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JCO Precis Oncol
JCO Precis Oncol
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JCO Precision Oncology
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Wolters Kluwer Health

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Commentaries
Diagnostic and Practical Challenges in Applying National Comprehensive Cancer Network Guidelines for Suspected Pathogenic TP53 Mosaicism
https://orcid.org/0000-0003-4186-7412
Nathan Daniel I. MD 1
https://orcid.org/0000-0002-2874-7777
Brander Tehilla MS, CGC 2
Gold Julie MS, CGC 2
Paul Deborah MS, CGC 3
Klein Paula 1
https://orcid.org/0000-0003-0136-6912
Cheng Kit MD 4
https://orcid.org/0000-0002-3959-1296
Liu Johnson M. MD 1
https://orcid.org/0000-0001-8590-6175
Marcellino Bridget K. MD, PhD 1
1 Division of Hematology and Medical Oncology, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY
2 Division of Medical Genetics and Genomics, Icahn School of Medicine at Mount Sinai, New York, NY
3 Department of Breast Services, The Blavatnik Family Chelsea Medical Center, Mount Sinai Beth Israel, New York, NY
4 Division of Medical Oncology and Hematology, Zuckerberg Cancer Center, New Hyde Park, NY
Bridget K. Marcellino, MD, PhD; e-mail: Bridget.marcellino@mssm.edu.
2024
11 7 2024
11 7 2024
8 e24000063 1 2024
12 4 2024
8 5 2024
© 2024 by American Society of Clinical Oncology
2024
American Society of Clinical Oncology
https://creativecommons.org/licenses/by-nc-nd/4.0/ Creative Commons Attribution Non-Commercial No Derivatives 4.0 License: https://creativecommons.org/licenses/by-nc-nd/4.0/

Benefits and limitations in using NCCN guidelines to distinguish TP53 CH from mosaic LFS.

OPEN-ACCESSTRUE
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pmcPathogenic variants (PV) in TP53 are presenting an increasing conundrum for practicing oncologists and hematologists because of the emerging use of next-generation sequencing (NGS) panels in solid malignancies and an increasing awareness of clonal hematopoiesis (CH).1,2

The TP53 gene, located on chromosome 17p, encodes the tumor suppressor protein p53 and is associated with pan-tumor pathogenicity.3 Germline variants in TP53 define Li-Fraumeni syndrome (LFS), which confers a significant lifetime risk of incident malignancies in men and women.4-6 Notably, breast cancer is the most common malignancy to develop in LFS, with median age of onset of 33 years.7

Distinct from germline variants, somatic variants arise at any point after the first zygotic division and can lead to distinct mutational profiles in different tissues, a phenomenon known as postzygotic mosaicism (PZM).8,9 Somatic PV in TP53 are likewise implicated in the pathogenesis of numerous cancers and generally portend worse prognoses.7 CH, the presence of somatic variants in hematopoietic stem and progenitor cells, is an example of PZM within the hematopoietic compartment. CH of indeterminate potential (CHIP), detectable CH without hematologic disease, occurs naturally with aging and increases the risk of all-cause mortality and hematologic malignancy.10

Given the range of diagnostic considerations and respective risks, it is imperative to understand the significance of a detected TP53 PV in peripheral blood (PB) or saliva on NGS platforms.1,2,11,12 Suspicion for germline origin may be raised by a high variant allele frequency (VAF), and heterozygosity is considered when the VAF is between 30% and 70%.1,13 A skin biopsy for fibroblast culture is the gold standard for germline confirmation.13,14 However, in patients with known solid malignancy and a high VAF TP53 PV in PB or saliva but negative fibroblast testing, there may be concern for PZM in multiple tissues, also known as mosaic LFS. In these cases, the uncertainty of the tissue distribution of the PV results in opaque risks for patients and confusion for providers in their appropriate management.

With their latest published guidelines, the National Comprehensive Cancer Network (NCCN) for the first time has recognized the significant implications this ambiguity carries for screening. In attempting to sort through each potential scenario, they provide a rubric for approximating a diagnosis and guidelines for subsequent screening, including the recommendation that suspected mosaic LFS be screened like true germline LFS.15,16 Such screening protocols are onerous and costly, including regular whole-body and brain magnetic resonance imaging (MRI), complete blood counts (CBC), breast MRI with mammography, upper and lower GI endoscopy, and comprehensive physical, dermatologic, and neurologic examination.16-18 Given the lack of certainty, it is vital to evaluate suspected cases critically to avoid undue financial and psychological burdens placed on individuals with potential CH.19 Herein, we contextualize these recommendations with several cases from our clinic that illustrate how this diagnostic dilemma unfolds in real-world settings and often leads to unsatisfactory conclusions and where additional tools are needed to decipher between these entities and design individualized management strategies.

This study was determined to be exempt from review by the local institutional review board, and all patient data are presented anonymously. Written informed consent was obtained from each patient.

Case 1: Conservative by Default

A 41-year-old woman of Asian descent was diagnosed with triple-positive Stage IB carcinoma of the left breast. Hereditary cancer screening on PB identified a TP53 PV reported as inconclusive heterozygosity as well as additional variants of unknown significance (VUS). Fibroblast testing did not identify the TP53 PV but did detect the other VUS (Table 1). Targeted sequencing for myeloid associated genes in PB performed 3 months later to quantify and capture clonal diversity in the context of mild thrombocytopenia detected the TP53 PV at VAF 10.7%. She was treated with neoadjuvant chemotherapy and dual human epidermal growth factor receptor 2 antibodies, and mastectomy showed pathologic complete response (pCR). Follow-up NGS from PB at the time of mastectomy confirmed the persistence of the TP53 PV and the same PV was found in breast tissue from both her diagnostic biopsy and from nontumor mastectomy tissue. As only bulk Sanger sequencing was performed on breast tissue, hematopoietic contamination could not be definitively ruled out and no VAF was reportable. Nevertheless, the presence of the PV in the blood, tumor, and normal breast tissue suggest mosaic LFS consistent with NCCN considerations. Testing of her child was negative for the PV, an important consideration for the heritable risks of LFS, but not dispositive of mosaicism in the proband. An additional consideration in this case was the absence of risk factors for CH, including her young age, lack of smoking history, and the presence of the TP53 PV without a previous history of cytotoxic chemotherapy.20 In line with current NCCN guidelines, she was recommended for conservative LFS surveillance protocols including consideration of risk reducing prophylactic contralateral mastectomy.

TABLE 1. Summary of Genomic Data

Case	Tissue (platform)	Gene	Reported Variant	VAFa	Classificationb	
1
A 41-year-old woman with breast cancer treated with neoadjuvant chemotherapy and mastectomy with pCR, found to have TP53 PV in PB with same PV identified in breast tumor and nontumor tissue.	Peripheral blood (MyRisk, Myriad)	TP53	c.733G>A	(p.Gly245Ser)	10-13c	Pathogenic	
BRCA2	c.3924A>C	(p.Glu1308Asp)		VUS	
MSH2	c.548A>G	(p.Gln183Arg)		VUS	
MUTYH	c.53C>T	(p.P18Leu)		VUS	
MUTYH	c.74G>A	(p.Gly25Asp)		VUS	
RAD51D	c.196G>A	(p.Val66Met)		VUS	
Fibroblast (MyRisk, Myriad)	TP53	Not detected				
BRCA2	c.3924A>C	(p.Glu1308Asp)		VUS	
MSH2	c.548A>G	(p.Gln183Arg)		VUS	
MUTYH	c.53C>T	(p.P18Leu)		VUS	
MUTYH	c.74G>A	(p.Gly25Asp)		VUS	
RAD51D	c.196G>A	(p.Val66Met)		VUS	
Peripheral blood +3 months (NeoType Myeloid Disorders Profile, NeoGenomics)	TP53	c.733G>A	(p.Gly245Ser)	10.7	Pathogenic	
Breast—tumor (custom TP53 assay via Sanger sequencing, NeoGenomics)	TP53	c.733G>A	(p.Gly245Ser)		Pathogenic	
Breast—nontumor (custom TP53 assay via Sanger sequencing, NeoGenomics)	TP53	c.733G>A	(p.Gly245Ser)		Pathogenic	
2
A 64-year-old woman with breast cancer treated with mastectomy and chemotherapy. New primary treated with contralateral mastectomy and chemotherapy. Found to have TP53 PV in PB with increasing VAF and cytopenias.	Peripheral blood (Invitae Common Hereditary Cancers Panel, Invitae)	TP53	c.422G>A	(p.Cys141Tyr)	HETd	Pathogenic	
Fibroblast (Invitae Li-Fraumeni Syndrome Test, Invitae)	TP53	Not detected	
Peripheral blood—pretreatment (Legacy Myeloid Molecular Profile, NeoGenomics)	TP53	c.422G>A	(p.Cys141Tyr)	43.1	Pathogenic	
DNMT3A	c.2408G>A	(p.Arg803Lys)		VUS	
Peripheral blood (Guardant360, Guardant)	TP53	c.422G>A	(p.Cys141Tyr)	47.1	Pathogenic	
TP53	c.455C>T	(p.Pro152Leu)	0.6	Pathogenic	
TP53	c.488A>G	(p.Tyr163Cys)	0.3	Pathogenic	
TP53	c.273G>A	(p.Trp91a)	0.2	Pathogenic	
TP53	c.102del	(p.Leu35fs)	0.6	Pathogenic	
Peripheral blood—post-treatment (Legacy Myeloid Molecular Profile, NeoGenomics)	TP53	c.422G>A	(p.Cys141Tyr)	48.6	Pathogenic	
DNMT3A	c.2408G>A	(p.Arg803Lys)	11.4	VUS	
3
A 47-year-old woman with breast cancer, TP53 PV detected in breast, PB, and bone marrow but not in fibroblasts.	Peripheral blood—diagnosis (Breast/Gyn Cancer Panel, GeneDx)	TP53	c.844C>T	(p.Arg282Trp)	34.9	Pathogenic	
Peripheral blood—diagnosis (custom ddPCR assay, Northwell Labs)	TP53	c.844C>T	(p.Arg282Trp)	39.7	Pathogenic	
Breast—tumor (NGS Solid Tumor Panel, GeneDx)	TP53	c.844C>T	(p.Arg282Trp)	29.8	Pathogenic	
Fibroblast (custom assay, Northwell Labs)	TP53	Not detected	
Peripheral blood +3 months (custom Sanger sequencing assay, Northwell Labs)	TP53	c.844C>T	(p.Arg282Trp)		Pathogenic	
Peripheral blood +3 months (custom ddPCR assay, Northwell Labs)	TP53	c.844C>T	(p.Arg282Trp)	38.3	Pathogenic	
Peripheral blood +8 months (custom TP53 assay, Northwell Labs)	TP53	c.844C>T	(p.Arg282Trp)	38.9	Pathogenic	
Peripheral blood +8 months (custom ddPCR assay, Northwell Labs)	TP53	c.844C>T	(p.Arg282Trp)	39.9	Pathogenic	
Bone marrow (OnkoSight Myeloid Malignancies Panel, GenPath)	TP53	c.844C>T	(p.Arg282Trp)		Pathogenic	
NOTE. Brief case summaries, tissue source, and mutational variant details from each analyzed sample.

Abbreviations: CBC, complete blood counts; ddPCR, digital droplet polymerase chain reaction; HET, heterozygous; NGS, next-generation sequencing; PB, peripheral blood; pCR, pathologic complete response; PV, pathogenic variant; VAF, variant allele frequency; VUS, variant of unknown significance.

a VAF is reported where available as a percentage (%).

b Variant classifications are reported according to American College of Genetics and Genomics category at the time of sequencing and reported here.

c Range estimate as provided by testing laboratory, initially reported as inconclusive for heterozygosity.

d Presumed zygosity reported in lieu of specific VAF or range estimate.

Case 2: Using Clinical Context

A woman of Guyanese and East Asian descent was diagnosed with Stage II triple-negative invasive ductal breast cancer of the right breast at age 57 years. She was treated with adjuvant anthracycline- and taxane-based chemotherapy and mastectomy but was diagnosed with a contralateral new primary 2 years later. At that time, she underwent genetic testing from PB that showed a PV in TP53 reported as heterozygous without specific VAF but not identified in confirmational fibroblast testing. No breast tissue sample was available for sequencing and no offspring were available for cascade testing. She was treated with neoadjuvant and adjuvant platinum and taxane based chemotherapy and left mastectomy. Targeted sequencing on a panel of myeloid associated genes identified the TP53 PV at 43% VAF and an additional VUS in DNMT3A. TP53 VAF increased to 48.6% on follow-up sequencing 2 years later. Liquid biopsy testing for tumor monitoring identified four additional PV in TP53 at VAF <1% (Table 1). Applying NCCN guidelines does not narrow down the differential diagnosis as CH is possible, the PV status of her tumor is unknown, and a second primary cancer is suspicious for mosaicism. This underscores the difficulty in applying these recommendations retroactively to patients for whom genomic evaluation was not available at the time of their diagnosis. Given her age, history of chemotherapy, and apparent clonal expansion, we concluded that CH is a likely diagnosis and recommended management accordingly. During 5 years of follow-up, she has undergone serial CBC every 3-4 months, which have showed cytopenias attributed to concurrent antineoplastic therapy, and she was recently referred to a dedicated CH clinic. A bone marrow biopsy is planned to rule out a diagnosis of clonal cytopenia of undetermined significance (CCUS) or other hematologic disease after completion of therapy, along with annual NGS from PB to assess growth of her TP53-mutant clone in the context of her previous exposure to cytotoxic chemotherapy.20-24

Case 3: A Question of Consequence

A 47-year-old woman of European descent was found to have Stage I hormone receptor–positive invasive breast ductal carcinoma. Family history was notable for breast cancer diagnosed at age 30 years in a paternal grandmother. NGS for hereditary cancer risk was performed on PB and identified a PV in TP53 at 35%. She was referred to genetic counseling to assist in distinguishing the etiology of the PV. Breast biopsy tissue was sent and found to be positive for the same PV in TP53 at 30% VAF; however, fibroblast testing was negative for the PV (Table 1). Although hematopoietic contamination of the breast tissue could not be ruled out with available testing, NCCN guidelines indicate that several potential diagnoses were possible. In our assessment, the high VAF observed in breast tissue and existing breast cancer diagnosis made mosaic LFS a likely diagnosis; however, CH was also a consideration because of the higher VAF in the PB than in tested tumor. Testing of both parents was negative for the identified PV, and testing was offered for her child, which she deferred until adulthood. A CBC showed cytopenias, and so she was referred for a bone marrow biopsy that confirmed the same PV and VAF in her aspirate as her PB. After shared decision making regarding the concern for new primary breast cancer in patients with LFS, she underwent prophylactic bilateral mastectomy followed by estrogen blockade and there has been no recurrence of disease or new cytopenias.25 NCCN recommendations for management agree with this approach, but do not address individual cases where there is no consensus diagnosis and might further suggest expanding screening to include imaging, colonoscopy, and skin examinations.18 Follow-up digital droplet polymerase chain reaction (ddPCR) testing on all PB samples identified a consistent VAF of 38%-40%.26,27 Remarkably, despite the presence of a high VAF TP53 PV in her PB and concern for a mosaic LFS state, she has remained free of hematologic cancer and carcinoma recurrence over 8 years of follow-up, further underscoring the unknown individual variant and patient-level factors that contribute to outcomes with TP53 PV.

Discussion

In summary, the interpretation and consequence of TP53 PV on sequencing assays in solid tumors is rife with uncertainty. The NCCN is commendable for issuing recommendations to clarify management strategies for these cases. Without question, germline TP53 PV confirmed on fibroblast testing consistent with LFS should be referred for appropriate screening protocols. A significant challenge exists when fibroblast testing for a PV found in PB or saliva is negative, and these cases require further discrimination between CH or mosaic LFS to determine proper screening.

It is notable that each presented case is of breast cancer; however, this is reflective of both (1) the high prevalence of breast cancer in LFS and (2) the real-world referral patterns of young women with breast cancer being screened for hereditary cancer testing from PB and subsequently referred for evaluation.28 However, it is imperative to maintain high suspicion in other tumor types, as both LFS and CH may be represented in a wide range of primary malignancies.

Even before the issuance of these NCCN guidelines, we have found clinician judgment, family history, clinical context, and collaboration with clinical geneticists and genetic counselors key in weighing the likelihood of each scenario. Certain canonical TP53 variants are highly associated with LFS, while less well-characterized variants may be found in individuals from ancestral backgrounds not well represented in genomic databases.29 Sequencing existing or planned biopsies of tumor or nontumor tissue can inform a screening protocol, although contamination from hematopoietic elements and inability to comprehensively map the somatic landscape of each organ can limit the specificity and sensitivity of this approach, respectively. Choosing an optimal sequencing platform is also important, since Sanger sequencing does not provide VAF for comparative purposes between tissues, as noted in Case 1, and more sensitive techniques such as ddPCR may be helpful in precisely measuring clonal burden. Despite the current limitations of available technologies, future application of commercial immunophenotypic single-cell analysis or microdissection of bulk tissue before sequencing may aid in clarifying the presence of a PV in a distant tissue site beyond the PB. Until then, LFS screening for suspected mosaic LFS is the most conservative and default recommendation, in line with screening of other mosaic genetic conditions.30,31 Yet, it is doubtful that every suspicious TP53 PV in PB is likely to be found in other tissues, let alone in every tissue that is a target for cancer screening, and the VAF ranges at which mosaic LFS ought to be considered are conspicuously wide (0%-100%).18 Although the NCCN guideline is an important step in raising awareness of these challenges, shared decision making to develop an individualized screening protocol feels inadequate in the face of such uncertainty, and the financial costs for testing to differentiate diagnostic possibilities can be burdensome without insurance coverage.19 Long-term prospective data on patients with TP53 mosaicism is critical to ably advise and inform these patients on their respective site-specific cancer risks and develop refined guidelines to enable expanded insurance coverage for all similar cases.

With respect to hematologic malignancy, the risk of TP53 CH is highly context-dependent and of greatest concern after cytotoxic or radiotherapy but to a milder degree without extrinsic selective pressures.20,24,32,33 Our practice to monitor CHIP and CCUS is to collect CBC every 3-6 months, repeat NGS annually, and perform bone marrow examination to investigate any unexplained cytopenias. TP53 CH is also an emerging risk factor for cardiovascular disease underscoring the complex longitudinal follow-up necessary for these patients, and given the poor outcomes of TP53-related myeloid malignancies, these patients should likewise be referred to specialized CH clinics for surveillance and clinical study.20,34,35

NGS offers much promise in the screening, diagnosis, and treatment of malignancies, and yet we are only scratching the surface of the implications of the volume of data available. There is much to learn about how TP53 PV on PB assays should inform future screening programs for at-risk individuals. The recent NCCN guidelines offer an important first step, but evidence and the technology needed to obtain evidence is lacking the certainty sought by our patients to relieve the specter of malignancy.

PRIOR PRESENTATION

SUPPORT

AUTHOR CONTRIBUTIONS

Conception and design: Daniel I. Nathan, Johnson M. Liu, Bridget K. Marcellino

Administrative support: Johnson M. Liu

Provision of study materials or patients: Deborah Paul, Paula Klein, Kit Cheng, Johnson M. Liu

Collection and assembly of data: Daniel I. Nathan, Deborah Paul, Paula Klein, Kit Cheng, Johnson M. Liu, Bridget K. Marcellino

Data analysis and interpretation: Daniel I. Nathan, Tehilla Brander, Julie Gold, Kit Cheng, Johnson M. Liu, Bridget K. Marcellino

Manuscript writing: All authors

Final approval of manuscript: All authors

Accountable for all aspects of the work: All authors

AUTHORS' DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

The following represents disclosure information provided by authors of this manuscript. All relationships are considered compensated unless otherwise noted. Relationships are self-held unless noted. I = Immediate Family Member, Inst = My Institution. Relationships may not relate to the subject matter of this manuscript. For more information about ASCO's conflict of interest policy, please refer to www.asco.org/rwc or ascopubs.org/po/author-center.

Open Payments is a public database containing information reported by companies about payments made to US-licensed physicians (Open Payments).

Presented in part at the Scientific Workshop at the American Society of Hematology Annual Meeting, San Diego, CA, December 8, 2023.

Supported by the National Cancer Institute of the National Institutes of Health under award number CA225617 (D.I.N.).

Julie Gold

Employment: Everly Health

Kit Cheng

Honoraria: GlaxoSmithKline, bioTheranostics

Johnson M. Liu

Employment: New York Blood Center

No other potential conflicts of interest were reported.
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