Somatic DICER1 pathogenic variants in a well-differentiated fetal lung adenocarcinoma diagnosed in a man with familial adenomatous polyposis

doi:10.21203/rs.3.rs-7013135/v1

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Case Report

Somatic DICER1 pathogenic variants in a well-differentiated fetal lung adenocarcinoma diagnosed in a man with familial adenomatous polyposis

https://doi.org/10.21203/rs.3.rs-7013135/v1

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Well-differentiated fetal lung adenocarcinoma (WDFLAC) is a rare pulmonary carcinoma characterized by its resemblance to the fetal lung. It was thought to be caused by somatic pathogenic variants (PVs) in the genes CTNNB1 or APC, which lead to the nuclear accumulation of beta(β)-catenin and down-stream activation of key oncogenes. However, recent studies have shown the involvement of another gene: DICER1, whereby germline and somatic DICER1 PVs are known to cause tumours with fetal-type morphologies. Here, we report the second case of WDFLAC in a male non-smoker with familial adenomatous polyposis (FAP), and the first case with confirmed inactivating PVs in both APC and DICER1. A review of the literature also showed that out of the 17 WDFLAC cases for which sequencing of DICER1, CTNNB1 and/or APC was performed, 13 had PVs in DICER1 and either CTNNB1 or APC, with one patient having all three genes mutated. Taken together, this suggests that the development of WDFLAC may be the summative effect of WNT and DICER1 dysfunction. It may thus be beneficial to test for DICER1 variants in diagnostically challenging cases and, if a germline DICER1 PV is found, additional screening for DICER1-associated tumors could be put into place for clinical management.

Well-differentiated fetal lung adenocarcinoma

familial adenomatous polyposis

DICER1

APC

WNT

β-catenin

case report

Well-differentiated fetal lung adenocarcinoma (WDFLAC) is a rare type of pulmonary malignancy known for its resemblance to the fetal lung at ten to fifteen weeks’ gestational age (1). In 2002, Nakatani et al.(1) postulated, based on nuclear accumulation of β-catenin in this tumour, that continued upregulation of the wingless-type MMTV integration site family (WNT) signaling pathway played a role in WDFLAC tumorigenesis. Nuclear expression of β-catenin is known to cause the activation of key oncogenes such as MYC and Cyclin D1 (2).

Familial adenomatous polyposis (FAP) is an autosomal dominant disease caused by deleterious mutations in the adenomatous polyposis coli (APC) gene (2). In the canonical WNT pathway, APC binds β-catenin and recruits it to the β-catenin destruction complex, leading to the protein’s degradation (3). Individuals with FAP are often heterozygous for APC predicted truncating pathogenic variants (PVs), which, when accompanied by a “second hit” on the wild-type allele, leads to the development of adenomatous colorectal polyps at an early age, in turn conferring risk of early-onset colorectal cancer (CRC) (2). Though FAP contributes to < 1% of CRC cases, somatic APC PVs are found in > 80% of colorectal tumours (3). FAP also increases the risk of desmoid tumors, thyroid carcinoma and hepatoblastoma (2), but rarely is it implicated in lung cancer. However, in 1995, WDFLAC was reported in a single patient with FAP (4).

Over a decade after Nakatani et al.’s finding, de Kock et al.(5) discovered an association between WDFLAC and DICER1, a gene involved in other types of rare lung tumours associated with an embryonic lung phenotype (6). We report here a second case of WDFLAC found in a young male non-smoker with FAP, and the first with molecular analysis that confirms the role of pathogenic variants in both APC and DICER1. We also review the existing literature on the molecular genetic alterations found in WDFLAC.

Prior to the patient’s lung cancer diagnosis, he was being followed in the Gastroenterology service for his FAP. The patient had a strong family history on his maternal side of polyposis, as well as young-onset colorectal cancer (Fig. 1a), which led to his genetic testing at age 11. He was found to carry the familial APC PV c.3379C > T (p.Q1127*). After his first colonoscopy the following year revealed the presence of multiple polyps, he underwent a proctocolectomy and J-pouch reconstruction at age 13. Post-operatively, the patient had semi-regular duodenoscopies and pouchoscopies, several of which resulted in the removal of tubular adenomas. He had been otherwise unaffected until age 26, when he presented to the emergency room with left chest and shoulder pain. This was accompanied by dyspnea and a persistent cough, still present after finishing a round of antibiotics for left upper-lobe pneumonia. A CT scan found an FDG-avid lung mass, and a subsequent biopsy revealed a cancer, which was diagnosed as pT3N0 WDFLAC (Figs. 1b-c) following a left upper lobectomy. He received one cycle of cisplatin (161 mg or 75 mg/m²) & pemetrexed (1075 mg or 500 mg/m²) prior to self-discontinuing due to adverse symptoms. The patient has been in remission as of time of publication.

Clinical molecular testing of the WDFLAC (AmpliSeq Focus Panel), which included CTNNB1, revealed no PVs in DNA or RNA. Immunohistochemical (IHC) staining of the tumour also showed intact nuclear expression of mismatch repair (MMR) proteins. Meanwhile, the β-catenin IHC showed nuclear accumulation of the protein in tumour cells not seen in the normal epithelial cells of the lung (Fig. 1d). As neither DICER1 nor APC were included in the clinical panel, DNA from the patient’s blood lymphocytes, normal lung tissue and tumour tissue were extracted as described previously (5) and sequenced to look for variants in both genes. Two DICER1 hits were found solely in the tumour: one in the RNase IIIb domain (c.5425G > T (p.G1809W); at variant allele frequency (VAF) 0.27), and the other a truncating loss-of-function mutation (c.3697delG (p.D1233Mfs*4); VAF 0.55) (Fig. 1e). The patient’s tumour also had a loss of heterozygosity (LOH) in APC at a VAF of 0.87, at the position of his germline PV (Fig. 1e).

The patient’s APC germline PV at amino acid 1127 lies upstream of the first 20 amino-acid repeat that comprises one of the main β-catenin binding sites in the protein. It is unusual for a predicted truncating germline PV at this position in APC to be accompanied by LOH at the same locus, as there is a selective pressure to retain at least one of the 20 amino-acid repeats between residues 1265 and 1400 for effective tumour formation in the colon(7). The only other reported patient with FAP and WDFLAC never underwent germline or somatic genetic testing (4), so their tumour’s second APC somatic hit cannot be determined. While none of the WDFLAC cases presented by de Kock et al.(5) and Chong et al.(6) had confirmed co-diagnoses of FAP, the latter article did report one case with a clear loss of heterozygosity for APC at p.G871* (VAF 0.77), which, similar to our current patient, would also produce a protein missing its key β-catenin binding sites. Due to these findings, we postulate that LOH of the wild-type APC allele at residue 1127, accompanying the germline pathogenic variant, predisposed this patient to develop a WDFLAC.

A new report investigating biallelic inactivation of tumour suppressor genes (TSGs) across multiple cancer databases showed that amongst rare, mutated TSGs found in non-canonical primaries, biallelic loss of APC was commonly present in prostate adenocarcinomas and lung adenocarcinomas (LUADs) (8). Though the percentage of LUADs with LOH in APC was not always significantly higher than LUADs with wild-type APC, β-catenin protein levels were significantly elevated in the former. There was also a degree of mutual exclusivity in mutations of WNT pathway genes implicated in LUADs (8). This is in line with our finding of wild-type CTNNB1 in our patient’s tumour, but Chong et al.(6) did note a patient with both mutated CTNNB1 and APC. However, this patient did appear to retain one wild-type copy of APC, so the CTNNB1 mutation may have played a more significant role in tumour formation. These results, nonetheless, do lead to the question of why there are not higher rates of lung cancer in FAP patients. It points to the possibility of spatiotemporal factors, or that other pathways take precedence over WNT in LUAD pathogenesis.

The DICER1 NGS results corroborate our earlier findings on WDFLAC (5, 6) and that of several other groups (Table 1(9–12)). The two pathogenic DICER1 hits in the patient’s tumour, as well as its “fetal”-type morphology, are characteristic of other DICER1-related tumours (13). There are WDFLAC cases, however, where neither WNT-pathway nor DICER1 mutations were found (6), so there may be other pathways implicated in its development. However, an interaction between the two does appear to be exist in other types of tumours. Chong et al.(6) analyzed sequencing data to show that DICER1 and CTNNB1 pathogenic mutations often co-occur in the related tumor, pulmonary blastoma. To et al.(14) were able to establish directionality in the interaction within ovarian cancer metastases, showing that β-catenin downregulates the expression of DICER1 mRNA in highly metastatic ovarian cancer lines. Contrastingly, in Illiou et al.’s (15) study where they generated DICER1-impaired CRC cell lines, these lines were shown to have greater nuclear accumulation of β-catenin compared to their wild-type counterparts, though it must be noted that they already had deleterious mutations in CTNNB1 or APC.

Table 1
Findings from previous genetic studies of low-grade/well-differentiated fetal lung adenocarcinoma (WDFLAC), with a focus on those which tested for both the presence of WNT pathway and *DICER1* gene variants. All listed variants are somatic unless otherwise mentioned.
Reference	Genes Tested	Case	Genetic Variant(s) (VAF)
de Kock et al. (5)	CTNNB1, DICER1	1a	CTNNB1 p.S33C; DICER1 p.Y1180* (germline) and p.D1709G
Liu et al. (9)	CTNNB1, DICER1, KRAS	2a	CTNNB1 p.S33C (0.41); DICER1 p.S1470Lfs*19 (0.32) and p.D1709N (0.38)
Zhang et al. (10)	Whole-exome sequencing (WES)	3a	MYCN p.T58M (0.14)
		3b	CTNNB1 p.G34R (0.27); DICER1 p.D1810Y (0.25)
		3c	CTNNB1 p.S33C (0.29); DICER1 p.E1813G (0.37)
Li et al. (11)	1021 cancer-related genes (custom probes)	4a	CTNNB1 p.S33C (0.43); DICER1 p.Q488* (0.40) and p.D1709V (0.37)
		4b	CTNNB1 p.S37F (0.38); DICER1 p.D1810F (0.36) and p.A1710del (0.293); MYCN p.P44L (0.45); RARA p.S287L (0.64); EGFR p.D247G (0.35); C15orf23 p.T47K (0.15)
		4c	CTNNB1 p.D32Y (0.48); DICER1 p.D1709A (0.84)
		4d	CTNNB1 p.S37F (0.38); DICER1 p.5096-1G > T (0.38) and p.D1810Y (0.35)
Yanagawa et al. (12)	CTNNB1, DICER1, KRAS, BRAF, PIK3CA and EGFR	5a	CTNNB1 p.S33F
Yanagawa et al. (12)	CTNNB1, DICER1, KRAS, BRAF, PIK3CA and EGFR	5b	CTNNB1 p.S37F; DICER1 p.D1709G
Chong et al. (6)	592 genes (Agilent SureSelect XT Panel)	6a	CTNNB1 p.D32Y (0.43); DICER1 p.Q1580* (0.47) and p.E1813D (0.38); APC p.R1640fs (0.41)
		6b	DICER1 p.Q1614* (0.23) and p.E1813Q (0.26); CDKN2A p.E88* (0.31); KRAS p.G12 (0.2); STK11 p.G171fs (0.35)
		6c	CTNNB1 p. S37F (0.38); DICER1 p.W400* (0.33) and p.D1810Y (0.31)
		6d	DICER1 p.G1809L (0.55); APC p.L2718_E2719delinsF* (0.54); FUBP1 p.Q628* (0.31); WRN p.Y1091* (0.35); TP53 p.E294* (0.65)
		6e	CTNNB1 p. L2718_E2719delinsRF (0.27) and p. S33C (0.09); DICER1 p.W1831* (0.29) and p.D1709N (0.56); STK11 p.D68fs (0.58); U2AF1 p.S34F (0.4)
		6f	DICER1 p.D1810Y (0.56); APC p.G871* (0.77); TP53 p.R273L (0.79)
* We also reviewed the recent study by Sun et al. (PMID: 38831114), but we noticed some inconsistencies in their findings. Firstly, their reported WDFLAC patient demographics (male, > 50 years old) were atypical, as WDFLAC is mostly diagnosed in females under 40. Their WES results also showed that BLTP1 was the only commonly mutated gene across four of the eight cases, a gene which has not been previously linked to WDFLAC. Two of the BLTP1 mutations were silent, and all the variants were sub-clonal mutations with a VAF < 0.20. Lastly, our internal enrichment analysis of their data revealed that only cigarette-smoking-affiliated genes were enriched in the set, which coincides with the reported patient histories and suggests that most of the reported variants are unrelated to the etiology of WDFLAC. Given the inconclusive results of the study, we chose to exclude it from our report.

Taken together with the results of our current case, DICER1 and WNT pathway PVs appear to play a synergistic role in the tumorigenesis of several tumours and influence the cell morphology of non-small cell lung cancers. We believe the absence of full-length APC in this patient’s lung tumor may have set the stage for development of a WDFLAC, driven by somatic DICER1 PVs. As such, in diagnostically challenging cases, testing for DICER1 variants could provide value. Furthermore, if a germline DICER1 PV is found, screening for DICER1-associated tumors, which often present in childhood, could be instigated.

The subject of this report signed a consent form that allows us to publish details of his case.

Additional Information

Acknowledgements

We would like to thank Dr. Pierre-Olivier Fiset for his valuable input and recommendations in the preparation of this report.

Authors’ contributions

AA wrote the original draft and created most of the figures and tables. PT created Figures 1b-1d and wrote their captions. A-LC extracted DNA from the patient’s samples and helped review and edit the paper. NB chose the best representative tissue samples for DNA extraction. DS performed the enrichment analysis described in the notes section of Table 1. GC analyzed APC and DICER1 NGS data. SCB originally diagnosed the patient. WDF provided supervision for the project. All authors contributed to reviewing and editing the manuscript.

Ethics approval and consent to participate

The patient was enrolled into our genetic predispositions biobank study after signing an informed consent document (MP-37-2019-4865) approved by the McGill University Health Centre REB Panel. This study was performed in accordance with the Declaration of Helsinki.

Data availability

Enrichment analysis was performed using whole exome sequencing data from Sun et al. (PMID: 38831114, in the article’s Supplementary Data 1).

Funding information and Conflict of Interest Statement

This research as well as biobanking of biological material and data was made possible in part through a collaboration with the Réseau de recherche sur le cancer (RRCancer) financially supported by the the Oncopole, the FRQ cancer division, which receives funding from Merck Canada Inc., GSK, Pfizer and the Ministère de l'Économie, de l'Innovation et de l'Énergie du Québec. The RRCancer is affiliated to the Canadian Tumor Repository Network (CTRNet).

This work was also funded in part by a research grant to Dr. William Foulkes by CIHR (FDN-148390). William D. Foulkes reports receiving funds from Astra Zeneca for an unrelated project on cascade testing.

None of the aforementioned organizations were involved in the preparation of this manuscript. The other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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No competing interests reported.

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Somatic DICER1 pathogenic variants in a well-differentiated fetal lung adenocarcinoma diagnosed in a man with familial adenomatous polyposis

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