Genomic landscape of adenoid cystic carcinoma of the breast

Authors

Luciano G Martelotto

Maria R De Filippo

Charlotte KY Ng

Rachael Natrajan

Laetitia Fuhrmann

Joanna Cyrta

Salvatore Piscuoglio

Huei-Chi Wen

Raymond S Lim

Ronglai Shen

Anne M Schultheis

Y Hannah Wen

Marcia Edelweiss

Odette Mariani

Göran Stenman

Timothy A Chan

Pierre-Emmanuel Colombo

Larry Norton

Anne Vincent-Salomon

Jorge S Reis-Filho

Britta Weigelt

Doi

10.1002/path.4573

PMID: 26095796 · DOI: 10.1002/path.4573 · Journal: J Pathol (2015)

TL;DR

Martelotto et al. performed whole-exome sequencing on 12 adenoid cystic carcinomas (AdCCs) of the breast — a rare triple-negative breast cancer subtype — combined with FISH and RT-PCR for the MYB-NFIB fusion. They found that 10/12 (83%) cases harbored the MYB-NFIB fusion, that breast AdCCs have a low mutation rate (0.27 non-silent mutations/Mb), and that, unlike common triple-negative/basal-like breast cancers, they lack TP53 and PIK3CA mutations. The mutational landscape resembled that of salivary gland AdCCs (mutations in MYB, FBXW7, SF3B1, FGFR2, PRKD1) far more than common-type BRCA, with recurrent 12q losses and intra-tumor genetic heterogeneity despite simple genomes (PMID:26095796).

Cohort & data

  • 12 breast AdCCs (fresh/frozen and FFPE) retrieved from Institut Curie (Paris) and Memorial Sloan Kettering Cancer Center (acbc_mskcc_2015).
  • All cases were triple-negative (ER−/PR−/HER2−); histologic grade 1 (58%) or grade 2 (42%); growth patterns: tubular-cribriform (67%), cribriform (17%), solid-cribriform (8%), solid-trabecular (8%).
  • Cancer type: ACBC (adenoid cystic carcinoma of breast), a special histologic subtype of BRCA.
  • Tumor and matched normal DNA underwent whole-exome-seq (Agilent SureSelect Human All Exon v4) on Illumina HiSeq 2000; median depth 78× (range 43–129×); mean 88% of target ≥10× coverage.
  • Variant calling: bwa alignment to GRCh37; mutect for SNVs; VarScan2 (varscan), strelka, and Scalpel for indels; targeted amplicon resequencing for orthogonal validation.
  • Copy number analysis: VarScan2 and gistic (GISTIC2.0); absolute for tumor purity, ploidy, and clonal heterogeneity.
  • MYB rearrangement assessment by fish (ZytoLight SPEC MYB Dual Color Break Apart Probe).
  • MYB-NFIB fusion transcripts assessed by RT-PCR and quantitative-rt-pcr; NanoString digital gene expression for 5′/3′ ratio analysis.
  • WES data deposited in NCBI SRA under accession SRP053134.

Key findings

  • MYB-NFIB fusion prevalence: 10/12 breast AdCCs (83%) harbored the MYB-NFIB fusion gene by FISH/RT-PCR; the most common chimeric transcripts were MYB exon 14 — NFIB exon 8c (n=6) or NFIB exon 9 (n=3), with one case (AdCC2T) showing MYB exon 9 — NFIB exon 8c (PMID:26095796).
  • Low mutation burden: median 12.5 non-synonymous mutations per tumor (range 6–23), corresponding to 0.27 non-silent mutations/Mb — significantly lower than basal-like breast cancers (1.41/Mb; Mann-Whitney U, p<0.001) or TNBCs (1.38/Mb; p<0.001) in TCGA, and similar to salivary gland AdCCs (0.31/Mb; p>0.1).
  • No mutations in TP53, PIK3CA, RB1, BRCA1, or BRCA2 — sharp contrast to common-type triple-negative/basal-like breast cancers.
  • 167 validated non-synonymous somatic mutations affecting 160 genes (130 missense, 18 nonsense, 10 frameshift, 6 essential splice site, 3 in-frame indels).
  • Recurrent mutations (only two): TLN2 missense mutations in 2/12 cases (AdCC2T, AdCC12T; 17%) and MYB missense mutations in 2/12 cases (AdCC1T, AdCC32T; 17%). In AdCC1T the MYB mutation occurred in the exon 13 splice site of the MYB allele participating in the MYB-NFIB fusion.
  • Cancer-related genes affected by non-passenger mutations: BRAF (1/12, 8%), FBXW7, SF3B1, FGFR2, RASA1, PTPN11, MTOR, SMARCA5 (chromatin remodeling).
  • Salivary gland overlap: 12/107 (11.2%) genes with non-passenger mutations were also mutated in salivary gland AdCCs, including SF3B1, FBXW7, FGFR2, MYB, and PRKD1.
  • Pathway enrichment: IGF1 and FGF signaling (p<0.001), EMT (p<0.001), and Ephrin receptor signaling (p<0.001) by Ingenuity Pathway Analysis and ConsensusPathDB.
  • Copy number alterations: low genetic instability; no amplifications or homozygous deletions. Recurrent losses of 12q12-q14.1 in 5/12 cases (all MYB-NFIB fusion-positive) and gains of 17q21-q25.1 in 3/12 cases. Regions typical of common-type TNBCs (8q gain, 5q loss) were absent.
  • Clonal heterogeneity: ABSOLUTE analysis showed ploidy ~2n across all cases; many mutations were clonal (cancer cell fraction ≥80%), but subclonal mutations (CCF 9–79%) affected known cancer genes including FBXW7, MTOR, KMT2D (MLL2), ARAF, and CDH1.
  • MYB-NFIB-negative cases: the 2 fusion-negative tumors (AdCC11T, AdCC12T) did not show elevated 5′ MYB / 3′ NFIB expression; however, AdCC12T showed overall MYB and 5′ MYB mRNA levels significantly higher than other fusion-negative samples (p<0.001), suggesting an alternative mechanism for MYB activation.

Genes & alterations

  • MYB — partner of t(6;9) MYB-NFIB fusion in 10/12 (83%) breast AdCCs; recurrently mutated by missense in 2/12 (17%) cases; one mutation occurred in the exon 13 splice site of the MYB-NFIB fusion allele.
  • NFIB — 3′ partner of MYB-NFIB chimeric transcripts (NFIB exon 8c or 9 fused most often to MYB exon 14).
  • TLN2 — missense mutations in 2/12 (17%) breast AdCCs (AdCC2T, AdCC12T); cytoskeletal protein involved in actin filament assembly and cell migration.
  • BRAF — mutation in 1/12 (8%) in this cohort; previously reported in 12% (3/25) of an independent breast AdCC cohort by the same group.
  • FBXW7 — non-passenger mutation; also recurrent in salivary gland AdCCs; some mutations were subclonal by ABSOLUTE.
  • SF3B1 — non-passenger mutation; shared with salivary gland AdCCs.
  • FGFR2 — non-passenger mutation; shared with salivary gland AdCCs; part of FGF-pathway enrichment.
  • SMARCA5 — chromatin remodeling gene affected by somatic mutation.
  • MTOR — non-passenger mutation in PI3K/mTOR pathway; subclonal in at least one case.
  • RASA1 — non-passenger mutation; rarely mutated in basal-like breast cancers.
  • PTPN11 — non-passenger mutation; rarely mutated in basal-like breast cancers.
  • PRKD1 — non-passenger mutation; shared with salivary gland AdCCs.
  • ARAF — subclonal mutation in at least one case.
  • KMT2D (MLL2) — subclonal mutation; chromatin remodeling.
  • CDH1 — subclonal mutation; cell adhesion.
  • TP53 — absent; sharp contrast to ~50–80% mutation rate in common-type basal-like/TNBC.
  • PIK3CA — absent; sharp contrast to ~10% mutation rate in common-type basal-like/TNBC.

Clinical implications

  • Histologic subtyping within triple-negative breast cancer matters: breast AdCCs have a favorable outcome relative to common-type TNBCs and a distinct mutational landscape, so “TNBC” is an operational umbrella term that encompasses biologically distinct lesions.
  • Agents targeting drivers of common-type TNBC (e.g. TP53/PIK3CA-pathway-directed therapies) are likely to have limited value in breast AdCC patients given the absence of these mutations.
  • The recurrent MYB-NFIB fusion and the enrichment for FGF, IGF1, and EMT signaling pathway mutations suggest potential alternative therapeutic angles for breast AdCC, though no specific drug claims are made in this paper.
  • Intra-tumor heterogeneity at diagnosis — even in tumors with simple genomes — implies that subclonal driver mutations (in FBXW7, MTOR, KMT2D, ARAF, CDH1) may underlie therapeutic escape and should be considered when designing targeted treatment strategies.

Limitations & open questions

  • Small cohort (n=12), reflecting the rarity of breast AdCCs (~0.1% of invasive breast cancers); however, this remains the largest breast AdCC cohort subjected to massively parallel sequencing as of 2015.
  • Normal breast tissue as germline source could yield false-negative somatic calls if contaminated by tumor; mitigated here by pathology review and microdissection.
  • Mutational signatures could not be derived because of the low somatic mutation count per tumor — this limits inference about underlying mutagenic processes in MYB-NFIB-driven cancers.
  • Mechanistic basis of MYB-NFIB oncogenesis (especially the 3′ NFIB role) remains uncharacterized; cell lines harboring the fusion are needed.
  • MYB-NFIB-negative breast AdCCs (AdCC11T, AdCC12T): alternative mechanisms of MYB activation/overexpression remain undefined; the authors recommend whole-genome sequencing of fusion-negative AdCCs.
  • Differences from salivary gland AdCCs were also observed (no NOTCH1/NOTCH2/SPEN mutations; no DNA-damage-response enrichment in breast AdCCs) — further studies needed to determine whether these genetic differences explain the divergent clinical behaviors of breast vs salivary AdCCs.
  • The clinical significance of intra-tumor genetic heterogeneity in a low-instability, indolent tumor remains to be elucidated.

Citations from this paper used in the wiki

  • “AdCCs have a low mutation rate (0.27 non-silent mutations/Mb), lack mutations in TP53 and PIK3CA, and display a heterogeneous constellation of known cancer genes affected by somatic mutations” (Abstract).
  • “10/12 breast AdCCs (83%) harbored the MYB-NFIB fusion gene” (Results, MYB-NFIB fusion gene prevalence).
  • “MYB and TLN2 were affected by somatic mutations in two cases each” (Abstract).
  • “the mutation rate found in breast AdCCs was more similar to that reported for pediatric malignancies and salivary gland AdCCs (0.31 non-silent mutations/Mb; Mann-Whitney U-test, p>0.1)” (Results, Spectrum of somatic mutations).
  • “The most frequent copy number alterations were losses of 12q12-q14.1 in 5/12 cases (all MYB-NFIB fusion gene-positive) and gains of 17q21-q25.1 in 3/12 cases” (Results, Landscape of somatic gene copy number alterations).
  • “subclonal mutations also affected known cancer genes, such as FBXW7, MTOR, MLL2 (KMT2D), ARAF or CDH1” (Results, Clonal heterogeneity).
  • “Whole exome sequencing data have been deposited into the NCBI Sequence Read Archive, under accession code SRP053134” (Methods).

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