Inter-tumor genomic heterogeneity of breast cancers: comprehensive genomic profile of primary early breast cancers and relapses

Background The breast cancer genome dynamically evolves during malignant progression and recurrence. We investigated the genomic profiles of primary early-stage breast cancers and matched relapses to elucidate the molecular underpinnings of the metastatic process, focusing on potentially actionable alterations in the recurrences. Methods A mono-institutional cohort of 128 patients with breast cancers (n = 68 luminal B HER2, n = 6 luminal B HER2+, n = 1 HER2+ non-luminal, n = 56 triple negative) and at least one recurrence in a timeframe of 17 years was evaluated. Next-generation sequencing comprehensive genomic profiling was performed on 289 formalin-fixed paraffin-embedded (FFPE) samples, including primary tumors and matched relapses. Correlations of genomic aberrations with clinicopathologic factors and time to breast cancer relapse were analyzed. Results Genomic data were available for 188 of 289 FFPE samples that achieved the sequencing quality parameters (failure rate 34.9%), including 106 primary tumors and 82 relapses. All primary and relapse samples harbored at least one genomic alteration, with a median number of six alterations per sample (range 1–16). The most frequent somatic genomic alterations were mutations of TP53 (primary tumors = 49%, relapses = 49%) and PIK3CA (primary tumors = 33%, relapses = 30%). Distinctive genomic alterations of primary tumors were significantly associated with molecular subtypes. TP53, PIK3R1, and NF1 somatic alterations were more frequently detected in triple negative tumors (p value < 0.05); CCND1, FGF3, and FGFR1 copy number gains were recurrently identified in luminal cases (p value < 0.05). Moreover, TP53 mutations and MYC amplification were significantly and independently associated with a shorter time to relapse (p value < 0.05). Molecular subtype changes between primary tumors and relapses were seen in 10 of 128 (7.8%) cases. Most driver genomic alterations (55.8%) were shared between primary tumors and matched recurrences. However, in 39 of 61 cases (63.9%), additional private alterations were detected in the relapse samples only, including 12 patients with potentially actionable aberrations. Conclusions Specific genomic aberrations of primary breast cancers were associated with time to relapse. Primary tumors and matched recurrences showed a core of shared driver genomic aberrations but private actionable alterations have been identified in the relapses.


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Conclusions: Specific genomic aberrations of primary breast cancers were associated with time to relapse. Primary tumors and matched recurrences showed a core of shared driver genomic aberrations but private actionable alterations have been identified in the relapses.
Keywords: Breast cancer, Recurrence, Genomic heterogeneity, TP53, MYC, Next-generation sequencing, Comprehensive genomic profile Background Breast cancer is the most commonly diagnosed cancer and the leading cause of tumor-related mortality in women worldwide [1]. Nearly 20-30% of patients with early-stage breast cancer experience local or distant recurrence even after standard loco-regional and adjuvant treatments [2]. Although great efforts have been made to identify prognostic biomarkers for risk stratification [3][4][5][6], the biological underpinnings of the recurrence are still poorly characterized, and predictive biomarkers leading to individualized treatments in the recurrence setting are still needed.
Large-scale next-generation sequencing technologies have provided valuable insights into the genomic landscape of breast cancers. The most recurrent alterations affect PIK3CA and TP53 genes. CCND1 copy number gain is more frequent in estrogen receptor (ER)-positive breast cancer. MYC amplification and homologous recombination deficiency, including BRCA1 pathogenic variants, have been reported more frequently in triple negative (ER−/PR−/HER2−) tumors [7,8]. FGFR1 amplification has been associated with poor prognosis in hormone receptor-positive, lymph node-positive breast cancers [9]. HER2 amplification [10] or PIK3CA and ESR1 mutations [11][12][13] have been linked to response to therapy. However, a heterogeneous spectrum of driver alterations characterizes the molecular portrait of invasive breast tumors and their clinical implications remain to be fully elucidated.
The dynamic evolution of the breast cancer genome from pre-invasive stages to metastasis is ruled by phenomena of spatial and temporal heterogeneity [14]. Spatial heterogeneity can involve distinct areas within a tumor causing differences at morphologic, genomic, transcriptomic, and proteomic levels. Temporal heterogeneity indicates the variations between primary and metastasis caused by the metastatic process itself as well as the therapeutic interventions administered. Therefore, given the intra-tumor genetic heterogeneity, a clonal selection event, as well as the onset of additional alterations, may occur during tumor evolution over time or in response to therapy.
In this study, we analyzed a single-institution cohort of 128 patients with early-stage breast cancer and at least one (regional or distant) recurrence in 17 years. We performed comprehensive genomic profiling of primary and matched relapsed tumors aiming to (i) define the repertoire of genetic alterations of primary and metastatic/recurrent breast cancers and (ii) their association with specific clinico-pathological features and (iii) identify additional and potentially actionable alterations in the metastasis/recurrence site.

Study population
The study population included a mono-institutional cohort of 128 patients with early-stage breast cancer that underwent surgery at the European Institute of Oncology of Milan between 1999 and 2019. All the patients had at least one loco-regional or distant relapse in a timeframe of 17 years. The median age at diagnosis was 50 years (range 24-79 years). The median time to relapse was 42.5 months, range 1-200 months. Primary and relapsed tumors were classified according to immunohistochemical surrogates, following the St. Gallen criteria [15]. Estrogen receptor and progesterone receptor (PR), HER2, and proliferation index (Ki-67) were evaluated by immunohistochemistry, and for HER2 equivocal cases (immunohistochemical score 2+), reflex fluorescence in situ hybridization (FISH) analysis was performed. The tumors were classified as luminal B HER2− (hormone receptors+/HER2−), luminal B HER2+ (hormone receptors+/HER2+), HER2+ non-luminal (hormone receptors−/HER2+), and triple negative (hormone receptors −/HER2−). The recurrence sites were divided into locoregional (axillary lymph-node, skin or soft tissue of chest wall) or distant (liver, lung, distant lymph node, pleura, distant soft tissue, ovary, bone) relapses. The clinicopathological characteristics of the study population are reported in Table 1. All patients gave written informed consent regarding the storage of any biological specimens collected in the course of diagnosis and the use of these samples for research purposes. The study was conducted in accordance with the 1964 Helsinki Declaration and later amendments.

Comprehensive genomic profiling of primary tumors and relapses
Primary tumors and recurrences were evaluated using large multi-genes NGS panels detecting different types of genetic alterations such as single nucleotide variants (SNVs), insertion/deletions (InDels), copy number variants (CNVs), and fusion genes. Representative formalin-fixed paraffin-embedded (FFPE) tumor tissue blocks of primary tumors and recurrences were retrieved from the archives of the Division of Pathology of the European Institute of Oncology. In the first phase of the study, 202 FFPE blocks were analyzed with the Foundation One test, including 315 genes (Roche Pharma AG, Grenzach-Wyhlen, Germany). We then performed a NGS panel in-house (Oncomine Comprehensive Assay v.3, ThermoFisher Scientific, Waltham, MA, USA), for 87 additional samples, following the manufacturer's instructions. Briefly, the nucleic acids (DNA and RNA) were extracted automatically using Promega Maxwell RSC DNA or RNA FFPE kit (Promega, Madison, WI, USA) and then quantified, as previously reported [16]. Ten nanograms of genomic DNA and 10 ng of RNA were used for the library preparation and the subsequent chip loading, both performed automatically on the Ion Chef System (ThermoFisher Scientific, Waltham, MA, USA). The sequencing run was done on Ion S5 System (ThermoFisher Scientific, Waltham, MA, USA) and data were analyzed using the Ion Reporter Analysis Software v5.10. Only mutations with a variant allele frequency (VAF) equal/superior to 5% and with adequate quality metrics (read depth > 100; VAF × read depth > 25; p value = 0.00001) were reported. Copy number variants were evaluated for samples with a Median of the Absolute values of all Pairwise Difference (MAPD) < 0.5 [17].
The mutations were classified as driver alterations, including all the alterations belonging to level I, II, and III class, as previously described [18] or variants of uncertain significance (VUS) if they were annotated as unknown in cancer gene mutation databases, including Catalogue of Somatic Mutations in Cancer (COSMIC) [19], cBioPortal for Cancer Genomics [20], and Clin-Var-NCBI-NIH [21], or considered damaging by "in silico" predictors only, available at VarSome website [22]. Furthermore, we evaluated the clinical actionability of driver alterations using OncoKB levels of evidence V2 ranking [23]. Variants classified as polymorphism, benign, likely benign, or neutral were not reported. The co-occurrence of selected gene alterations was evaluated using the mutual exclusivity analysis of cBioPortalbioinformatics tool [20].
For this analysis, only alterations occurring in genes targeted by both the FoundationOne and Oncomine Comprehensive Assays (Additional file 1 for the complete gene list) were evaluated. Overall, 188 of 289 (65.1%) samples analyzed with large NGS panels achieved the quality parameters required by the specific assay, including 106 primary and 82 recurrence tumors. Moreover, for 70 of 128 (54.7%) patients, both primary and matched relapse sample were successfully profiled with the same NGS panel.

Statistical analysis
Patient clinico-pathological characteristics were reported with median and interquartile range (IQR) for continuous variables and absolute and relative frequencies for categorical variables. Cohen's Kappa test was used to assess the driver gene alteration agreement between primary and relapse samples. Genes with a Kappa coefficient over 60% and a mutation frequency over 10% were The study populations included 128 women affected by breast cancer and relapsed in a timeframe of 17 years IHC immunohistochemistry, pT pathologic stage classification of primary tumor, pN pathologic stage classification of regional lymph nodes, IBC invasive breast carcinoma, NA not available *N = 133, 5 patients had multiple primary tumors **N = 135, 6 patients had multiple recurrences considered in this analysis. Hamming distance for binary variables was applied to implement a heatmap of the selected driver genes. Univariate logistic models were used to assess the associations of gene aberrations with molecular subtype (triple negative vs luminal B) and recurrence site (distant vs loco-regional). The results were shown using the R package "EnhancedVolcano" for Volcano plot implementation. The association of driver gene alterations with time to recurrence was analyzed with a Log-rank test. We also evaluated the association of the total number of mutations in primary breast cancer samples as categorical variables, with the median value as cutoff. Multivariate Cox proportional hazard models were chosen considering backward and forward selection of variables and adjusting for known prognostic factors (molecular subtype and pT). Hazard ratios (HR) with 95% confidence intervals (95% CI), from multivariate models, were reported. Since the survival analyses were carried out only for patients with a relapse, hazard ratios represented a measure of the association with time to relapse and they should not be interpreted as probability of relapse. Molecular subtype changes between primary breast cancer and relapse samples were displayed in a Chord Diagram from the R package "circlize." Finally, Fisher exact tests were employed to estimate whether gene aberrations in primary and recurrence samples were significantly different by molecular subtype (triple negative vs luminal B). Only gene alterations with a frequency over 5% in both primary breast and relapse groups were used in this analysis. Due to the explorative nature of this study, a multiplicity correction was omitted.

Results
Molecular portraits of primary early breast cancers and correlations with clinico-pathological characteristics The most frequently mutated genes were TP53 and PIK3CA (Fig. 1a), with alterations spanning the whole coding sequence of TP53 and involving hotspot regions in PIK3CA (Additional file 3). Moreover, recurrent copy number gains were identified in MYC, CCND1, FGF19, FGF3, and FGFR1 (Fig. 1a). Among them, CCND1, FGF19, and FGF3 genes mapped on the same cytogenetic band, 11q13.3, and showed a statistically significant co-occurrence (p value < 0.001 and q < 0.01). No other significant driver alterations co-occurrence was found. Distinctive genomic alterations were significantly associated with molecular subtypes (Additional files 4 and 5). In particular, TP53, PIK3R1, and NF1 mutations were detected more frequently in triple negative tumors (odds ratio > 2.71, p value < 0.05). CCND1, FGF3, and FGFR1 copy number gains were recurrently identified in luminal cases (odds ratio < 0.36, p value < 0.05) (Additional file 4).
Moreover, TP53 mutations and MYC copy number gain were significantly associated with shorter time to relapse, both in univariate and multivariate analyses, adjusted for known prognostic factors (p value < 0.05) (Fig. 2, Table 2). Also, an increased number of alterations, including both driver and VUS variants, was associated with a shorter time to relapse in univariate analysis, even if not statistically significant (p value 0.06, Fig. 2).
TP53 mutations and MYC copy number gain findings in primary breast cancers were prognostic factors independently associated with shorter time to relapse, along with a high primary tumor pathologic stage (pT = 2-4) and triple negative molecular subtype.
Similarly to the primary tumors, specific alterations identified in the recurrence samples were associated with Fig. 1 Distribution and co-occurrence of recurrent driver genomic alterations. Oncoprint plots showed genes altered in more than 5% of breast cancers samples. a Primary tumors (n = 106). b Recurrences (n = 82). Each gene was reported in rows; each case was reported in columns. Significant co-occurrent and recurrent copy number gains involved CCND1, FGF19, and FGF3 genes (p value < 0.001 and q < 0.01, according to mutual exclusivity analysis). Oncoprinter tool -cBioportal (https://www.cbioportal.org/oncoprinter) was used to create graphs and perform mutual exclusivity analysis molecular subtypes (Additional files 4 and 5), including TP53 and NF1 alterations in triple negative tumors (odds ratio > 2.71 p value < 0.05) and CCND1, FGFR1, and ESR1 aberrations in luminal cases (odds ratio < 0.36 p value < 0.05).

The evolution of genomic landscape between primary and matched breast cancer relapses
In 70 cases, we successfully analyzed primary tumors and matched recurrences with the same NGS panel. Among these, 61 patients had a single primary and relapse specimen (Fig. 3). We found that 55.8% of driver alterations were shared between primary tumors and recurrences, with a median number of two aberrations in common per sample (range 0-7). Including the variants of unknown significance, the prevalence of shared alterations was 61.2% (median = 5; range 0-11) (Additional file 7). Moreover, the most recurrent driver alterations identified in the primary samples were maintained in the recurrence, including mutations of TP53 and PIK3CA and copy number gain of CCND1, FGF19, FGF3, and FGFR1 (Fig. 4). The number of shared aberrations was significantly associated with the time to relapse. Considering the median relapse time (50 months) of the cases successfully profiled, those with early and late recurrences showed a different proportion of shared aberrations (66.7% versus 56%, respectively) and driver (62.9% versus 49.3% respectively) alterations (Additional file 8). Indeed, in 39 of 61 patients (63.9%), additional private alterations were detected in the recurrence samples only, affecting breast cancer-related genes as ERBB2, ESR1, FGFR1, or NF1, and including 12 patients (19.7%) with clinically relevant alterations according to the OncoKB levels of evidence V2 ranking (levels 1-3) (Additional files 9 and 10). Nine cases had multiple primary and/or recurrence samples available for the analysis (Additional file 11). Although spatial and temporal heterogeneity was seen, most driver alterations were retained in the different recurrence samples.

Discussion
In the present study, we performed a comprehensive genomic profile of 106 primary breast cancers and 82 recurrences, including 70 cases with matched primary and relapse samples. We identified specific molecular characteristics of primary tumors associated with time to relapse. Moreover, we showed that a backbone of recurrent driver molecular alterations of primary tumors was retained in the recurrences. However, additional private genomic aberrations were detected in relapse samples, including clinically relevant genes and potentially actionable targets. Overall, more than 95% of the specimens under investigations, including both primary and recurrences, harbored at least one driver mutation, with a median number of three driver alterations per sample. As previously reported in larger series [24,25], we found heterogeneous genomic profiles, with few recurrent molecular aberrations, including mutations of TP53 and PIK3CA and copy number gains MYC, CCND1, FGF19, FGF3, and FGFR1. Specific alterations were significantly associated with breast cancer subtypes in both primary tumors and recurrences. TP53 and NF1 mutations were more frequently identified in triple negative breast cancers whereas CCND1, FGF3, FGF19, ESR1, and FGFR1 copy number gains were recurrent in tumors of luminal subtype. In this latter group, a significant association between ESR1 alterations and recurrence was seen, probably reflecting the mechanism of acquired resistance to endocrine therapies [26].
TP53 mutations and MYC copy number gains in primary tumors were significantly associated with the time to relapse. This association was retained in the multivariable analysis adjusted for known prognostic factors. The clinical value of somatic TP53 mutations has been largely evaluated according to a specific mutation type, protein domain involved, gene expression data (i.e., PAM 50), and hormone receptor status [27][28][29]. Moreover, MYC deregulation plays a critical role in cell proliferation and tumor progression, and it has been associated with an aggressive clinical behavior and poor prognosis in breast cancer [30][31][32][33][34][35]. As previously reported [36], our data suggest that MYC and TP53 alterations may represent independent poor prognostic factors in early-stage breast cancer. Moreover, an increased number of total mutations in primary tumors could be associated with a shorter time to relapse. Even if different NGS panels have been used in this study and the data should be further validated, these findings are consistent with the observation that a high tumor mutation burden may correlate with a poor prognosis in various cancer types [37][38][39].
To focus on the dynamic evolution of breast cancer genome, we firstly compared the overall data between primary and recurrence tumors. A trend of an increasing number of driver alterations of breast cancer-related genes was observed in recurrence samples as compared to primary tumors, including alterations in FGFR1, ESR1, NF1, BRCA1, and PTEN genes. These data may have a clinical impact since the additional burden of Fig. 3 Distribution of driver and VUS alterations in 61 matched primary tumors and relapses. Each column represents one case, with primary tumors in blank columns and matched relapses in dashed columns. The cases were grouped according to the molecular subtype (luminal B, triple negative or HER2-non luminal) and the recurrence site (distant vs loco-regional) color-coded as in the legend. The most frequently altered genes (occurring in more than 5% of samples) and the type of alterations (driver or VUS) were reported in rows and color-coded according to the legend. The total number of alterations affecting each sample was shown in the lower part of the figure alterations included actionable or druggable genes [13,40,41] or genes related to therapy resistance. In particular, NF1 alterations have been reported in association with endocrine therapy resistance in lobular breast cancer [42]. Recently, Pearson et al. showed that NF1 mutations were frequently acquired in breast cancer at progression and were associated with shorter survival in hormone receptor-positive breast cancers relapsing during adjuvant endocrine therapy [43]. However, in our series, mutations of NF1 were more frequently detected in tumors of triple negative subtype. Further studies are needed to confirm these findings and to unveil the biological and clinical significance of this alteration in triple negative breast cancers.
We evaluated in detail the evolution of breast cancer biology at the single patient level with the analysis of matched primary and relapse samples. Molecular subtype changes from primary tumors to recurrences were seen in 7.8% of cases. These proportions were lower but consistent with previously reported meta-analysis data of larger cohorts [44][45][46][47][48][49][50][51]. We observed a high level of concordance (55.8%) of genomic aberrations between primary and matched relapse specimens. However, the proportion of shared aberrations was lower in cases with later recurrence (49.3%). Indeed, Yates and colleagues showed that the number of mutations was similar in primary and synchronous metastasis but a high number of mutations accumulated during breast cancer evolution and can be detected in samples from late relapses [52]. Moreover, 63.9% of cases had private alterations in their recurrence, including 19.7% of patients with clinically actionable aberrations (e.g., affecting ERBB2, BRCA2, PIK3CA) that may be targeted by available biological drugs [23].
This study has several limitations. First, given the failure rate of NGS performed on nucleic acids extracted from old FFPE samples, we were able to evaluate only 65.1% of the cases. As known, stringent NGS quality Fig. 4 Concordance of the driver genetic alterations identified in primary tumors and matched relapses. Only genes altered in more than 10% of the study population and with a concordance of at least 60% between matched primary tumor and relapse were reported in the heatmap, with primary tumors on X-axis and matched recurrence samples on Y-axis. Darker blue color indicated a higher level of gene driver alteration concordance. The recurrent co-occurrence of copy number gains of CCND1, FGF19, and FGF3 genes was pin-pointed metrics are needed to obtain robust results when longterm stored FFPE specimens are investigated. In our cohort, 73 (25.3%) specimens had an archival time longer than 10 years, and 79 (27.3%) cases longer than 5 years. Second, using two different NGS panels (FoundationOne and Oncomine Comprehensive Assay), only data about genes included in both panels were considered and only matched primary tumors and recurrences investigated with the same panel could be analyzed. Third, given the retrospective nature of this study including a heterogeneous cohort of patients with breast cancer, we were unable to perform detailed survival analysis or to test the effect of therapy on molecular alterations acquired at progression. Finally, this is an exploratory hypothesisgenerating study evaluating the genomic profile of primary breast cancers and breast cancer relapses to investigate inter-tumor genomic heterogeneity. Although the most recurrent driver alterations of primary tumors were detected in matched relapses, we showed that additional and potentially actionable alterations may be detected in the recurrence sample only, as showed by OncoKB levels assessment. Given that we analyzed samples from a retrospective cohort of patients, we could not assess the clinical impact of these findings. However, our data may suggest that inter-tumor genomic heterogeneity of breast cancers might be of clinical relevance and the genomic profile of breast cancer relapses might guide patients tailored treatments. Further ad hoc investigation is needed to confirm our findings and evaluate their clinical impact.

Conclusions
In conclusion, this study showed that the presence of TP53 mutations and MYC copy number gain in primary early-stage breast cancers were independently associated with time to relapse. A trend of association between the number of genomic alterations and time to relapse was seen and required further investigation. Although shared driver aberrations were identified in primary tumors and matched recurrences, comprehensive genomic profiling of relapse samples may reveal additional private and actionable alterations.