, and increases the rate of false negatives and positives.All in all, these considerations raise the intriguing question of how many CPS were potentially overlooked in the tu-mor-only sequencing group. Furthermore, the identification of a cancer predisposing germline variant influences clinical Do it once, but do it rightL. Yasin and A. Borkhardthttps://doi.org/10.3324/haematol.2024.285246Haematologica | 109 September 20242773EDITORIAL  L. Yasin and A. Borkhardt care and long-term follow-up in what is termed as “per-sonalized cancer surveillance and prevention”. The overall risk for the development of subsequent malignancies in long-term survivors of childhood cancer who are carriers of a cancer predisposing germline variant is at least 3 to 4 times higher compared to their non-carriers.8 The other critical observation in this study comes from the fact that within the immediate family members of those 16 patients with a proven CPS, no early-onset cancer was diagnosed. Thus, the child with the hematologic cancer became a red flag for the family. Out of the 33 that went for subsequent panel testing, a CPS for 12 family members was revealed. Although this obviously came as an unwel-come surprise, it also offers future strategies for surveil-lance and removes anxiety from all other family members. Of note, the de novo mutation rate for many CPS genes is still unknown and family-based sequencing is required to fill this knowledge gap.9In an ideal world, both pre- and post-test genetic counseling should be offered not only to individuals whose germline DNA were molecularly profiled, but also to those with tumor-only sequencing. However, in certain regions and communities, the existing hurdles in accessing genetic counseling ser-vices need to be addressed. This is particularly the case for countries where the integration of genetic counseling into the healthcare systems remains a challenge.In summary, even if cancers that arise in carriers of patho-genic germline alleles may not directly depend on or may even be unrelated to the specific germline variant,10 the arguments for continuing with tumor-only sequencing are poor, especially in the light of recent cost-effective sequencing options. DisclosuresNo conflicts of interest to disclosure.ContributionsBoth authors wrote the editorial.Figure 1. Comparison of tumor-only and paired tumor-normal sequencing for cancer-predisposition syndromes. Whereas paired tumor-normal sequencing enables a clear distinction of germline and somatic variants, tumor-only gene sequencing causes ad-ditional bioinformatic challenges. CPS: cancer-predisposition syndrome.Haematologica | 109 September 20242774EDITORIAL  L. Yasin and A. Borkhardt References 1. Newman H, Clark ME, Wong D, et al. Genomic profiling of pediatric hematologic malignancies and diagnosis of cancer predisposition syndromes: tumor-only versus paired tumor-normal sequencing. Haematologica. 2024;109(9):3024-3030. 2. Wagener R, Elitzur S, Brozou T, Borkhardt A. Functional damaging germline variants in ETV6, IKZF1, PAX5 and RUNX1 predisposing to B-cell precursor acute lymphoblastic leukemia. Eur J Med Genet. 2023;66(4):104725. 3. Schienda J, Church AJ, Corson LB, et al. Germline sequencing improves tumor-only sequencing interpretation in a precision genomic study of patients with pediatric solid tumor. JCO Precis Oncol. 2021;5:PO.21.00281. 4. Terraf P, Pareja F, Brown DN, et al. Comprehensive assessment of germline pathogenic variant detection in tumor-only sequencing. Ann Oncol. 2022;33(4):426-433. 5. Li MM, Chao E, Esplin ED, et al. Points to consider for reporting of germline variation in patients undergoing tumor testing: a statement of the American College of Medical Genetics and Genomics (ACMG). Genet Med. 2020;22(7):1142-1148. 6. Asmann YW, Parikh K, Bergsagel PL, et al. Inflation of tumor mutation burden by tumor-only sequencing in under-represented groups. NPJ Precis Oncol. 2021;5(1):22. 7. Parikh K, Huether R, White K, et al. Tumor mutational burden from tumor-only sequencing compared with germline subtraction from paired tumor and normal specimens. JAMA Netw Open. 2020;3(2):e200202. 8. Chen C, Qin N, Wang M, et al. Cancer germline predisposing variants and late mortality from subsequent malignant neoplasms among long-term childhood cancer survivors: a report from the St Jude Lifetime Cohort and the Childhood Cancer Survivor Study. Lancet Oncol. 2023;24(10):1147-1156. 9. Kuhlen M, Taeubner J, Brozou T, Wieczorek D, Siebert R, Borkhardt A. Family-based germline sequencing in children with cancer. Oncogene. 2019;38(9):1367-1380. 10. Srinivasan P, Bandlamudi C, Jonsson P, et al. The context-specific role of germline pathogenicity in tumorigenesis. Nat Genet. 2021;53(11):1577-1585.

Do it once, but do it right

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Do it once, but do it right

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