Homologous recombination DNA repair defects in PALB2- associated breast cancers

Abstract

Mono-allelic germline pathogenic variants in the Partner And Localizer of BRCA2 (PALB2) gene predispose to a high-risk of breast cancer development, consistent with the role of PALB2 in homologous recombination (HR) DNA repair. Here, we sought to define the repertoire of somatic genetic alterations in PALB2-associated breast cancers (BCs), and whether PALB2-associated BCs display bi-allelic inactivation of PALB2 and/or genomic features of HR-deficiency (HRD). Twenty-four breast cancer patients with pathogenic PALB2 germline mutations were analyzed by whole-exome sequencing (WES, n = 16) or targeted capture massively parallel sequencing (410 cancer genes, n = 8). Somatic genetic alterations, loss of heterozygosity (LOH) of the PALB2 wild-type allele, large-scale state transitions (LSTs) and mutational signatures were defined. PALB2-associated BCs were found to be heterogeneous at the genetic level, with PIK3CA (29%), PALB2 (21%), TP53 (21%), and NOTCH3 (17%) being the genes most frequently affected by somatic mutations. Bi-allelic PALB2 inactivation was found in 16 of the 24 cases (67%), either through LOH (n = 11) or second somatic mutations (n = 5) of the wild-type allele. High LST scores were found in all 12 PALB2-associated BCs with bi-allelic PALB2 inactivation sequenced by WES, of which eight displayed the HRD-related mutational signature 3. In addition, bi-allelic inactivation of PALB2 was significantly associated with high LST scores. Our findings suggest that the identification of bi-allelic PALB2 inactivation in PALB2-associated BCs is required for the personalization of HR-directed therapies, such as platinum salts and/or PARP inhibitors, as the vast majority of PALB2-associated BCs without PALB2 bi-allelic inactivation lack genomic features of HRD.

Keywords: Breast cancer; Cancer genetics; Cancer genomics.

Fig. 1

Non-synonymous somatic mutations in PALB2-associated breast cancers. Heatmap depicting the somatic genetic alterations identified in the 24 PALB2-associated breast cancers analyzed by whole-exome (n = 16) or targeted MSK-IMPACT (n = 8) massively parallel sequencing. Somatic mutations affecting the 410 cancer genes present in MSK-IMPACT, in decreasing overall mutational frequency observed in PALB2-associated breast cancers are plotted. Cases are shown in columns, and genes in rows. Estrogen receptor (ER) and HER2 status, PALB2 germline mutation type, presence of a second somatic PALB2 mutation or loss of heterozygosity (LOH) of the PALB2 wild-type allele, large-scale state transition (LST) score, dominant mutational signature and sequencing platform are indicated in the phenobar (top), color-coded according to the legend. Note that mutational signatures and LST scores could not be assessed in tumors subjected to MSK-IMPACT sequencing due to the limited number of mutations present. Clonal somatic PALB2 mutations or clonal LOH of the PALB2 wild-type allele are indicated by yellow boxes. Somatic mutations are color-coded according to the legend, and LOH of the wild-type allele of mutated genes other than PALB2 is represented by a diagonal bar. Indel small insertion/deletion; LOH loss of heterozygosity, LST large-scale state transition, N/A not assessable, SNV single nucleotide variant, WES whole-exome sequencing

Fig. 2

Repertoire of copy number alterations in PALB2-associated breast cancers. Copy number alterations in the 24 PALB2-associated breast cancers analyzed by whole-exome (n = 16) or targeted MSK-IMPACT (n = 8) massively parallel sequencing. Cases are represented in rows and chromosomes in columns along the x-axis. Immunohistochemical features, PALB2 germline mutation type, presence of a somatic PALB2 mutation or loss of heterozygosity (LOH) of the PALB2 wild-type allele, large-scale state transition (LST) score, mutational signature and sequencing platform are provided in the phenobar (left), color-coded according to the legend. Dark red, amplification; light red, copy number gain; dark blue, homozygous deletion; light blue, copy number loss; white, no change. LOH loss of heterozygosity, LST large-scale state transition; N/A not assessable, SNV single nucleotide variant, WES whole-exome sequencing

Fig. 3

HRD genomic features in breast cancers with and without bi-allelic PALB2 inactivation. a Mutational signatures of all somatic SNVs in the 16 PALB2-associated breast cancers sequenced by whole-exome sequencing (left) as inferred by deconstructSigs based on the 30 signatures represented in COSMIC, and a bar plot indicating the proportion of the major mutational signatures identified in each case (right), in decreasing proportion of each signature. The dominant mutational signatures were assigned according to Alexandrov et al., following the consensus of at least two of three approaches (deconstructSigs based on 30 signatures from COSMIC, based on the 12 signatures known to occur in breast cancer, and NMF method based on 30 signatures from COSMIC) where signature 1 relates to aging and signature 3 to defective homologous recombination DNA repair, and are shown for cases with bi-allelic PALB2 alterations (top) and mono-allelic PALB2 alterations (bottom). The number of SNVs is shown in parentheses. Sig signature, SNV single nucleotide variant. b Large-scale state transition (LST) scores of the four PALB2-associated breast cancers with mono-allelic PALB2 alterations and the 12 PALB2-associated breast cancers with bi-allelic PALB2 alterations. The median LST scores, and the 75th and 25th percentiles are displayed at the top and bottom of the boxes, respectively. Each dot corresponds to the LST score and the mutational signature of a given case. Dominant mutational signatures are color-coded according to the legend. Comparisons of LST scores between groups were performed using the Mann–Whitney U test. c Average deletion length (nucleotides) in PALB2-associated breast cancers with mono-allelic PALB2 alterations (n = 3) and with bi-allelic PALB2 alterations (n = 11). Only PALB2-associated breast cancers harboring small insertions and deletions were included in the analysis. The median value of deletion length, and the 75th and 25th percentiles are displayed at the top and bottom of the boxes, respectively. Comparisons of deletion lengths between groups were performed using the Mann–Whitney U test. d Number of genes affected by copy number alterations (CNAs) of the four PALB2-associated breast cancers with mono-allelic PALB2 alterations and the 12 PALB2-associated breast cancers with bi-allelic PALB2 alterations. The median value of the number of genes with CNAs, and the 75th and 25th percentiles are displayed at the top and bottom of the boxes, respectively. Comparisons were performed using Fisher’s exact test

Fig. 4

Comparison of PALB2-associated breast cancers and non-BRCA1/2/PALB2-associated breast cancers. a, b Heatmap depicting the most recurrently mutated genes affecting 410 cancer genes identified in PALB2-associated breast cancers and non-BRCA1/2/PALB2-associated breast cancers from TCGA. Cases are shown in columns, genes in rows. Multi-Fisher’s exact test comparisons of mutational frequencies of the mutated genes were performed between a the 24 PALB2-associated breast cancers and the 683 ER+/HER2−, ER+/HER2+ and ER−/HER2− non-BRCA1/2/PALB2-associated breast cancers from TCGA, and b the 18 ER+/HER2− PALB2-associated breast cancers and the 441 ER+/HER2− non-BRCA1/2/PALB2-associated breast cancers from TCGA. P-value of each comparison is shown on the right side of the heatmap, with statistically significant P-values in bold. Indel, small insertion/deletion; SNV, single nucleotide variant. c Box and whisker plots showing the large-scale state transition (LST) scores of the PALB2-associated breast cancers with mono-allelic/bi-allelic PALB2 alterations, ER−/HER2− and ER+ non-BRCA1/2/PALB2-associated breast cancers, and ER−/HER2− and ER+/HER2− non-BRCA1/2/PALB2-associated breast cancers where LST scores could be inferred. The median value of LST scores, and the 75th and 25th percentiles are displayed at the top and bottom of the boxes, respectively. Each dot represents the LST score and/or mutational signature of a given case. Mutational signatures are color-coded according to the legend. *of the 601 ER−/HER2− and ER+ non-BRCA1/2/PALB2-associated breast cancers, the 34 cases lacking LST scores but displaying mutational signatures are not shown, three of these cases display signature 3. **of the 491 ER+/HER2− non-BRCA1/2/PALB2-associated breast cancers, the 29 cases lacking LST scores but displaying mutational signatures are not shown, three of these cases display signature 3. P-values of the comparisons of LST scores are shown using Fisher’s exact tests. N/A signatures not assessable, LST large-scale state transition

Fig. 5

Comparison of PALB2-associated breast cancers and BRCA1 and BRCA2 breast cancers. a, b Heatmap depicting the most recurrently mutated genes affecting 410 cancer genes identified in PALB2-associated breast cancers and BRCA1 and BRCA2 breast cancers from TCGA. Cases are shown in columns, and genes in rows. Multi-Fisher’s exact test comparisons of mutational frequencies of the recurrently mutated genes were performed between a the 16 PALB2-associated breast cancers with bi-allelic PALB2 alterations and 17 BRCA1 breast cancers bi-allelic BRCA1 alterations, and b the 16 PALB2-associated breast cancers with bi-allelic PALB2 alterations and 16 BRCA2 breast cancers with bi-allelic BRCA2 alterations. P-value of each comparison is shown on the right side of the heatmap, with statistically significant P-values in bold. Indel, small insertion/deletion; SNV, single nucleotide variant. c Boxplots showing the large-scale state transition (LST) scores of the 12 PALB2-associated breast cancers with bi-allelic PALB2 alterations, 17 BRCA1 and 16 BRCA2 breast cancers with bi-allelic BRCA1 and BRCA2 alterations, respectively. The median value of the LST scores, and the 75th and 25th percentiles are displayed at the top and bottom of the boxes, respectively. Each dot corresponds to the LST score and/or mutational signature of one case. Mutational signatures are color-coded according to the legend. P-values of the comparisons of LST scores are shown using Fisher’s exact tests. N/A signatures not assessable, LST large-scale state transition