Targeted next-generation sequencing assays using triplet samples of normal breast tissue, primary breast cancer, and recurrent/metastatic lesions

Studies using next-generation sequencing (NGS) have demonstrated remodeling of genes in metastatic cancer compared with the primary tumor from the same individual, possibly as a result of subclonal diversification or mutational evolution [1‐10]. Whole-exon or whole-genome analyses can yield valuable information but are costly and time consuming. Whole-exon sequencing analysis of metastatic breast cancers showed drug-targetable mutations in genes such as ERBB4, NOTCH3, and ALK [11]. A targeted NGS assay of metastatic breast cancers and their associated primary tumor using a general cancer panel can also detect drug-targetable mutations. Vasan et al. reported that 84% (43/51) of metastatic breast cancers showed at least one genomic alteration that could be targeted by currently available drugs [12]. In 2016, Muller et al. reported that 45% (10/22) of metastatic breast cancers contained molecular targets for currently available therapies, including for off-label use [13]. In these studies, comprehensive cancer panels that were not breast cancer specific were used. To our knowledge, there has been no study using breast cancer-targeted NGS of paired samples of primary breast cancer and recurrent/metastatic cancer.

The aim of the present study was to perform breast cancer-targeted NGS to compare gene mutations and copy number variations (CNVs) in samples of primary breast cancer and recurrent/metastatic lesions from the same individuals. Becuse sequencing analyses of matched tumor and normal tissue are essential [14], we performed an NGS study using three or more samples from each patient, consisting of normal breast tissue, primary breast cancer, and recurrent/metastatic lesion(s). This study aimed to clarify the practical possibility of breast cancer-targeted NGS and improve understanding of subclonal diversification or mutational evolution of metastatic breast cancers.

We showed that older FFPE materials often had lower DNA quality and could not be analyzed. The depth discrepancy between molecular-tagged sites and all sites is interesting. The Spearman’s correlation coefficients with age were − 0.586 for tagged sites and − 0.412 for all sites, suggesting that the molecular tag might be sensitive to age-related DNA damage. PAXgene tissue fixation and/or low-temperature paraffin block storing at 4 °C or − 20 °C could improve DNA quality [17].

For 66 of 107 cases (61.7%), the tumor content in samples of primary tumors and/or recurrent/metastatic tumors was less than 20%. The cell blocks of fluid materials frequently included many inflammatory cells and reactive mesothelial cells together with cancer cells; hence, the extraction of tumor DNA from these cell blocks was often inefficient. A primary breast cancer containing abundant stromal lymphoplasmacytic cell infiltration or a severe desmoplastic reaction would also inhibit effective cancer genome recovery. Thus, the development of a more efficient microdissection system is desirable.

We demonstrated that targeted NGS using the QIAseq Human Breast Cancer Panel could detect the driver mutations in all cases of breast cancer examined, except for one case with a germline TP53 mutation that did not meet the classic criteria for Li–Fraumeni syndrome [18] or the Chompret criteria [19].

The most frequently detected mutation shared between primary and metastatic lesions was in TP53. Breast cancer patients with a somatic TP53 mutation have a poor prognosis [20, 21]; unfortunately, TP53 mutations are not presently targetable. Phosphatidylinositide 3-kinase (PI3K) inhibitor could be a good therapeutic option for cases with PIK3CA mutations [22‐24]. Alpelisib has been reported to improve the survival patients with PIK3CA-altered, ER-positive, HER2-negative breast cancer [25, 26]. Alpelisib is already approved by the US Food and Drug Administration. The Lotus trial showed that ipatasertib, an oral AKT inhibitor, improved the progression-free survival of breast cancer patients with PIK3CA/AKT/PTEN mutations [27]. IPATunity130, a pivotal randomized phase III trial evaluating ipatasertib (IPAT) + paclitaxel for PIK3CA/AKT1/PTEN-altered advanced triple-negative or hormone receptor-positive HER2-negative breast cancer, is ongoing (http://​ascopubs.​org/​doi/​abs/​10.​1200/​JCO.​2018.​36.​15_​suppl.​TPS1117). Breast cancers with a PIK3CA mutation have a good prognosis [21]. CDH1-mutated breast cancer cells are sensitive to ROS1 tyrosine kinase inhibitors including foretinib or crizotinib [28]. ESR1 mutations of breast cancer are often reported after aromatase inhibitor and/or tamoxifen therapy [29]. The SoFEA (Study of Faslodex Versus Exemestane With or Without Arimidex) trial showed that cases with ESR1 mutations had better survival when treated with fulvestrant compared with exemestane [30].

Of the additional mutations detected, ERBB2 p.S310F is notable because it results in activation of HER2 without gene amplification or protein overexpression [31]. A tumor with this mutation is likely to be sensitive to neratinib, as are those with G660D, R678Q, E693K, and Q709 mutations [32]. The neratinib HER Mutation Basket Study (SUMMIT) has already started [33]. A PBRM1 mutation may evoke immunotherapy resistance [34].

Cases with PTEN loss could be treated with PI3K/AKT inhibitor [35]. Case K18 and K28 (triple-negative cancers) with CCND1 gene amplification might be sensitive to CDK4/6 inhibitors [36]. Meanwhile, erdafitinib and cetuximab might be effective for cases K30 and K32 in which the metastatic lesions contained FGFR and EGFR amplifications [37, 38]. Case K30 and K32, which had decreased ATM might be sensitive to topotecan or the poly-(ADP-ribose) polymerase inhibitor olaparib [39]. CDKN2A or RB1 downregulation could be target of palbociclib [40].

The present study with only 93 genes analyzed showed actionable mutations or CNVs in 73% (8/11) of recurrent/metastatic breast cancer lesions. This is comparable to the findings of previous studies including MSK-IMPACT (61%), the study by Vasan et al. (84%), and the study by Muller et al. (45%) [12, 13, 41].

The major limitations of the present study are its small scale and subtype bias, for which problems with DNA availability are responsible. Patients with luminal breast cancer often show late recurrence/metastasis, such as up to 18 years after diagnosis in our series. The primary tumor blocks of such cases are too old and rarely maintain sufficient DNA quality. In contrast, triple-negative breast cancer usually recurs shortly after surgery. Another limitation is the determination of the cutoff level for CNV. The relationship between drug sensitivity and CNV remains to be elucidated. Moreover, additional immunohistochemistry might be helpful for cases with altered CNVs of EGFR or FGFR. We could detect the somatic driver mutations or germline mutations in five triple-negative cancers; however, the genes covered by the QIAseq Human Breast Cancer Panel might be inadequate for analysis of triple-negative breast cancers because these cancers are known to have highly variable mutations [42].

Our targeted NGS assay using the QIAseq Human Breast Cancer Panel showed that recurrent/metastatic breast cancer lesions sometimes gained additional mutation and CNV. This method assists the identification of drug-targetable mutations and changes in CNV levels. The performance of an expanded study including an analysis of drug sensitivity is to be encouraged.