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Pathology - Research and Practice

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PATHOLOGY RESEARCH PRACTICE

Whole genome profiling of primary and metastatic adrenocortical carcinoma unravels significant molecular events*

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Taylor Kalomeris a,1, Majd Al Assaad b,c,1, Jesus Delgado-de la Mora b,C, Gunes Gundem d, Max F. Levine ª, Baris Boyrazb, Jyothi Manohar ”, Michael Sigouros ”, Juan S. Medina-Martínez ª, Andrea Sboner b,c,e, Olivier Elemento ce, Theresa Scognamiglio b, Juan Miguel Mosquera b,c,

a Department of Pathology and Laboratory Medicine, NewYork-Presbyterian Hospital/Weill Cornell Medical Center, 1300 York Ave, New York, NY 10065, USA

b Department of Pathology and Laboratory Medicine, Weill Cornell Medicine, 1300 York Ave, New York, NY 10065, USA

Englander Institute for Precision Medicine, Weill Cornell Medicine, 413 East 69th Street, New York, NY 10021, USA

d Isabl Inc., 175 Greenwich Street, Fl 38, New York, NY 10007, USA

e Institute for Computational Biomedicine, Weill Cornell Medicine, 1305 York Avenue, New York, NY 10021, USA

ARTICLE INFO

Keywords:

Adrenocortical carcinoma Whole genome sequencing Tumor mutational burden Homologous recombination deficiency Oncocytic subtype

ABSTRACT

Adrenocortical carcinoma (ACC) is a rare, aggressive malignancy with limited treatment options and poor prognosis, with a 5-year survival rate of about 15 %. This study used whole genome sequencing to characterize the genomic landscape of five patients, one of them with both primary and metastatic samples. Key driver mutations were detected, including APC, JAK1, RFWD3 as well as other genes. Notably, a primary tumor harbored a RAD51 biallelic deleterious translocation, associated with homologous recombination deficiency signature. Large-scale copy neutral loss of heterozygosity (LOH) was identified in four tumors, three had TP53 mutations, with structural variants impacting genes as RB1, CDKN2A, and NF1. A genomic signature specific to mismatch repair was observed in a sample with MHS6 mutation. Two tumors presented novel fusions at TERT locus, including TERT :: ZNF521. Comparative analysis between conventional and oncocytic ACC subtypes revealed no significant differences in mutation load, microsatellite instability, or specific gene enrichment. This comprehensive WGS analysis broadens the spectrum of genomic alterations in ACC, highlighting potential molecular targets and differences across subtypes that may inform future therapeutic strategies.

1. Introduction

Adrenal cortical carcinoma (ACC) is a rare, aggressive endocrine carcinoma, arising from the adrenal cortex, with an annual estimated incidence of about one to two cases per million people [1,2]. ACC ac- counts for up to 14 % of incidentally detected adrenal neoplasms and often becomes invasive or metastasizes by the time of diagnosis [3]. The median survival time following onset and diagnosis is 3-4 years and is reduced to as little as 15 months in patients with metastatic disease [4-8].

Histopathologically, ACC’s can present as well circumscribed or infiltrative tumors, composed of nests or sheets of cells, frequently with

areas of necrosis [9]. Morphologic subtypes include conventional, myxoid, sarcomatoid and, oncocytic, the latter of which has previously shown genomic characteristics associated with a better prognosis [10, 11]. Immunophenotypically, ACC tumor cells are typically positive for steroidogenic factor (SF1), synaptophysin, Cam 5.2, inhibin, calretinin and Melan A (A103) [12].

The diagnosis of ACC is complex, particularly in early stages (I-II) when differentiating it from adrenocortical adenoma. The differentia- tion relies on a combination of variables and the application of different algorithms, depending on the case’s morphology. The Weiss score is most commonly used for classifying and prognosticating adrenal cortical neoplasm. It is based on nine pathologic criteria and, three are needed to

* Presented in part at the 112th Annual Meeting of the United States and Canadian Academy of Pathology in March 2023.

* Correspondence to: Department of Pathology and Laboratory Medicine Weill Cornell Medicine, New York, NY 10065, USA. E-mail address: jmm9018@med.cornell.edu (J.M. Mosquera).

1 T.K. and M.A.A. contributed equally to this work

https://doi.org/10.1016/j.prp.2024.155725

diagnose conventional ACC [13,14]. Other classifications can be applied in specific cases, such as the Lin-Weiss-Bisceglia system for the oncocytic subtype and the reticulum algorithm or Helsinki scoring system, which has shown to effectively predict metastatic potential in ACC [13,15-17].

In terms of treatment, the primary resection is the mainstay for localized ACC; however, recurrence occurs in almost 50 % of patients within 2-5 years [18-20]. For patients with advanced (stage III) or metastatic (stage IV) tumors, resection is not feasible, and treatment is limited to etoposide, doxorubicin, cisplatin and mitotane [21].

Currently, there is no established second-line therapy, and clear target therapies have yet to be identified or confirmed [22]. This gap in treatment options underscore the need for a deeper understanding of the molecular underpinnings of ACC’s. While some cases are associated with genetic syndromes as MEN1, Li-Fraumeni and Beckwith-Wiedemann, most ACC’s occur sporadically [23-25], and the roles of common drivers like TP53, CTNNB1, ZNRF3 and IGF2 [22,26-30], remains un- clear [31]. Recent studies involving genomic, methylation, and tran- scriptome analyses have investigated the existence of molecular subgroups of ACC’s, distinction between primary and metastatic sam- ples, and the molecular landscape linked to specific morphological subtypes [10,22,28]. These efforts primarily aim to identify prognostic alterations and potential targets for ACC therapies [32].

In this study, we performed whole-genome sequencing (WGS) on four primary and three metastatic tumors from five patients diagnosed with ACC. These included two oncocytic cases, one of which had both primary and metastasis. We employed the Isabl GxT analytic platform [33,34] to uncover mutations and structural variants affecting cancer-associated genes, genomic signatures that may have biologic significance and potential therapeutic targets. We then correlated the genomic data with pathologic and clinical annotation. These findings could help us understand the relationship between the pathology and genetics of ACC, providing crucial information to aid in treatment efforts including the development of targeted therapies.

2. Material and methods

2.1. Case selection and pathologic examination

The study was performed under institutional review board protocols WCM IRB# 1305013903 and # 1007011157. Seven tumor samples from five patients, three women and two men ranging from 27 to 63 years old at diagnosis, were studied. Patient demographics, clinical presentation, tumor size, treatment, follow-up interval, and outcome are summarized in Table 1. The hematoxylin and eosin (H&E) stained slides were reviewed by study pathologists.

2.2. Whole genome sequencing and data analysis

Whole genome sequencing (WGS) was performed at the New York Genome Center on tumor/normal pairs, as recently described [34,35].

Briefly, DNA from tumor and normal sample pairs was extracted from unstained, formalin-fixed paraffin-embedded (FFPE) tissue and quanti- fied by Maxwell® 16 FFPE Plus DNA kit (Promega, Cat# AS1135) on the Maxwell® 16 instrument (Promega, Madison, WI). Its quality and quantity were assessed using the Agilent Tapestation 4200 (Agilent Technologies) and Qubit Fluorometer (ThermoFisher).

Targeting 500 bp fragments, WGS libraries were prepared using the KAPA Hyper Library Preparation Kit (KAPABiosystems KK8502, KK8504) in accordance with the manufacturer’s instructions. The extracted DNA fragments were sheared using a Covaris LE220 sonicator, end-repaired, adenylated, ligated to Illumina sequencing adapters. Then they underwent size selection using bead-based methods and were amplified. Final libraries were quantified, assessed for quality and then sequenced on an Illumina Novaseq6000 sequencer using 2 × 150 base pair cycles. The sequencing reads were then aligned to a reference genome (hg19) using Burrows-Wheeler aligner [36]. We employed the Isabl GxT analytic platform and manually curated structural variants (SV) and single base substitution (SBS) molecular signatures that involved oncogenes and tumor suppressor genes [33]. The cutoff value for high tumor mutational burden (TMB) was set at 10 mutations per megabase (mut/mb). For microsatellite instability (MSI), a score of 10 was used as the cutoff for MSI-High, and for homologous recombination deficiency (HRD), a score 0.5-1 was considered high, a score from 0.1 to 0.5 was deemed probable, and a score less than 0.1 was considered negative [37,38].

2.3. RNA sequencing

RNA sequencing (RNA-seq) was performed on case 5 only, and the corresponding data analysis was conducted as previously described [39, 40]. Briefly, bulk RNA was isolated, and its integrity was confirmed. CDNA synthesis was conducted from total RNA and subsequent sequencing was executed on a HiSeq 2000 platform as paired ends [39, 40]. All sequencing reads were independently aligned using STAR_2.4.0f1 [41] for aligning against the human genome sequence build hg19 for sorting and indexing the reads. Expression levels (FPKMS) were estimated using Cufflinks (2.0.2), and the GENCODE v19 GTF file was employed for annotation [42].

2.4. Visualization of WGS/RNA-seq data

WGS data results and integration of RNA-seq results and integration of RNA-seq results was visualized in the interactive web-based interface portal at https://cornell.isabl.io/ [33,43].

Table 1 Patient demographics and clinical findings of five cases of adrenocortical carcinoma interrogated by whole-genome sequencing.
Case	Sex/ Age	Initial, clinical presentation	Initial imaging	Sites	Tx	Outcome
1	F/50	Non-functional; RUQ abdominal pain, nausea.	CT scan showing 6 cm R adrenal mass	R adrenal, pelvic/ liver mets	surgical resection, adjuvant cisplatin/etoposide, radiotherapy	Deceased
2	F/27	Functional status unknown; chest pressure and early satiety	Unknown	R adrenal, lung, bone, liver, brain mets	Surgical resection, mitotane monotherapy, radiation, temozolomide	Unknown
3	M/ 63	Non-functional; no initial symptoms, routine CT chest performed for strong family history of lung cancer	CT scan showed 4.6 cm L adrenal mass	L adrenal	Surgical resection only	Alive
4	M/	Functional (hypercortisolism); truncal weight gain,	CT scan showing 8.2 cm R	R adrenal, bone and	Surgical resection, lost to follow	Deceased
4	52	increasing HTN, increased 24-hour free cortisol	adrenal mass	liver mets	up
5	F/52	Non-functional; GERD, weight loss, epigastric fullness	CT scan showing 18 cm retroperitoneal mass	L kidney, retroperitoneum	Surgical resection, lost to follow up	Unknown

RUQ: right upper quadrant, F: female, M: male, HNT: hypertension, GERD: gastroesophageal reflux disease, R: right, L: left, Mets: Metastasis

3. Results

3.1. Histopathology and clinical characteristics of the adrenocortical carcinoma study cohort

The cohort consisted of seven tumor samples from five patients (four primary and three metastases). Metastatic sites included the pelvis for Case 1 and brain and lungs in a metachronous manner for Case 2. The median age at the time of primary diagnosis was 52 (range: 27-63). Two out of the five patients underwent chemotherapy following the diagnosis including both patients with metastatic disease.

Representative histopathology images are shown in Fig. 1. The samples from two cases (Case 1 and Case 3) showed oncocytic morphology while the remaining three cases showed conventional his- tology. Two samples showed high grade morphology, one at the primary site (Case 4) and one at the metastatic site (Case 1), defined by a mitotic count > 16 per 10 mm2. All conventional cases met the Weiss criteria while oncocytic cases met the Lin-Weiss-Bisceglia criteria for adrenal cortical carcinoma (Table 2) [11,13-15].

3.2. Whole genome characterization of primary and metastatic adrenocortical carcinoma demonstrates novel molecular events and potential therapeutic targets

Whole genome sequencing (WGS) was performed on the seven tumor/normal pairs, with three normal samples derived from blood and two from frozen non-neoplastic tissue. Sequencing coverage, TMB, MSI score, selected coding single nucleotide variants (SNVs) and copy number alterations (CNAs), selected molecular single based substitution (SBS) signatures, biomarkers and potential treatments are presented in Table 3. For the entire cohort, median values included a TMB of 5.2 muts/mb, structural variants (SVs) of 140, and 76 % tumor purity.

For the list of somatic variants in cancer-associated genes that were identified across the 7 tumor samples, see Supplementary Table 1. Of these 72 were SVs and 15 were SNVs that included 15 driver mutations in NRG1, MSH6, APC, TET2, MGA and RFWD3, among other genes. TP53 was mutated in tumor samples from 3 of 5 patients. Relevant copy number alterations (CNAs) included the targetable alterations CCNE1 amplification and CDKN2A deletion in one sample each. (Fig. 2A). Ten germline variants were detected, none of them annotated as pathogenic/ likely pathogenic in ClinVar [44,45]. The single based substitution (SBS) molecular signatures are illustrated in Fig. 2B.

In the patient with both primary and metastatic samples (Case 1), the post-treatment metastatic sample showed a significantly higher TMB of 10.1 muts/mb compared to 4.4 muts/mb in the pre-treatment primary tumor, an acquired mutator phenotype. In the metastatic sample, additional alterations were documented, including likely drivers muta- tions in NRG1, JAK1 and TET2; the acquisition of three additional sig- natures (SBS1, SBS92 and SBS8) and structural variants (SVs) in genes such as IRF4 and PCLO. This patient received cisplatin/etoposide chemotherapy following the diagnosis of metastatic disease. Histopa- thology of the primary tumor showed diffuse sheets of oncocytic cells with mild pleomorphism and focal areas of necrosis. The metastatic tumor showed a similar morphology, with increased cellular pleomor- phism (Fig. 1 and Fig. 3).

Case 2 had two metastatic samples with high TMB (7.8 muts/mb and 12.6 muts/mb) and a splice site variant in MSH6 in both samples, accompanied by mismatch repair (MMR) signatures (SBS6 and SBS21) (see Table 3). An indeterminate HRD status (HRD score = 0.16) was identified in case 5, a primary ACC that harbored a deleterious biallelic translocation impacting RAD51, increased number of tandem duplica- tions (characteristic damage signature associated with HRD), and SBS3 signature (associated with HRD) (Fig. 4).

Large-scale copy neutral loss of heterozygosity (CNLOH) was

Fig. 1. Representative histopathology of adrenocortical carcinomas that underwent whole genome characterization. A, B. Tumor samples of (A) primary and (B) metastatic sites from one patient (Case 1), both showing oncocytic morphology, the latter with increased pleomorphism. C, D. Tumor samples from a patient (Case 2) with metastatic adrenocortical carcinoma to the brain (C) and lung (D). E, F. Primary tumors from two patients with right (E, Case 4) and left (F, Case 3) adrenocortical carcinoma, which show conventional and oncocytic morphology, respectively. (Hematoxylin & Eosin stain, 200x original magnification).

Table 2 Histopathologic analysis of seven samples of adrenocortical carcinoma from five patients.
Case	Sites evaluated	Tumor Size (cm)	Subtype	Mitoses (10 mm2)	Necrosis	LVI/ CI	Nuclear grade	>1/3 diffuse architecture	<25% clear cells	Atypical mitosis	IHC
1 P	Right adrenal	8	Oncocytic	22	Y	Y/N	N/R	N/R	N/R	Y	+inhibin, +Melan
1 M	Pelvic mass	3.5									A,
											-panCK
2	*Lung and brain	16	Conventional	>4	Y	Y/	N/R	Y	Y	Y	+Melan A,
	metastases					N/R					+calretinin,
											Inhibin (focal +),
3	Left adrenal	4.9	Oncocytic	6	Y	N/N	N/R	N/R	N/R	Y	+synaptophysin. +inhibin, +Melan A, synaptophysin (focal +), calretinin (focal +), -panCK,
4	Right adrenal	6	Conventional	>16	Y	N/Y	III/IV	Y	Y	Y	-chromogranin +inhibin, +Melan A
5	Left adrenal/	25	Conventional	N/R	Y	N/R	III/IV	Y	Y	N	+inhibin, +Melan
	retroperitoneal					/N					A, +vimentin,
	mass										-S100,
	mass										-chromogranin, -panCK

* For Case 2, the primary resection specimen was received as a consult. Only metastatic samples were sequenced. Cases with conventional morphology classified using Weiss criteria while oncocytic cases classified by Lin-Weiss-Bisceglia histological system. M = metastatic site, P = primary site, LVI = lymphovascular invasion, CI = capsular invasion, IHC = immunohistochemistry, hpf = high power field, Y = yes, N = no, NR = not reported.

Table 3 Selected whole-genome sequencing results of four primary adrenocortical carcinomas and three metastases.
Case	Site	TMB (mut/mb)	MSI Score	Number of SVs	Selected Targets	Select Molecular Signatures and Biomarkers	Treatments
1	Primary	4.4	4.64	140	CCNE1 amplification, RB1 deletion	MMR, Chemotherapy	RP-6306, BLU-222
	Met	10.1	4.75	80	-	High TMB	Pembrolizumab
2	Met	7.8	0	33	ATM c.8122 G>A	High TMB	Olaparib,
2					MSH6 c.3801+1 G>A	SBS6, SBS21	Pembrolizumab
	Met	12.6	4.76	51	ATM c.8122 G>A	High TMB	Olaparib,
					MSH6 c.3801+1 G>A	SBS6, SBS21	Pembrolizumab
3	Primary	5.2	4.51	54	CDKN2A deletion	-	Abemaciclib,
3	Primary	5.2	4.51	54	CDKN2A deletion		Palbociclib
4	Primary	1.3	0.01	312	APC p.Q1625* APC p.L1624fs*26	-	-
5	Primary	2.5	3.22	662	TERT :: ZNF521 fusion RAD51 deleterious rearrangement	Indeterminate HRD	-

Met: metastasis, TMB: tumor mutational burden, MSI: microsatellite instability, SNV: single nucleotide variant,

CNA: copy number alteration, SV: structural variants, HRD: homologous recombination deficiency, AMP; amplification, SBS6, SBS21: single based substitution signatures 6 and 21 (associated to MMR)

identified in four tumor samples. Of these, three had a small mutation in TP53 while SVs affected RB1, CDKN2A, and NF1 in one case each. Notably, two tumors harbored rearrangements at the telomerase reverse transcriptase (TERT) locus resulting in novel TERT:PPP2R1A and TERT :: ZNF521 fusions, the latter validated by RNA-seq (Fig. 4).

3.3. Comparative genomics of conventional and oncocytic subtypes of adrenocortical carcinoma

We conducted a comparative genomic analysis of the conventional (4 samples from 3 cases) and oncocytic (3 samples from 2 cases) subtypes of ACC. No statistically significant differences (p > 0.05) were found between the subtypes for several metrics, including MSI Score (4.63 vs 2.00), HRD Score (0.049 vs 0.030), TMB (6.03 vs 6.57), total SNVs (14495.75 vs 17379.33), total Indels (2979.00 vs 1619.33) and total SVs (264.50 vs 91.33). Furthermore, no significant differences in the contribution of specific mutation signatures between subtypes were found (p > 0.05). Gene enrichment analysis identified no statistically significant enrichment in either subtype, with the exceptions of TRIP13 (p =0.048, q=0.048) and NIPBL (p =< 0.01, q =< 0.01), which

alterations were enriched in oncocytic ACC.

To further investigate clinical and genomic differences, we interro- gated whole-exome sequencing (WES) data from a publicly available database of 91 primary ACC [46-48]. We compared 87 conventional cases with 4 tumors of the oncocytic subtype. This analysis, conducted via the cBioPortal platform (https://www.cbioportal.org), revealed a trend of better overall survival probability in patients with oncocytic ACC, but it did not reach statistical significance (Logrank Test p-value= 0.087) (Figure S1). Other variables analyzed were not significantly different (p > 0.05), such as age at diagnosis (median 48 vs. 55 years), gender distribution (34.5 % vs. 50 % male), TMB (median of 0.9 vs. 0.7 mut/mb), mutational count (median 27.0 vs. 20.5 mutations), and MSIsensor Score (0.57 vs. 0.35). Additionally, no gene was found to be enriched in either subtype, including TRIP13 and NIPBL.

4. Discussion

In this study, we performed a comprehensive whole genome profiling of primary and metastatic ACC and integrated the clinical and pathologic characteristics of these patients. In addition to previously

Fig. 2. Most frequent mutations and molecular signatures in adrenocortical carcinoma. A. The oncoplot illustrates molecular alterations identified by whole- genome sequencing in 7 samples from 5 patients. B. Stacked bar chart illustrating the contribution of the six most prevalent single base substitution (SBS) mutational signatures in 7 samples from 5 patients. Each color represents a different signature, and the percentage indicates the contribution of each signature within the sample. P = primary adrenocortical carcinoma; M = metastatic adrenocortical carcinoma; TMB high = high tumor mutational burden.

Signature

Case 1P

Case 2M

Case 1M

Case 3P

Case 2M

Case 5P

Case 4P

SBS1 (Aging)

SBS18 (Damage by ROS)

SBS40 (Clock-like)

TP53

71%

SBS6 (MMR)

TMB

29%

22.1%

SBS8 (HR/NER deficiency)

CLPTM1L

19.1%

SBS92 (Tobacco chewing)

29%

CCNE1

29%

DLG2

29%

Contribution (%)

15.1%

KDM6A

29%

8.7%

RB1

23.4%

29%

MGA

29%

21.6%

TERT

29%

12.2%

24.1%

CDKN2A

12.4%

14%

MTAP

9.1%

14%

MIR4457

27.6%

14%

Alterations

11.0%

29.2%

NF1

14%

25.1%

Rearrangement

23.4%

7.4%

RAD51

14%

Truncating Variant

15.1%

Inframe Variant

9.5%

5.4%

8.9%

DAXX

14%

Deep Deletion

Tmb High

BCOR

Case 1P

Case 1M

Case 2M-Lung

Case 2M-Brain

Case 3P

Case 4P

Case 5P

14%

Non-Synonymous Variant

APC

Amplification

14%

Fusion

Fig. 3. Whole genome sequencing of adrenocortical carcinoma elucidates an acquired mutator phenotype in metastatic disease. A. The primary adreno- cortical carcinoma (histopathology, circos plot and WGS results overview are shown) has a low TMB (4.4/mb) and a TP53 mutation (Case 1). The patient developed metastatic disease and received cisplatin/etoposide chemotherapy. (Hematoxylin & Eosin stain, 200x original magnification). B. The metastatic sample to pelvic soft tissue (histopathology, circos plot and WGS results overview are shown) has high TMB (10.1/mb) and shows oncocytic morphology with high-grade transformation. (Hematoxylin & Eosin stain, 400x original magnification). WGS = whole-genome sequencing; TMB = tumor mutational burden.

—

…

—

documented alterations in ACC [11,22,26-30], we report an expanded list of cancer gene mutations in MSH6, APC, JAK1 and RFWD3, some of which are, or may be, tumor drivers.

Previous studies have explored the differences between primary and metastatic ACC’s samples, frequently identifying that metastases ac- quire additional alterations during progression, often related to gene mutations-particularly in genes associated with the mismatch repair (MMR) pathway [10,49]. In our two metastatic samples, one of which was paired with its primary tumor, we observed a high TMB (TMB-H)

with low MSI levels. In Case 2, we identified a somatic MSH6 splice site mutation along with SBS6, a molecular signature specifically associated with MMR. Additionally, alterations in genes such as FLT4, MGA, TET2, and NOTCH2 in this case are likely subclonal and passenger mutations, resulting from DNA damage associated with MMR deficiency.

Previous studies have shown that genomic profiles differ between primary and recurrent or metastatic ACC, with metastases displaying heterogeneity across cases but relative homogeneity within the same individual [10,49]. We observed similar findings in two of our cases

Fig. 4. Whole-genome sequencing of metastatic adrenocortical carcinoma detects novel structural variants impacting TERT. A. Histopathology of Case 5 demonstrates focal necrosis and cystic degeneration (Hematoxylin & Eosin, 200x original magnification). B. Circos plot highlights whole-genome sequencing results. C. Analytics suggest homologous recombination deficiency (HRD), supported by contribution of SBS3 molecular signature (associated to HRD), structural variant (SV) affecting RAD51 and tandem duplication SV phenotype. D. Fusion circos shows structural variants including a novel TERT:ZNF521 fusion.

1005 indels

wieton

nepeut deletion

m-homology deletion

—

complex

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5946 SNVs of signatures:

50512

purity ploidy

2.1

copy number

662 rearrangements

t. digication

deletion

CAT . THC

translocation

00€

5887

SBS3

SBS6

Contribution (%)

SBS9

5852: APOBEC

SAS3: MRD

ShSố: MMR

SBS12

5099: POLI somatic hypermutation

SBS13

50512

50513: APOGEC

SBS16

SAS40: Clock-Jke

50556: Chemotherapy

58589

SBS40

50593

€

SBS85

SBS86

SBS89

100

SBS93

(Cases 1 and 2). This heterogeneity has clinical relevance, as a subgroup of patients with advanced or metastatic ACC with TMB-H may poten- tially benefit from immune-therapy such as pembrolizumab [50]. Pre- vious studies in ACC have shown that pembrolizumab is safe for advanced cases [51]; however, efficacy results have been mixed: one report documented a long-term complete response in a patient with TMB-H and a deleterious MSH2 mutation, while another case showed no response in a TMB-H patient without MMR deficiency [52]. Additional studies have demonstrated modest efficacy of pembrolizumab in ACC, regardless of MSI status [51]. The exact role of TMB-H in guiding tar- geted therapy for ACC remains uncertain, and further studies are needed to clarify its potential.

In ACC, several well-recognized morphological subtypes exist, including conventional, oncocytic, myxoid and sarcomatoid [11]. However, it remains unclear whether these subtypes differ significantly in their genomic landscape and whether subtype classification impacts patient prognosis. A previous study reported a better prognosis for the oncocytic subtype of ACC compared to other subtypes, however, in a multivariate model, this variable did not independently influence pa- tient prognosis [10]. Next-generation sequencing revealed that the conventional and myxoid subtype, compared to the oncocytic subtype, have a higher TMB, a greater frequency of RB1 and CDKN2A inactiva- tion, and a higher occurrence of CDK4 gain-of-function alterations. These genetic changes are associated with cell cycle progression and may contribute to ACC progression [10].

Based on this, we explored our current series but, in contrast to prior studies [10], we did not find significant differences between oncocytic and conventional subtypes across multiple variables, including TMB. The only notable finding was the enrichment of alterations in TRIP13 and NIPBL genes in the oncocytic subtype. When such analysis was

expanded to a publicly available WES cohort of 92 ACC [46-48], no significant differences were identified, although a trend towards better survival was observed in oncocytic ACC.

The discrepancies between our findings and previous reports [10] could be attributed to several factors: (1) sample size limitations (studies on ACC’s remain small due to its rarity), and (2) differences in sequencing techniques, with targeted NGS used in prior studies versus WES [10] and WGS [22,46-48]. Future studies with larger sample sizes and or harmonized sequencing approach could help clarify these issues.

Alterations of telomere regulation pathway have also been impli- cated in ACC pathogenesis, and TERT driver mutations and focal am- plifications were previously identified [22,53]. In a study by Gupta et al., TERT promoter rearrangements were reported in 2/165 cases (1.2 %), including one case that possessed a CCDC47 :: TERT fusion [54]. We found novel TERT :: PPP2R1A and TERT:ZNF521 novel gene partners with TERT in two cases, further providing evidence for the role of TERT rearrangements in the biology of a small subset of ACC. TERT fusions have also been documented in other neoplasms including epithelioid trophoblastic tumors [55], metastatic Leydig cell tumors [56] and ju- venile granulosa cell tumors [57].

We determine the HRD status of our ACC cases using a novel WGS- based classifier that incorporates small deletions with microhomology, structural variant (SV) deletions, and SV duplications. Although one tumor (Case 5) had features of HRD, the score was indeterminate. Limited cases of ACC’s associated with HRD have been reported, in the context of germline mutations in BRCA2 and CHEK2 [58,59], as well as somatic alterations in other genes [22]. Interestingly, none of the latter were pathogenic BRCA1/2 variants, but SV impacting RAD52 and copy losses in multiple DNA repair genes, including CHEK2 and ATM. One of such cases with an SV in RAD52 received Olaparib (PARP-inhibitor),

resulting in a partial response and disease stabilization for 8.5 months in a patient with stage IV disease. The patient later showed a mixed response to carboplatin and paclitaxel, with disease progression occur- ring 5 months after treatment initiation [22].

Further WGS analysis of an expanded cohort of metastatic ACC would determine the frequency of HRD, which will identify who may benefit from selected target therapies, such as PARP inhibitors [60-62]. Finding therapeutic strategies to effectively treat this rare disease is an unmet clinical need. The current survival rate in patients diagnosed with ACC is 3-4 years, with a reduced rate in advanced or metastatic disease. Hence, visualization of the entire genome with detection of relevant molecular signatures and molecular phenotypes, as well as structural variants on cancer genes emphasizes the importance of using whole genome profiling to aid in identifying potential targets in ACC.

The main limitation of our study is the small sample size. Yet, it is the second WGS-based profiling of ACC to date and, remarkably, potential targetable biomarkers were detected in 3 of 5 patients. Future WGS studies of larger annotated cohorts of this rare and aggressive malig- nancy will help us understand further the genomic landscape of meta- static ACC, leading to better detection of new and potential biomarkers that may improve survival outcomes.

Funding sources

The authors received no specific funding for this work.

CRediT authorship contribution statement

Juan S. Medina-Martínez: Data curation, Writing - review & edit- ing. Michael Sigouros: Data curation, Project administration. Olivier Elemento: Writing - review & editing. Juan Miguel Mosquera: Conceptualization, Formal analysis, Methodology, Project administra- tion, Writing - review & editing. Andrea Sboner: Writing - review & editing. Majd Al Assaad: Data curation, Formal analysis, Methodology, Project administration, Writing - original draft, Writing - review & editing. Theresa Scognamiglio: Data curation, Writing - review & editing. Taylor Kalomeris: Data curation, Formal analysis, Methodol- ogy, Writing - original draft, Writing - review & editing. Gunes Gun- dem: Data curation, Formal analysis, Writing - review & editing. Jesus Delgado-de la Mora: Data curation, Formal analysis, Methodology, Writing - review & editing. Jyothi Manohar: Data curation, Writing - review & editing. Baris Boyraz: Data curation, Formal analysis, Writing - review & editing. Max F. Levine: Data curation, Writing - review & editing.

Declaration of Competing Interest

The authors declare the following financial interests/personal re- lationships which may be considered as potential competing interests: Max F. Levine, Gunes Gundem and Juan S. Medina-Martinez reports a relationship with Isabl, Inc. that includes: employment. If there are other authors, they declare that they have no known competing financial in- terests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by the Englander Institute for Precision Medicine. Whole-genome sequencing was performed at the New York Genome Center, supported by an Agreement with Illumina, Inc., and Weill Cornell Medicine. Project support for this research was also pro- vided in part by the Center for Translational Pathology (Ruben Diaz, Leticia Dizon, Bing He) from the Department of Pathology and Labora- tory Medicine at Weill Cornell Medicine. Figure 3 was partially created with BioRender.com.

Author contributions

J.M.M. conceptualized the study. M.S., J.M., and A.S. curated the data. J.M.M., T.S., M.F.L., G.G., B.B., and J.S.M.M. conducted the formal analysis. T.K., M.A, J.D.M. and M.F.L. wrote - original draft. T.K, M.A, J. D.M., O.E., and J.M.M. wrote - review and editing. All authors read and approved the final paper.

Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.prp.2024.155725.

Data availability

The analyzed molecular data is available in the supplementary documents. Raw data are available upon reasonable request from the corresponding authors.

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