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NPJ Precis Oncol
NPJ Precis Oncol
NPJ Precision Oncology
2397-768X
Nature Publishing Group UK London

39232122
684
10.1038/s41698-024-00684-w
Case Report
Efficacy of PARP inhibitor therapy after targeted BRAF/MEK failure in advanced melanoma
Phillipps Jordan 1
http://orcid.org/0009-0000-4271-7868
Nassief George 1
Morecroft Renee 1
Adeyelu Tolulope 2
Elliott Andrew 2
Abdulla Farah 2
http://orcid.org/0000-0002-6842-2563
Vanderwalde Ari 2
Park Soo 3
Butt Omar 1
Zhou Alice 1
http://orcid.org/0000-0002-7178-8777
Ansstas George gansstas@wustl.edu

1
1 https://ror.org/01yc7t268 grid.4367.6 0000 0004 1936 9350 Division of Medical Oncology, Department of Medicine, Washington University in Saint Louis, Saint Louis, MO 63130 USA
2 https://ror.org/04wh5hg83 grid.492659.5 0000 0004 0492 4462 Caris Life Sciences, Phoenix, AZ USA
3 https://ror.org/0168r3w48 grid.266100.3 0000 0001 2107 4242 University of California San Diego, San Diego, CA USA
5 9 2024
5 9 2024
2024
8 18729 2 2024
28 8 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
Modern advancements in targeted therapy and immunotherapy have significantly improved survival outcomes for advanced melanoma; however, there remains a need for novel approaches to overcome disease progression and treatment resistance. In recent years, PARPi therapy has shown great promise both as a single regimen and in combination with other therapeutics in melanoma. Here, we describe three unique cases of advanced BRAF V600 mutated melanoma that progressed on targeted BRAF/MEK agents that subsequently exhibited partial to near-complete responses to combinatory PARPi and BRAF/MEK inhibitors. This highlights both a potential synergy underlying this combinatory approach and its efficacy as a treatment option for patients with advanced melanoma refractory to targeted and/or immunotherapies. Prospective clinical trials are needed to explore this synergic effect in larger melanoma cohorts to investigate this combination for treating refractory advanced melanoma.

Subject terms

Melanoma
Cancer therapy
issue-copyright-statement© Springer Nature Limited 2024
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pmcIntroduction

Melanoma, the deadliest type of skin cancer, accounts for nearly 2% of all global cancer diagnoses and remains the fifth most common cancer in the US1. Alarmingly, the prevalence of the disease has increased drastically in recent years, with over 100,000 new cases and 8,000 deaths expected in 20242. However, melanoma mortality, especially from advanced/metastatic disease, has dropped significantly due to modern advancements in immunotherapy and targeted therapies (e.g., BRAF, MEK, MAPK)1–4. Activating mutations in BRAF, which are present in about 50% of metastatic melanoma, are recognized as predictive biomarkers for targeted therapies in patients with advanced melanoma. Furthermore, nearly 50% of patients with advanced melanoma treated with immunotherapies, such as combination ipilimumab and nivolumab, remain alive at 7.5 years, a vast improvement over the prior era of cytotoxic chemotherapies and cytokine treatment5. In addition, the NEMO trial demonstrated that binimetinib, a MEK inhibitor, contributed to a significantly longer progression-free survival (2.8 months) compared to dacarbazine (1.5 months) in patients with NRAS-mutant advanced melanoma6. However, resistance and intolerance to both immunotherapy and targeted therapies are common and remain a challenge in advanced melanoma treatment7–9. Thus, novel therapeutics or combinatory approaches are required to overcome resistance and disease progression, especially in cases of advanced disease.

Poly (ADP-Ribose) Polymerase inhibitors (PARPi) have emerged as a promising treatment in cancers with alterations in homologous recombination repair (HRR) genes, a type of DNA damage repair (also termed HR-DDR). PARPi treatments work by creating double-stranded DNA (dsDNA) breaks that, under normal physiology, are repaired via homologous recombination (HR) mechanisms. However, homologous recombination deficient (HRD) cells cannot repair dsDNA breaks and thus undergo cell death via synthetic lethality. HRD can occur due to the presence of HR-DDR pathway alterations, such as gene mutations in BRCA1, BRCA2, ATM, CHEK2, and PALB210–12. HR-DDR mutations in melanoma are common, with frequencies ranging from 18.1% to upwards of 41%13,14. In general, patients with advanced melanoma without HR-DDR mutations respond poorly to PARPi15–18, however, there are reports of favorable responses to PARPi in patients harboring HR-DDR mutations17–19. Furthermore, the addition of PARPi to immune checkpoint inhibitors (ICI), such as anti-PD1 therapy, has been proposed due to the immune priming effects of PARPi therapy, such as increasing PD-L1 expression and genomic instability, which further promote immune response to ICI and antitumor activity20,21. Clinically, this synergism was seen in a study that observed combinatory ICI and PARPi therapy to demonstrate a consistent objective response rate in a cohort of patients with ovarian cancer22. In addition, the synergism between PARPi and BRAF/MEK inhibition has been explored preclinically in melanoma showing promise in using this combinatory approach23,24. These findings warrant further research on the efficacy and safety of PARPi as standalone therapy or in combination with other therapeutic options in treating patients with melanoma.

Here, we report three unique cases of advanced melanoma refractory to BRAF/MEK inhibition that subsequently demonstrated favorable clinical responses to PARPi therapy. We also assessed treatment response through longitudinal, tumor-informed circulating tumor DNA (ctDNA) monitoring when possible. These results help contextualize the efficacy of combinatory PARPi and targeted therapy within a framework of overcoming treatment resistance in patients with advanced melanoma.

Results

We identified three patients at Siteman Cancer Center of Washington University School of Medicine who presented for management of metastatic melanoma. All three patients harbored an activating mutation in the MAP kinase pathway and most (two of the patients) harbored an alteration in HR pathways. All patients initially progressed on immunotherapy as well as BRAF/MEK inhibitor (BRAF/MEKi) therapy, after which a PARPi was added with significant clinical responses (one complete response [CR] and two partial responses [PR] based on Recist 1.1 criteria25). Treatments were well-tolerated. Patient demographics and clinical data are reported in Table 1.Table 1 Patient demographics and clinical data

	Patient 1	Patient 2	Patient 3	
Demographics	60-year-old, Male, Caucasian (non-Hispanic)	70-year-old, Male, Caucasian (non-Hispanic)	55-year-old, Female, Caucasian (ethnicity not specified)	
PMHx	None	Barrett’s esophagus, hypertension, hyperlipidemia, adrenal adenoma (unchanged)	Migraines, hypertension	
Family History	No melanoma or non-melanoma skin cancers	No melanoma or non-melanoma skin cancers	No melanoma or non-melanoma skin cancers	
Melanoma Location and Pathology	Primary: right shin

Metastasis: LN (right iliac, retroperitoneal)

Subtype: nodular

	Primary: mid-upper back Metastasis: brain, lung, heart, adrenal glands, superficial soft tissue/muscle and pelvic

Subtype: NOS

	Primary: posterior right calf

Metastasis: LN (right femoral) and lung

Subtype: nodular

	
Stage at diagnosis	Stage IIIC (pT3, pN3b, M0)	Stage IV	Stage IIB (pT3b, N0)	
Therapies before BRAF/MEKi therapy and response	1. WLE and SLNB

2. Adjuvant pembrolizumab 200 mg q3 weeks then recurrence after 33.2 months

3. Ipilimumab 3 mg/kg q3 weeks /nivolumab 1 mg/kg q2 weeks for 0.7mths (discontinued due to grade 3 ICI-associated CIPD)

4. Resection and retroperitoneal LN dissection

	1. Ipilimumab 3 mg/kg q3 weeks + nivolumab 1 mg/kg q4 weeks (discontinued after 1 month due to grade 3 ICI-associated pneumonitis)	1. WLE of right leg and SLNB

2. Resection of right femoral LN due to recurrence (Stage IIIC)

3. High dose adjuvant ipilimumab 10 mg/kg q3 weeks (PD after 3 months)

4. Pembrolizumab 200 mg q3 weeks for 19mths (no response)

5. Atezolizumab 840 mg q2 weeks + oral cobimetinib 40 mg PO QD on clinical trial (PR)

6. Atezolizumab 1200 mg every 3 weeks off trial for 4.5mths

	
BRAF/MEKi therapy	Regimen: binimetinib 45 mg PO BID

Duration: 9.1 months

Response: PD

AE: acneiform rash

	Regimen: encorafenib 450 mg PO QD + binimetinib 45 mg PO BID

Duration: 9.5 months

Response: MR

AE: hypothyroidism

	Regimen: encorafenib 450 mg PO QD + binimetinib 45 mg PO BID

Duration: 2.3 months

Response: MR

AE: drug-induced diabetes mellitus

	
Interval therapy	None	1. Gamma knife to the right parietal lesion

2. LITT to the right temporal and occipital lobe lesions

	1. Nivolumab 1 mg/kg q4 weeks + encorafenib/binimetinib (MR after 3.7 months)

2. interval T-VEC + nivolumab (PD after 4.6 months)

3. Restarted nivolumab + encorafenib/binimetinib (PD as a new left lower lobe pulmonary nodule after 18 months)

	
PARPi therapy	Regimen: olaparib 300 mg PO BID + binimetinib, radiation therapy to abdominal lymph nodes

Time to response: 6 months

Duration: 15 months and ongoing

Response: CR

Adverse effect: none

	Regimen: olaparib 300 mg PO BID + encorafenib/binimetinib with intermittent brain radiation therapy and LITT

Time to response: 2.6 months

Duration: 12.6 months and ongoing

Response: PR

Adverse effect: none

	Regimen: olaparib 300 mg PO BID + encorafenib/binimetinib + nivolumab

Time to response: 3.5 months

Duration: 7 months and ongoing

Response: PR

Adverse effect: nausea and fatigue (Olaparib reduced to every other day)

	
PMHx past medical history, BRAF/MEKi BRAF/MEK inhibitor, PARPi PARP inhibitor, PR partial response, CR complete response, NOS not otherwise specified, PD progressive disease, MR mixed response, WLE wide local excision, SLNB sentinel lymph node biopsy, LN lymph node, ICI immune checkpoint inhibitor, CIPD chronic inflammatory demyelinating polyradiculoneuropathy, LITT laser interstitial thermal therapy, T-VEC talimogene-laherparepvec, AE adverse effects.

Patient 1

A 60-year-old Caucasian male presented for management of a primary nodular melanoma of the right shin with lymph node (right iliac and retroperitoneal) metastases. He noticed a pigmented lesion on his right shin which ulcerated and underwent wide local excision (WLE) and sentinel lymph node biopsy (SLNB) which revealed Stage IIIC (pT3 pN3b M0) disease. Molecular profiling detected mutations in NRAS, TERT promoter, EZH2, ARID2, and CHD2 with a low genomic loss of heterozygosity (LOH) and tumor mutational burden (TMB) (Table 2). Before MEK inhibitor monotherapy, he received adjuvant pembrolizumab (200 mg q3 weeks), followed by ipilimumab (3 mg/kg q3 weeks) /nivolumab (1 mg/kg q2 weeks) due to metastatic recurrence in the right iliac and retroperitoneal lymph nodes. Unfortunately, after 2 cycles, he developed grade 3 immune checkpoint inhibitor (ICI)-associated neuropathy and proceeded to surgical resection. Due to the NRAS mutation, the patient was subsequently started on binimetinib (45 mg PO BID) with no evidence of radiographic response by 9.1 months and persistent ctDNA-positivity (Fig. 1A). Olaparib(300 mg PO BID), a PARPi, was added to binimetinib along with radiation therapy (35 Gy in 5 fractions) to abdominal lymph nodes, resulting in a partial response (PR) after 6 months and a complete response (CR) after 15 months, which has persisted at last follow up and correlated with sustained ctDNA clearance (Fig. 1A). No serious adverse events to MEKi or PARPi therapy occurred.Table 2 Melanoma tumor genetic and molecular profiling

	Patient 1	Patient 2	Patient 3	
Mutations	NRAS- pathogenic variant p.Q61K, exon 3; VAF 37%

TERT promoter- pathologic variant; VAF 33%

EZH2- pathological variant p.Y646N, exon 16; VAF 32%

ARID2- pathogenic variant p.W264, exon 8; VAF 17%

	BRAF- pathologic variant p.V600K, exon 15; VAF 70%

ATM-deletion

IDH1-pathologic variant p.R132C, exon 4; VAF 45%

MGA-pathologic variant p.R2396, exon 19; VAF 43%

MRE11-deletion

TERT promoter-pathologic variant, exon 0; VAF 63%

ARID2- variant of uncertain significance p.R652K, exon 15; VAF 38%

RET- variant of uncertain significance p.5444 F, exon 7; VAF 42%

POLE-deletion

	BRAF- pathologic variant p.V600K, exon 15; VAF 33%

NTRK1- variant of uncertain significance p.G307E, exon 8; VAF 21%

TERT promoter-pathogenic variant; VAF 67%

B2M-pathogenic variant p.L85fs, exon 2; VAF 39%

WRN-pathogenic variant p.Q1010, exon 25; VAF 19%

	
MSI	Stable	Stable	Stable	
MMR status	Intact	Intact	Intact	
TMB (mutations per Mb)	Low (6)	High (21)	High (11)	
LOH	Low (8%)	Low (5%)	Low (12%)	
PD-L1	80%	0%	0%	
TMB tumor mutational burden, MSI microsatellite instability, MMR mismatch repair, LOH loss of heterozygosity, PD-L1 Programmed death ligand 1, VAF Variant allele frequency, Mb megabase.

Fig. 1 Timeline correlating treatment and clinical response (using ctDNA and imaging) for patients 1 and 3.

A Patient 1. Top: CT-imaging 3 months after Olaparib initiation demonstrating resolution of a right common iliac node (previously 17 mm in short axis). Bottom: Graph depicting a timeline of administered systemic therapy and ctDNA changes during surveillance. White circle – ctDNA-negative; Black circle – ctDNA-positive; red triangle – progressive disease on imaging; light green triangle – partial response on imaging; dark green triangle – complete response on imaging; light purple rectangle – Binimetinib; light blue rectangle – combinatory Binimetinib and Olaparib; vertical dark blue line – initiation of radiotherapy. B Patient 3. Top: CT-imaging 3 months after Olaparib initiation demonstrating interval decrease in the size of a left lower lobe pulmonary nodule measuring 1.1 cm (previously measured 1.4 cm). Bottom: Graph depicting a timeline of administered systemic therapy and ctDNA changes during surveillance. Black circle – ctDNA-positive; red triangle – progressive disease on imaging; light green triangle – partial response on imaging; dark green triangle – complete response on imaging; light yellow rectangle – combinatory T-VEC and Nivolumab; purple rectangle – combinatory Encorafenib, Binimetinib, and Nivolumab; bright blue rectangle – combinatory Olaparib, Encorafenib, Binimetinib, and Nivolumab.

Patient 2

A 70-year-old Caucasian male presented for management of a mid-upper back primary melanoma with widespread metastases (Table 1). He presented to the emergency department with aphasia and was found to have a left temporal brain lesion with biopsy revealing malignant melanoma (type not otherwise specified). Notably, a mutation in ATM (HR-DDR gene) was present amongst other mutations including BRAF V600K, TERT promoter, and ARID2; LOH was low and TMB was high (Table 2). He started treatment with ipilimumab (3 mg/kg q3 weeks)/nivolumab (1 mg/kg q4 weeks); however, this was discontinued after one cycle due to grade 3 ICI-associated pneumonitis. Encorafenib (450 mg PO QD)/binimetinib (45 mg PO BID) was then initiated with an overall mixed response and treatment-associated hypothyroidism after 9.5 months of therapy. He then underwent treatment of three brain metastases (gamma knife radiosurgery for parietal lesion; laser interstitial thermal therapy (LITT) for right temporal and occipital lesions). He then started olaparib (300 mg PO BID) and radiation therapy (stereotactic radiosurgery [SRS, 1 fraction, 21 Gy] to a new frontal lesion; LITT and fractionated SRS [5 fractions, 30 Gy] to right occipital and parietal lesions). He achieved a partial response after 2.6 months of PARPi therapy with intermittent local therapies to brain metastases, which has persisted at his last follow-up (12.6 months). No other adverse events to BRAF/MEKi or PARPi therapy occurred.

Patient 3

A 55-year-old Caucasian female presented for management of a Stage IIB nodular melanoma of the right calf. She underwent WLE and SLNB but later had recurrence in the right femoral lymph nodes that was treated with lymph node dissection and high-dose adjuvant ipilimumab (10 mg/kg q3 weeks) for BRAF wild-type disease. Unfortunately, she developed a second recurrence (new right groin mass) which was refractory to pembrolizumab (200 mg every 3 weeks) after 19 months of therapy. Subsequently, she was enrolled in a clinical trial with atezolizumab (840 mg q2 weeks)/cobimetinib (40 mg PO QD) followed by off-trial atezolizumab (1200 mg q3 weeks) due to clinical benefit seen despite trial closure. A repeat lymph node biopsy revealed tumor mutations including BRAF V600K, NTRK1, TERT promoter, B2M, and WRN (HR-DDR gene) with a low LOH and high TMB (Table 2). Due to disease progression and a newly acquired BRAF mutation, she was started on encorafenib (450 mg PO QD)/ binimetinib (45 mg PO BID) but had a mixed response (after 2.3 months), thus nivolumab (1 mg/kg q4 weeks) was added to her regimen. She still only showed a mixed response after 3.7 months of therapy, so BRAF/MEKi was held while she received nivolumab with intralesional talimogene-laherparepvec (T-VEC), to which she experienced disease progression (after an additional 4.6 months). She then restarted combination therapy with nivolumab and encorafenib/binimetinib (as it was thought to have contained her disease), however, she again experienced disease progression (new left lower lobe pulmonary nodule) with a concurrent rise in ctDNA levels after 18 months (Fig. 1B). Consequently, olaparib (300 mg PO BID) was added to her regimen, and she achieved a partial response after 3.5 months along with a marked reduction in ctDNA levels (Fig. 1B), which has persisted at last follow-up. Notably, she was not taking her Olaparib as prescribed for two months (due to associated nausea and fatigue), so it was reduced to every other day at the last follow-up visit. No other adverse events to BRAF/MEKi or PARPi therapy were reported.

Discussion

This case series reviewed three patients with advanced melanoma refractory to targeted therapy (BRAF/MEK inhibition) who subsequently responded to the addition of PARPi therapy (2 PR, 1 CR). Treatment was generally well-tolerated, and responses were seen rapidly within 2.6–6 months of initiating PARPi therapy.

PARPi is an emerging class of cancer therapeutics, and patient selection remains crucial in personalizing care and overcoming prior treatment resistance. HR-DDR pathway gene mutations (e.g., BRCA1/2, CHEK2, ATM – see Table 3 for a comprehensive list) and high HRD status (measured as a composite score from a single or multiple genomic instability measures such as LOH26) have been identified as biomarkers for PARPi therapy in patients with melanoma. We have previously described successful clinical responses to PARPi in melanoma harboring HR-DDR gene mutations19 or high HRD status without HR-DDR gene mutations27,28. Additionally, we previously presented an advanced acral melanoma patient without traditional HR-DDR mutations achieving a complete response to PARPi following ICI failure. This patient had an EMSY amplification, which has been implicated in being involved in the HR-DDR pathway29. These reports suggest that a multifactorial basis underlies the response to PARPi therapy in melanoma. Interestingly, in the present study, all patients had low HRD status (as determined by genomic LOH), yet all patients still achieved significant clinical responses upon the addition of PARPi therapy, which corresponded to ctDNA clearance or a marked decrease in ctDNA levels when such testing was performed. Importantly, patients 2 and 3 harbored mutations in ATM and WRN, respectively, which are less commonly involved in the HR-DDR pathway and could potentially help explain the partial response to combinatory treatment. Nonetheless, these results provide real-world experience supporting the use of PARPi in patients with HR-DDR pathway gene mutations. Furthermore, these results provide clinical experience supporting a possible synergistic interaction between PARPi and targeted therapy (e.g., BRAF/MEK inhibition) in patients with advanced melanoma. However, more studies are needed to confirm this synergism preclinically and clinically in larger cohorts.Table 3 Common mutations affecting homologous recombination pathways

Homologous Recombination DNA Damage Response	
Genes29,45	ARID1A, ARID1B, ATM, ATR, ATRX, BAP1, BARD1, BLM, BRCA1, BRCA2, BRIP1, CHEK2, EMSY, FANCA/C/D2/E/F/G/L, MRE11, NBN, PALB2, RAD50, RAD51, RAD51B/C, WRN	

The synergism of PARPi and BRAK/MEK inhibitors is not well described in the literature, however, several preclinical studies evaluate this relationship. Frohlich et al. demonstrated that melanoma cells resistant to MAPK inhibitors (MAPKi) were highly susceptible to PARPi treatment in vitro and in vivo via a synergistic effect of synthetic lethality, causing reduced melanoma cell proliferation and migration23. Interestingly, the authors observed that MAPKi-resistant melanoma cells had exhibited lower basal ATM expression, theorizing that these cells had decreased HRR activity leading to a reduced repair of dsDNA caused by PARPi and suggesting melanoma cell ATM expression as a potential novel biomarker for PARPi therapy response23. Furthermore, Ferretti et al. observed that PARPi treatment had restored sensitivity to MAPKi in melanoma cells resistant to MAPKi regardless of DDR24. The authors noted that PARPi treatment causes transcriptomic and epigenetic changes that reverse epithelial-mesenchymal transition-like phenotype switching, a leading cause for therapeutic resistance24,30–32, redirecting melanoma cells to a proliferative and sensitive state to MAPKi. Additionally, Maertens et al. demonstrated that MAPKi suppressed several HR pathway genes in MAPKi-sensitive melanoma cells, ultimately inducing an increased HR-defect signature in treated cells. Importantly, this BRCA-ness phenotype is a biomarker for PARPi, supported by the authors observing increased cytotoxicity in melanoma MAPKi-sensitive cell lines after subsequent PARPi treatment (versus no potent cytotoxic effect when using PARPi monotherapy)33.

Furthermore, Maertens et al. analyzed transcriptional data between MAPKi-sensitive melanoma cell lines and Histone deacetylase (HDAC) inhibitors and found elevated MGMT gene expression (9-fold). Moreover, they discovered that MGMT-expressing cells were sensitive to this combinatory treatment, whereas cell lines lacking MGMT expression were not responsive to this treatment approach. The authors highlighted that MGMT expression could be utilized as a biomarker for response to combinatory MAPKi and HDAC inhibitors. Additionally, they observed high MGMT-expressing melanomas to possess broad DNA repair gene defects, and ultimately, an HRD gene signature33. Unfortunately, MGMT expression for the patients in this study is not available. Nonetheless, future studies are warranted to elucidate the role of MGMT as a biomarker in melanoma response to combinatory MAPKi and PARPi.

These preclinical findings, together with our clinical results, strongly suggest a synergistic effect of MAPKi and PARPi as a combinatory treatment option in patients with advanced melanoma, especially those with immunotherapy resistance. In addition, PARPi were found to increase cell-intrinsic immunity by creating cytoplasmic chromatin fragments that further activate the cGAS/STING pathway, increase cytokine production, and activate downstream IFN signaling in DNA damage response-deficient tumor cells34. Moreover, PARPi have shown promise as radiosensitizers in patients with various cancers, including breast cancer and melanoma35,36, which could also help explain the responses demonstrated by patients 1 and 2.

Interestingly, patients 1 and 2 harbored ARID2 mutations, which is a member of the SWI/SNF family of chromatin remodeling complexes. ARID2 was implicated to be indirectly involved in the DDR pathways37–39, and deficiencies in the gene have been associated with response to ICI in melanoma40. Interestingly, Moreno et al. highlighted in a study how lung cancer cells with ARID2 deficiencies were associated with sensitivity to PARPi treatment41. These results highlight the potential utility of ARID2 as a biomarker for PARPi response, however, more investigation is needed to confirm these results in larger melanoma cohorts.

In conclusion, the results of this study support a growing body of evidence on the efficacy and safety of using PARPi in treating advanced melanoma that is refractory to targeted BRAF/MEK inhibition and/or ICI. Our study is limited by a small sample size and its retrospective nature. Larger, randomized controlled trials are required to further elucidate synergistic mechanisms and establish the potential benefit of PARPi in patients with advanced melanoma refractory to immunotherapy and targeted therapies.

Methods

Materials

Formalin-fixed paraffin-embedded (FFPE) samples were prepared from patient biopsies and submitted to a commercial, CLIA-certified laboratory for molecular profiling (Caris Life Sciences, Phoenix, AZ). Samples were analyzed by next-generation sequencing (NGS) of DNA (whole exome sequencing, WES) and RNA (whole transcriptome sequencing, WTS), along with immunohistochemistry (IHC).

DNA next generation sequencing (NGS)

In preparation of the samples for molecular testing, tumor enrichment was done by harvesting targeted tissues using manual microdissection techniques. Genomic DNA was extracted from FFPE tissue samples and subjected to NGS using the NovaSeq 6000 Platforms (Illumina, Inc. San Diego, CA). A custom SureSelect XT assay (Agilent Technologies, Santa Clara, CA) was utilized to enrich exonic regions 592 whole-gene targets. For tumor sample sequenced on the Novaseq 6000 platform, more than 700 clinically relevant genes were assessed. All variants were detected with >99% confidence based on allele frequency and amplicon coverage, with an average sequencing depth of coverage of >500 and an analytic sensitivity threshold established of 5% for variant calling. Certified molecular geneticists examined the identified genomic variants and categorized them in alignment with the standards set by the American College of Medical Genetics and Genomics (ACMG).

TMB was measured by counting all non-synonymous missense, nonsense, in-frame insertion/deletion, and frameshift mutations found per tumor that had not been previously described as germline alterations in dbSNP151, Genome Aggregation Database (gnomAD) databases, or benign variants identified by Caris’s geneticists. High TMB (TMB-H) was defined by a cut-off of ≥10 mutation/megabase (mut/MB) based on the KEYNOTE-158 pembrolizumab trial, where it was shown that patients with ≥10 mut/MB had increased response rates compared to those with <10 mut/MB42. To calculate the genomic loss of heterozygosity (LOH), LOH in approximately 250k single nucleotide polymorphisms (SNPs) within segmented autosomal chromosomes was calculated. LOH was based on the percentage of all 552 segments with observed LOH (High ≥16%, Low <16%; if fewer than 3000 SNPs were read, the test was reported as indeterminate).

Whole transcriptomic sequencing

Formalin-fixed paraffin-embedded (FFPE) tissue sections mounted on glass slides underwent staining with nuclear fast red (NFR). Regions that contained a minimum of 10% tumor content were delineated for manual microdissection and subsequent mRNA extraction. Whole transcriptome sequencing (WTS) was executed using the Illumina NovaSeq platform (Illumina, Inc., San Diego, CA) along with the Agilent SureSelect Human All Exon V7 bait panel (Agilent Technologies, Santa Clara, CA), and the resulting data reported transcripts per million (TPM).

Immunohistochemistry (IHC)

IHC was conducted on complete sections of formalin-fixed paraffin-embedded (FFPE) tissues mounted on glass slides. The slides underwent staining employing automated staining methods as directed by the manufacturer. These procedures were meticulously optimized and confirmed to meet the standards outlined by CLIA/CAO and ISO. PD-L1 expression was determined using primary antibody SP142 (Spring Biosciences, Pleasanton, CA, USA), with a positive threshold of ≥2 + stain intensity and ≥ 5% percentage of cells stained.

Deficient mismatch repair/microsatellite instability-high (dMMR/MSI-H)

dMMR/MSI-H was determined by a combination of immunohistochemistry (IHC) using antibodies for MLH1 (M1 antibody), MSH2 (G2191129 antibody), MSH6 (44 antibody), and PMS2 (EPR3947 antibody) from Ventana Medical Systems (Tucson, AZ), and next-generation sequencing (NGS). The outcomes from these platforms are mostly in agreement, as previously described43. In instances where conflicting results emerged, the order of priority for determining the MSI/MMR status of the tumor was IHC, followed by NGS.

Personalized, tumor-informed ctDNA testing

Longitudinal ctDNA testing was performed in two of the three patients at the discretion of the treating provider. A personalized, tumor-informed, 16-plex PCR assay (SignateraTM, Natera, Inc.) was used for the detection and quantification of ctDNA, as previously described44. Briefly, whole-exome sequencing (WES) was performed on FFPE tumor tissue and matched normal blood samples. A set of up to 16 patient-specific somatic single nucleotide variants (SNVs) from WES results were selected for multiplex PCR (mPCR). The mPCR primers targeting the personalized SNVs were used to track ctDNA in the corresponding patients’ plasma samples. Plasma samples with ≥ 2 SNVs detected above a predefined confidence threshold were defined as ctDNA-positive. ctDNA concentration was reported in mean tumor molecules (MTM)/mL of plasma.

Patient consent

The authors have obtained written patient consent from all patients to publish the details in this study. This study was approved by the Washington University IRB.

Acknowledgements

The authors would like to thank the patients for their consent to publish their medical history. The authors would also like to thank Charuta Palsuledesai (and the Natera team) for their expertise on ctDNA methodology and compiling Fig. 1. This study received no funding.

Author contributions

J.P., G.N., and R.M. performed literature review, extracted/analyzed patient data, and wrote/revised the manuscript. T.A., A.E., F.A., A.V., S.P., O.B. provided expertise in editing and revising the manuscript. G.A. conceived the project idea and approved the final manuscript.

Data availability

The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.

Competing interests

The authors declare no competing interests.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

These authors contributed equally: Jordan Phillipps, George Nassief.
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References

1. Saginala K Barsouk A Aluru JS Rawla P Barsouk A Epidemiology of melanoma Med Sci. (Basel) 2021 9 63 34698235
Saginala, K., Barsouk, A., Aluru, J. S., Rawla, P. & Barsouk, A. Epidemiology of melanoma. Med Sci. (Basel) 9, 63 (2021).34698235
2. Melanoma Skin Cancer Research | Melanoma Studies. https://www.cancer.org/cancer/types/melanoma-skin-cancer/about/new-research.html.
3. Kahlon N Melanoma treatments and mortality rate trends in the US, 1975 to 2019 JAMA Netw. Open 2022 5 e2245269 10.1001/jamanetworkopen.2022.45269 36472871
Kahlon, N. et al. Melanoma treatments and mortality rate trends in the US, 1975 to 2019. JAMA Netw. Open 5, e2245269 (2022).36472871 10.1001/jamanetworkopen.2022.45269
4. Aggarwal P Knabel P Fleischer AB United States burden of melanoma and non-melanoma skin cancer from 1990 to 2019 J. Am. Acad. Dermatol. 2021 85 388 395 10.1016/j.jaad.2021.03.109 33852922
Aggarwal, P., Knabel, P. & Fleischer, A. B. United States burden of melanoma and non-melanoma skin cancer from 1990 to 2019. J. Am. Acad. Dermatol. 85, 388–395 (2021).33852922 10.1016/j.jaad.2021.03.109
5. Hodi FS Long-term survival in advanced melanoma for patients treated with nivolumab plus ipilimumab in CheckMate 067 JCO 2022 40 9522 9522 10.1200/JCO.2022.40.16_suppl.9522
Hodi, F. S. et al. Long-term survival in advanced melanoma for patients treated with nivolumab plus ipilimumab in CheckMate 067. JCO 40, 9522–9522 (2022).10.1200/JCO.2022.40.16_suppl.9522
6. Dummer R Binimetinib versus dacarbazine in patients with advanced NRAS-mutant melanoma (NEMO): a multicentre, open-label, randomised, phase 3 trial Lancet Oncol. 2017 18 435 445 10.1016/S1470-2045(17)30180-8 28284557
Dummer, R. et al. Binimetinib versus dacarbazine in patients with advanced NRAS-mutant melanoma (NEMO): a multicentre, open-label, randomised, phase 3 trial. Lancet Oncol. 18, 435–445 (2017).28284557 10.1016/S1470-2045(17)30180-8
7. Jerby-Arnon L A cancer cell program promotes T cell exclusion and resistance to checkpoint blockade Cell 2018 175 984 997.e24 10.1016/j.cell.2018.09.006 30388455
Jerby-Arnon, L. et al. A cancer cell program promotes T cell exclusion and resistance to checkpoint blockade. Cell 175, 984–997.e24 (2018).30388455 10.1016/j.cell.2018.09.006
8. Brastianos PK Pembrolizumab in brain metastases of diverse histologies: phase 2 trial results Nat. Med. 2023 29 1728 1737 10.1038/s41591-023-02392-7 37268724
Brastianos, P. K. et al. Pembrolizumab in brain metastases of diverse histologies: phase 2 trial results. Nat. Med. 29, 1728–1737 (2023).37268724 10.1038/s41591-023-02392-7
9. Pires da Silva I Site-specific response patterns, pseudoprogression, and acquired resistance in patients with melanoma treated with ipilimumab combined with anti-PD-1 therapy Cancer 2020 126 86 97 10.1002/cncr.32522 31584722
Pires da Silva, I. et al. Site-specific response patterns, pseudoprogression, and acquired resistance in patients with melanoma treated with ipilimumab combined with anti-PD-1 therapy. Cancer 126, 86–97 (2020).31584722 10.1002/cncr.32522
10. Boussios S Combined Strategies with Poly (ADP-Ribose) Polymerase (PARP) inhibitors for the treatment of ovarian cancer: a literature review Diagnostics (Basel) 2019 9 87 10.3390/diagnostics9030087 31374917
Boussios, S. et al. Combined Strategies with Poly (ADP-Ribose) Polymerase (PARP) inhibitors for the treatment of ovarian cancer: a literature review. Diagnostics (Basel) 9, 87 (2019).31374917 10.3390/diagnostics9030087
11. LaFargue CJ Dal Molin GZ Sood AK Coleman RL Exploring and comparing adverse events between PARP inhibitors Lancet Oncol. 2019 20 e15 e28 10.1016/S1470-2045(18)30786-1 30614472
LaFargue, C. J., Dal Molin, G. Z., Sood, A. K. & Coleman, R. L. Exploring and comparing adverse events between PARP inhibitors. Lancet Oncol. 20, e15–e28 (2019).30614472 10.1016/S1470-2045(18)30786-1
12. Phillipps J Zhou AY Butt OH Ansstas G PARP inhibition and immunotherapy: a promising duo in fighting cancer Transl. Cancer Res. 2023 12 2433 2437 10.21037/tcr-23-726 37859734
Phillipps, J., Zhou, A. Y., Butt, O. H. & Ansstas, G. PARP inhibition and immunotherapy: a promising duo in fighting cancer. Transl. Cancer Res. 12, 2433–2437 (2023).37859734 10.21037/tcr-23-726
13. Heeke AL Prevalence of homologous recombination-related gene mutations across multiple cancer types JCO Precis. Oncol. 2018 2018 PO.17.00286 30234181
Heeke, A. L. et al. Prevalence of homologous recombination-related gene mutations across multiple cancer types. JCO Precis. Oncol. 2018, PO.17.00286 (2018).30234181
14. Kim H The prevalence of homologous recombination deficiency (HRD) in various solid tumors and the role of HRD as a single biomarker to immune checkpoint inhibitors J. Cancer Res. Clin. Oncol. 2022 148 2427 2435 10.1007/s00432-021-03781-6 34510272
Kim, H. et al. The prevalence of homologous recombination deficiency (HRD) in various solid tumors and the role of HRD as a single biomarker to immune checkpoint inhibitors. J. Cancer Res. Clin. Oncol. 148, 2427–2435 (2022).34510272 10.1007/s00432-021-03781-6
15. Chan WY Brown LJ Reid L Joshua AM PARP inhibitors in melanoma-an expanding therapeutic option? Cancers (Basel) 2021 13 4520 10.3390/cancers13184520 34572747
Chan, W. Y., Brown, L. J., Reid, L. & Joshua, A. M. PARP inhibitors in melanoma-an expanding therapeutic option? Cancers (Basel) 13, 4520 (2021).34572747 10.3390/cancers13184520
16. Plummer R A phase II study of the potent PARP inhibitor, Rucaparib (PF-01367338, AG014699), with temozolomide in patients with metastatic melanoma demonstrating evidence of chemopotentiation Cancer Chemother. Pharm. 2013 71 1191 1199 10.1007/s00280-013-2113-1
Plummer, R. et al. A phase II study of the potent PARP inhibitor, Rucaparib (PF-01367338, AG014699), with temozolomide in patients with metastatic melanoma demonstrating evidence of chemopotentiation. Cancer Chemother. Pharm. 71, 1191–1199 (2013).10.1007/s00280-013-2113-1
17. Lau B Menzies AM Joshua AM Ongoing partial response at 6 months to olaparib for metastatic melanoma with somatic PALB2 mutation after failure of immunotherapy: a case report Ann. Oncol. 2021 32 280 282 10.1016/j.annonc.2020.11.006 33308898
Lau, B., Menzies, A. M. & Joshua, A. M. Ongoing partial response at 6 months to olaparib for metastatic melanoma with somatic PALB2 mutation after failure of immunotherapy: a case report. Ann. Oncol. 32, 280–282 (2021).33308898 10.1016/j.annonc.2020.11.006
18. Kiel, P. J., Radovich, M., Schneider, B. P. & Logan, T. F. Sustained exceptional response to poly (ADP-Ribose) polymerase inhibition plus temozolomide in metastatic melanoma with DNA repair deficiency. JCO Precis. Oncol.10.1200/PO.18.00150 (2018).
19. Khaddour K Ansstas M Visconti J Ansstas G Mutation clearance and complete radiologic resolution of immunotherapy relapsed metastatic melanoma after treatment with nivolumab and olaparib in a patient with homologous recombinant deficiency: any role for PARP inhibitors and checkpoint blockade? Ann. Oncol. 2021 32 279 280 10.1016/j.annonc.2020.10.602 33308899
Khaddour, K., Ansstas, M., Visconti, J. & Ansstas, G. Mutation clearance and complete radiologic resolution of immunotherapy relapsed metastatic melanoma after treatment with nivolumab and olaparib in a patient with homologous recombinant deficiency: any role for PARP inhibitors and checkpoint blockade? Ann. Oncol. 32, 279–280 (2021).33308899 10.1016/j.annonc.2020.10.602
20. Peyraud F Italiano A Combined PARP inhibition and immune checkpoint therapy in solid tumors Cancers (Basel) 2020 12 1502 10.3390/cancers12061502 32526888
Peyraud, F. & Italiano, A. Combined PARP inhibition and immune checkpoint therapy in solid tumors. Cancers (Basel) 12, 1502 (2020).32526888 10.3390/cancers12061502
21. Stewart RA Pilié PG Yap TA Development of PARP and immune-checkpoint inhibitor combinations Cancer Res. 2018 78 6717 6725 10.1158/0008-5472.CAN-18-2652 30498083
Stewart, R. A., Pilié, P. G. & Yap, T. A. Development of PARP and immune-checkpoint inhibitor combinations. Cancer Res. 78, 6717–6725 (2018).30498083 10.1158/0008-5472.CAN-18-2652
22. Konstantinopoulos PA Single-arm phases 1 and 2 trial of niraparib in combination with pembrolizumab in patients with recurrent platinum-resistant ovarian carcinoma JAMA Oncol. 2019 5 1141 1149 10.1001/jamaoncol.2019.1048 31194228
Konstantinopoulos, P. A. et al. Single-arm phases 1 and 2 trial of niraparib in combination with pembrolizumab in patients with recurrent platinum-resistant ovarian carcinoma. JAMA Oncol. 5, 1141–1149 (2019).31194228 10.1001/jamaoncol.2019.1048
23. Fröhlich LM PARP inhibitors effectively reduce MAPK inhibitor resistant melanoma cell growth and synergize with MAPK inhibitors through a synthetic lethal interaction in vitro and in vivo Cancer Res. Commun. 2023 3 1743 1755 10.1158/2767-9764.CRC-23-0101 37674529
Fröhlich, L. M. et al. PARP inhibitors effectively reduce MAPK inhibitor resistant melanoma cell growth and synergize with MAPK inhibitors through a synthetic lethal interaction in vitro and in vivo. Cancer Res. Commun. 3, 1743–1755 (2023).37674529 10.1158/2767-9764.CRC-23-0101
24. Ferretti LP Combinatorial treatment with PARP and MAPK inhibitors overcomes phenotype switch-driven drug resistance in advanced melanoma Cancer Res 2023 83 3974 3988 10.1158/0008-5472.CAN-23-0485 37729428
Ferretti, L. P. et al. Combinatorial treatment with PARP and MAPK inhibitors overcomes phenotype switch-driven drug resistance in advanced melanoma. Cancer Res 83, 3974–3988 (2023).37729428 10.1158/0008-5472.CAN-23-0485
25. Eisenhauer EA New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1) Eur. J. Cancer 2009 45 228 247 10.1016/j.ejca.2008.10.026 19097774
Eisenhauer, E. A. et al. New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). Eur. J. Cancer 45, 228–247 (2009).19097774 10.1016/j.ejca.2008.10.026
26. Abkevich V Patterns of genomic loss of heterozygosity predict homologous recombination repair defects in epithelial ovarian cancer Br. J. Cancer 2012 107 1776 1782 10.1038/bjc.2012.451 23047548
Abkevich, V. et al. Patterns of genomic loss of heterozygosity predict homologous recombination repair defects in epithelial ovarian cancer. Br. J. Cancer 107, 1776–1782 (2012).23047548 10.1038/bjc.2012.451
27. Khaddour K Ansstas M Ansstas G Clinical outcomes and longitudinal circulating tumor DNA changes after treatment with nivolumab and olaparib in immunotherapy relapsed melanoma with detected homologous recombination deficiency Cold Spring Harb. Mol. Case Stud. 2021 7 a006129 10.1101/mcs.a006129 34667064
Khaddour, K., Ansstas, M. & Ansstas, G. Clinical outcomes and longitudinal circulating tumor DNA changes after treatment with nivolumab and olaparib in immunotherapy relapsed melanoma with detected homologous recombination deficiency. Cold Spring Harb. Mol. Case Stud. 7, a006129 (2021).34667064 10.1101/mcs.a006129
28. Zhou A Determining PARP inhibition as a treatment strategy in melanoma based on homologous recombination deficiency–related loss of heterozygosity J. Natl Compr. Cancer Netw. 2023 21 688 693.e3 10.6004/jnccn.2022.7102
Zhou, A. et al. Determining PARP inhibition as a treatment strategy in melanoma based on homologous recombination deficiency–related loss of heterozygosity. J. Natl Compr. Cancer Netw. 21, 688–693.e3 (2023).10.6004/jnccn.2022.7102
29. Nassief G Butt O Zhou A Ansstas G PARPi therapy response in an acral melanoma patient with EMSY gene amplification JAAD Case Rep. 2024 48 59 61 10.1016/j.jdcr.2024.04.005 38778889
Nassief, G., Butt, O., Zhou, A. & Ansstas, G. PARPi therapy response in an acral melanoma patient with EMSY gene amplification. JAAD Case Rep. 48, 59–61 (2024).38778889 10.1016/j.jdcr.2024.04.005
30. Verfaillie A Decoding the regulatory landscape of melanoma reveals TEADS as regulators of the invasive cell state Nat. Commun. 2015 6 6683 10.1038/ncomms7683 25865119
Verfaillie, A. et al. Decoding the regulatory landscape of melanoma reveals TEADS as regulators of the invasive cell state. Nat. Commun. 6, 6683 (2015).25865119 10.1038/ncomms7683
31. Rambow F Marine J-C Goding CR Melanoma plasticity and phenotypic diversity: therapeutic barriers and opportunities Genes Dev. 2019 33 1295 1318 10.1101/gad.329771.119 31575676
Rambow, F., Marine, J.-C. & Goding, C. R. Melanoma plasticity and phenotypic diversity: therapeutic barriers and opportunities. Genes Dev. 33, 1295–1318 (2019).31575676 10.1101/gad.329771.119
32. Hoek KS In vivo switching of human melanoma cells between proliferative and invasive states Cancer Res. 2008 68 650 656 10.1158/0008-5472.CAN-07-2491 18245463
Hoek, K. S. et al. In vivo switching of human melanoma cells between proliferative and invasive states. Cancer Res. 68, 650–656 (2008).18245463 10.1158/0008-5472.CAN-07-2491
33. Maertens O MAPK pathway suppression unmasks latent DNA repair defects and confers a chemical synthetic vulnerability in BRAF-, NRAS-, and NF1-mutant melanomas Cancer Discov. 2019 9 526 545 10.1158/2159-8290.CD-18-0879 30709805
Maertens, O. et al. MAPK pathway suppression unmasks latent DNA repair defects and confers a chemical synthetic vulnerability in BRAF-, NRAS-, and NF1-mutant melanomas. Cancer Discov. 9, 526–545 (2019).30709805 10.1158/2159-8290.CD-18-0879
34. Chabanon RM PARP inhibition enhances tumor cell-intrinsic immunity in ERCC1-deficient non-small cell lung cancer J. Clin. Invest. 2019 129 1211 1228 10.1172/JCI123319 30589644
Chabanon, R. M. et al. PARP inhibition enhances tumor cell-intrinsic immunity in ERCC1-deficient non-small cell lung cancer. J. Clin. Invest. 129, 1211–1228 (2019).30589644 10.1172/JCI123319
35. Feng FY Targeted radiosensitization with PARP1 inhibition: optimization of therapy and identification of biomarkers of response in breast cancer Breast Cancer Res. Treat. 2014 147 81 94 10.1007/s10549-014-3085-5 25104443
Feng, F. Y. et al. Targeted radiosensitization with PARP1 inhibition: optimization of therapy and identification of biomarkers of response in breast cancer. Breast Cancer Res. Treat. 147, 81–94 (2014).25104443 10.1007/s10549-014-3085-5
36. Sun C PARP inhibitors combined with radiotherapy: are we ready? Front Pharm. 2023 14 1234973 10.3389/fphar.2023.1234973
Sun, C. et al. PARP inhibitors combined with radiotherapy: are we ready? Front Pharm. 14, 1234973 (2023).10.3389/fphar.2023.1234973
37. Lee H-S Park J-H Kim S-J Kwon S-J Kwon J A cooperative activation loop among SWI/SNF, gamma-H2AX and H3 acetylation for DNA double-strand break repair EMBO J. 2010 29 1434 1445 10.1038/emboj.2010.27 20224553
Lee, H.-S., Park, J.-H., Kim, S.-J., Kwon, S.-J. & Kwon, J. A cooperative activation loop among SWI/SNF, gamma-H2AX and H3 acetylation for DNA double-strand break repair. EMBO J. 29, 1434–1445 (2010).20224553 10.1038/emboj.2010.27
38. Ray A Human SNF5/INI1, a component of the human SWI/SNF chromatin remodeling complex, promotes nucleotide excision repair by influencing ATM recruitment and downstream H2AX phosphorylation Mol. Cell Biol. 2009 29 6206 6219 10.1128/MCB.00503-09 19805520
Ray, A. et al. Human SNF5/INI1, a component of the human SWI/SNF chromatin remodeling complex, promotes nucleotide excision repair by influencing ATM recruitment and downstream H2AX phosphorylation. Mol. Cell Biol. 29, 6206–6219 (2009).19805520 10.1128/MCB.00503-09
39. Niimi A Chambers AL Downs JA Lehmann AR A role for chromatin remodellers in replication of damaged DNA Nucleic Acids Res. 2012 40 7393 7403 10.1093/nar/gks453 22638582
Niimi, A., Chambers, A. L., Downs, J. A. & Lehmann, A. R. A role for chromatin remodellers in replication of damaged DNA. Nucleic Acids Res. 40, 7393–7403 (2012).22638582 10.1093/nar/gks453
40. Fukumoto T ARID2 Deficiency Correlates with the Response to Immune Checkpoint Blockade in Melanoma J. Invest. Dermatol. 2021 141 1564 1572.e4 10.1016/j.jid.2020.11.026 33333124
Fukumoto, T. et al. ARID2 Deficiency Correlates with the Response to Immune Checkpoint Blockade in Melanoma. J. Invest. Dermatol. 141, 1564–1572.e4 (2021).33333124 10.1016/j.jid.2020.11.026
41. Moreno T ARID2 deficiency promotes tumor progression and is associated with higher sensitivity to chemotherapy in lung cancer Oncogene 2021 40 2923 2935 10.1038/s41388-021-01748-y 33742126
Moreno, T. et al. ARID2 deficiency promotes tumor progression and is associated with higher sensitivity to chemotherapy in lung cancer. Oncogene 40, 2923–2935 (2021).33742126 10.1038/s41388-021-01748-y
42. Marabelle A Association of tumour mutational burden with outcomes in patients with advanced solid tumours treated with pembrolizumab: prospective biomarker analysis of the multicohort, open-label, phase 2 KEYNOTE-158 study Lancet Oncol. 2020 21 1353 1365 10.1016/S1470-2045(20)30445-9 32919526
Marabelle, A. et al. Association of tumour mutational burden with outcomes in patients with advanced solid tumours treated with pembrolizumab: prospective biomarker analysis of the multicohort, open-label, phase 2 KEYNOTE-158 study. Lancet Oncol. 21, 1353–1365 (2020).32919526 10.1016/S1470-2045(20)30445-9
43. Vanderwalde A Spetzler D Xiao N Gatalica Z Marshall J Microsatellite instability status determined by next-generation sequencing and compared with PD-L1 and tumor mutational burden in 11,348 patients Cancer Med. 2018 7 746 756 10.1002/cam4.1372 29436178
Vanderwalde, A., Spetzler, D., Xiao, N., Gatalica, Z. & Marshall, J. Microsatellite instability status determined by next-generation sequencing and compared with PD-L1 and tumor mutational burden in 11,348 patients. Cancer Med. 7, 746–756 (2018).29436178 10.1002/cam4.1372
44. Reinert T Analysis of plasma cell-free DNA by ultradeep sequencing in patients with stages I to III colorectal cancer JAMA Oncol. 2019 5 1124 1131 10.1001/jamaoncol.2019.0528 31070691
Reinert, T. et al. Analysis of plasma cell-free DNA by ultradeep sequencing in patients with stages I to III colorectal cancer. JAMA Oncol. 5, 1124–1131 (2019).31070691 10.1001/jamaoncol.2019.0528
45. Kim KB Prevalence of homologous recombination pathway gene mutations in melanoma: rationale for a new targeted therapeutic approach J. Invest. Dermatol. 2021 141 2028 2036.e2 10.1016/j.jid.2021.01.024 33610559
Kim, K. B. et al. Prevalence of homologous recombination pathway gene mutations in melanoma: rationale for a new targeted therapeutic approach. J. Invest. Dermatol. 141, 2028–2036.e2 (2021).33610559 10.1016/j.jid.2021.01.024
