Mutational profiling of SMARCA4 and SMARCB1 in ampullary adenocarcinoma: a preliminary study

Dear Editor,

Ampullary adenocarcinoma (AAC) accounts for only 0.2% of gastrointestinal malignancies and, although a surgical approach is suitable in 80% of cases, the 5-year survival rate is 30-50% with a high recurrence rate (50%) ¹. The most frequent histological subtypes are mixed, pancreatobiliary (PB), and intestinal (INT) ¹. Therapeutic approaches are limited, with approved gemcitabine-based regimen in PB and 5-fluororacil in INT AAC patients, respectively ¹. AAC molecular profiling predominantly highlighted KRAS mutations followed by TP53, WNT/APC, PIK3CA, SMAD4 then BRAF, CDKN2A, FBXW7, ERBB2, PDFGRA, SMARCB1, RB1 ¹. Among them, only BRAF point mutations (detected in 9.3% of cases) and NTRK aberrant transcripts (2.0% of cases) make AAC patients eligible for Idabrafenib plus trametinib (MEK inhibitors) and anti NTRK inhibitors, respectively ¹. In this scenario, the identification of novel specific molecular hallmark able to optimize the clinical management of AAC patients is crucial.

SMARC proteins are core subunits of the SWI1/SNF1 complex and are involved in chromatin remodeling and DNA repair ². SMARCA4 (BRAHMA RELATED GENE, BRG1), involved in ERBB, GnRH, and PI-3K/AKT/mTOR signaling pathways, acts in transcriptional modulation, DNA repair, and cell cycle checkpoint ². A high expression level of SMARCA4 has been described as a negative prognostic factor across different solid tumors ².

Article

SMARC proteins are core subunits of the SWI1/SNF1 complex and are involved in chromatin remodeling and DNA repair ². SMARCA4 (BRAHMA RELATED GENE, BRG1), involved in ERBB, GnRH, and PI-3K/AKT/mTOR signaling pathways, acts in transcriptional modulation, DNA repair, and cell cycle checkpoint ². A high expression level of SMARCA4 has been described as a negative prognostic factor across different solid tumors ². A plethora of SMARCA4 molecular alterations, including missense mutations, indels and copy number variations (CNVs), were identified ².

SMARCB1 (integrase interactor 1, INI1), involved in SWI1/SNF1 complex, plays a crucial role in tumor suppression through the epigenetic mechanisms ³. Moreover, SMARCB1 is directly involved in downregulating INK4A, WNT-signaling pathway, repressing retinoblastoma (RB) target genes and promoting MYC oncogene-mediated transactivation ³. SMARCB1 molecular alterations, depending on biallelic inactivation from deletions, large intragenic deletions/duplications, small out-of-frame intragenic deletion/insertions, splice-site, nonsense and truncating mutations, lead to the complete loss of function in malignant neoplasms ³. Interestingly, rare SMARCB1 missense mutations were identified in sporadic AAC cases, without any details about the molecular landscape ⁴.

Here, we aimed to overview the preliminary results from SMARCA4 and SMARCB1 integrating analysis (protein expression and dysfunctional gene alterations) in a pilot retrospective cohort of 9 AACs, constituted by 2 mixed histological type (22.2%), 2 BP (22.2%), and 5 INT (55.6%), respectively. Immunohistochemical analysis of SMARCA4 and SMARCB1 proteins was performed following standardized procedures with primary monoclonal antibodies against SMARCA4 (BRG1, 1:50; Abcam), and SMARCB1 (clone A-5 mouse, 1:100; Santa Cruz), on the automated platform Autostainer Link 48 (Dako, Carpinteria, CA, USA). After microdissection and extraction of neoplastic cellularity from unstained slides, DNA was sequencing with a comprehensive commercially available NGS panel (Oncomine™ Tumor Mutation Load Assay, Thermo Fisher Scientifics) that allows the simultaneous analysis of single nucleotide variants, copy number variations, and insertions/deletions in 409 cancer related genes (including SMARCA4 and SMARCB1) on NGS platform.

Immunohistochemistry showed a retained pattern (nuclear staining in neoplastic cells) in a single case (11%) and in 6 cases (66%) for SMARCA4 SMARCB1, respectively. A loss pattern (absence of nuclear immunostaining in neoplastic cells with preserved staining in non-neoplastic tissue) was identified in 8 cases (89%) for SMARCA4 (Fig. 1). In 3 cases, SMARCB1 evaluation was not reliable due to the absence of immunostaining in internal controls.

SMARCA4 gene alterations were detected in 77.7% (7 out of 9) of cases but removing a median of 10.1 (ranging from 1.0 to 20.0) synonymous mutations, potentially impacting SMARCA4 alterations were identified. Moreover, all SMARCA4 hotspot alterations were detected in bromodomain (BRK) domain (Tab. I). In vivo studies demonstrated that the BRK domain is crucial for interacting acetylated histone H3K14 suggesting a promising target for selective inhibition ⁵. Similarly, SMARCB1 molecular alterations were observed in 5 out of 9 (55.6%) cases (Tab. I). Among them, a median of 1.9 (ranging from 1.0 to 8.0) non-synonymous mutations was detected. In addition, concomitant molecular alterations in SMARCA4 and SMARCB1 were also identified in 5 out of 9 (55.6%) AAC patients. This finding has been described showing a SMARCA4 and SMARCB1 simultaneous loss pattern in undifferentiated endometrial carcinoma ² and concomitant SMARCB1 alteration in 2/56 malignant rhabdoid tumor ⁶ and in 3/19 ossifying fibromyxoid tumor ⁷. The higher frequency rate of SMARCA4/SMARCB1 concomitant molecular alterations in our series of AAC patients can suggests their eligibility in clinical trials investigating SMARCA4 deficient tumor patients administered with Immune Check Point Inhibitors (ICIs) ⁸. Moreover, in accordance with ClinVar public database, variant of uncertain significance (VUS) in 3 (33.3%) patients was found whereas a likely pathogenic alteration was detected in a single instance. In addition, a SMARCB1 pathogenic alteration was identified in a single patient (Tab. I).

Two patients (SMA#6, #9) did not meet quality metrics (average reads per amplicon < 100) from NGS analysis and showed a SMARCA4/SMARCB1 wild type molecular profile. In this regard, pre-analytical management of tissue sample represents a crucial issue strongly related to the successful rate of molecular analysis. In our series, three patients (SMA#2, #3, #5) (33.4%) showed TP53 molecular alterations. In addition, KRAS non-synonymous variants were detected in 4 out of 9 (44.4%) patients (SMA#1, #2, #3, #7). Moreover, APC and ERBB3 molecular alterations were also found in accordance with literature data, while CDKN2A variants were not detected.

SMARCA4 immunostaining matched with the SMARCA4 molecular profile in 66.0% of cases. In one case, SMARCA4 missense mutation showed a preserved nuclear immunostaining. SMARCB1 immunostaining matched with SMARCB1 genic status in 22.0% of cases. Beyond the 33.0% of cases not evaluable for immunostaining, a retained protein immunostaining (45.0%) was simultaneous inspected with SMARCB1 molecular alterations. This data can suggest investigating SMARCA4 and SMARCB1 in AAC by molecular assays. Of note, concomitant SMARCA4-SMARCA2 inactivating mutations were also identified in thoracic neoplasms, suggesting an evolutionary double hit model in sarcomatoid/undifferentiated tumors ⁹. Similarly, simultaneous SMARCA-SMARCB inactivating mutations play a synergic effect on dysfunctional chromatin remodeling supporting the hypothesis on the pivotal role of SMARC complex in regulating cell cycle ¹⁰.

Our study is affected by several limitations. The first is small series of AAC patients that requires further investigation to confirm molecular records; the second one is that functional studies and methylation-based assays should be used to validate molecular alterations detected in promising biomarkers by wide NGS panel. Finally, clinical information on survival and therapy is lacking. Further data to validate the evolutionary molecular landscape of SMARC deficient AAC patients to implement these findings in the clinical practice are needed.

In conclusion, SMARC investigation in AAC can suggest a peculiar molecular pathway, suitable for possible clinical management and targeted therapy in a subset of patients. Methods for SMARC protein and genic alteration assessment, by both immunohistochemistry and molecular assays need further validation with a pre-analytic standardization.

ACKNOWLEDGEMENTS

We wish to thank Dr. Chiara Di Giorgio (Fondazione IRCCS Casa Sollievo della Sofferenza) for reviewing this manuscript for English language.

CONFLICT OF INTEREST STATEMENT

P.P. received personal fees (as consultant and/or speaker bureau) unrelated to the current work from MSD, Astellas, Pierre Fabre, GSK, Diatech Pharmacogenetics, Incyte, Bristol. F.P. received personal fees (as consultant and/or speaker bureau) unrelated to the current work from Menarini. Roche and Agilent U.M. received personal fees (as consultant and/or speaker bureau) unrelated to the current work from Boehringer Ingelheim, Roche, MSD, Amgen, Thermo Fisher Scientifics, Eli Lilly, Diaceutics, GSK, Merck and AstraZeneca, Janssen, Diatech, Novartis and Hedera. G.T. reports personal fees (as speaker bureau or advisor) unrelated to the current work from Roche, MSD, Pfizer, Boehringer Ingelheim, Eli Lilly, BMS, GSK, Menarini, AstraZeneca, Amgen and Bayer. P.G. has received personal fees (as consultant and/or speaker bureau) unrelated to the current work from Boehringer Ingelheim, Roche, MSD, Amgen, Eli Lilly, AstraZeneca, Pfizer and Novartis.

FUNDING

This work is partly founded (PP) by Italian Minister of Health, Ricerca Corrente Program 2022-2024.

AUTHORS CONTRIBUTION

Conceptualization: PP, FP, PG and UM. Methodology: all authors. Software: all authors. Validation: all authors. Formal analysis: all authors. Investigation: all authors. Resources: all authors. Data curation: all authors. Writing – original draft preparation: PP, FP. Writing – review and editing: all authors. Visualization: all authors. Supervision: PG, UM. Project administration: PG, UM. Funding acquisition: PP.

ETHICAL CONSIDERATION

All information regarding human material was managed using anonymous numerical codes, and all samples were handled in compliance with the Helsinki Declaration.

History

Received: May 17, 2024

Accepted: June 13, 2024

Figures and tables

Figure 1: Case 8 – Coronal section showing ampullary adenocarcinoma (A; hematoxylin and eosin, X0,5); SMARCA4 loss pattern (B; immunohistochemistry, X25); SMARCB1 retained pattern (C; immunohistochemistry, X25).

Table I. SMARCA4 and SMARCB1 gene mutations grouped for functional protein effect.

ID	Mutations in SMARCA4*	Mutations in SMARCB1*	Non-sense mutations		Missense Mutations		Clinically significant mutations
			SMARCA4	SMARCB1	SMARCA4	SMARCB1
SMA1	4	0	0	0	1	0	N.O.
SMA2	47	16	1	2	18	5	SMARCA4 p.Arg905Cys (c.2713C > T) MAF: 9.4% - Type: VUS
SMA3	40	4	2	1	16	0	SMARCA4 p.Ala1002Val (c.3005C > T) MAF: 10.5% - Type: VUS
SMA4	14	6	0	1	5	0	SMARCB1 p.Trp51Ter (c.152G > A) MAF: 55.9% - Type: Pathogenic
SMA5	27	2	0	0	10	0	SMARCA4 p.Gly1644Ser (c.4930G > A) MAF: 16.0% - Type: VUS
SMA6	0	0	-	-	-	-	N.O.
SMA7	23	8	2	0	8	2	N.O.
SMA8	2	0	0	0	1	0	N.O.
SMA9	0	0	-	-	-	-	N.O.
MAF (Mutant Allelic Frequence); N.O. (Not Observed); VUS (Variant Uncertain Significance).
* Mutations count includes all synonymous, non-sense, missense and unknown mutations. Clinical significance of reported mutations refers to ClinVar database (last update at submission date).