Frequency of genetic alterations differs in advanced breast cancer between metastatic sites

About 20%–30% of breast cancer (BC) patients will develop distant metastases, preferentially in bones, liver, lung, and brain. BCs with different intrinsic subtypes prefer different sites for metastasis. These subtypes vary in the abundance of genetic alterations which may influence the localization of metastases. Currently, information about the relation between metastatic site and mutational profile of BC is limited. In this study, n = 521 BC metastases of the most frequently affected sites (bone, brain, liver, and lung) were investigated for the frequency of AKT1, ERBB2, ESR1, PIK3CA, and TP53 mutations via NGS and pyrosequencing. Somatic mutations were present in 64% cases. PIK3CA and TP53 were the most frequently mutated genes under study. We provide an analysis of the mutational profile of BCs and the affected metastatic site. Genetic alterations differed significantly depending on the organ site affected by metastases. TP53 mutations were mostly observed in brain metastases (51.0%), metastases outside of the brain revealed a much lower proportion of TP53 mutated samples. PIK3CA mutations are frequent in liver (40.6%), lung (36.8%), and bone metastases (35.7%), whereas less common in brain metastases (18.4%). The highest percentage of ESR1 mutations was observed in liver and lung metastases (about 30% each), whereas metastatic lesions in the brain showed significantly less ESR1 mutations, only in 2.0% of the cases. In summary, we found significant differences of mutational status in mBC depending on the affected organ and intrinsic subtype. Organotropism of metastatic cancer spread may be influenced by the mutational profile of individual BCs.

Different cancers show a specific pattern of metastasis involving distant organs.For example, most patients with advanced prostate cancer suffer from bone metastases, while liver metastases are predominantly observed in patients with pancreatic cancer. 8,9In contrast, other tumor types, such as BC, commonly metastasize to multiple organs (bone, brain, liver, and lung). 1,7,10,11The most common site of metastases is the bone which occurs in 70% of mBC patients. 12,13 is a heterogeneous disease with morphological, molecular, and clinical diversity. 14,155][16] Tumors with different subtypes show distinguishing clinical features and therapeutic responses, including metastatic spread. 17,18ch intrinsic subtype has different preferential sites of metastatic involvement. 19,20Luminal BCs commonly metastases to the bone, whereas HER2-positive BC is associated with a higher frequency of liver metastases. 21HER2-positive and triple-negative BC (TNBC) often metastasizes to the central nervous system. 20,21In addition intrinsic subtypes vary in the abundance of genetic alterations. 22,23e survival outcome of mBC patients is associated with the organ to which BC is metastasized. 24Each organ varies in its physical accessibility, vascular and nutrient supply, and stromal composition, thus placing demands on infiltrating cancer cells. 25Most of the previous studies encompassing larger cohorts of mBC patients were conducted on plasma samples (liquid biopsy). 4,19,26Mutation detection in circulating tumor DNA (ctDNA) may miss a considerable proportion of cases. 27There are only few studies which exploited DNA from metastatic biopsies, but mostly without differentiation between metastatic sites. 22,28,29Bertucci et al. differentiated BC metastases from lymph node, skin, liver, lung, and soft tissue, but not from brain and bone. 29erefore, information about the relation between metastatic site and mutational profile of BC is limited.
In this study BC organ metastases of the most frequently affected sites, including bone, brain, liver, and lung were investigated for the prevalence of AKT1, ERBB2, ESR1, PIK3CA, and TP53 mutations.
The aim of the study was to compare mutation frequencies of these genes with the metastatic site and clinicopathological parameters.
TP53 is one of the most frequently mutated genes in invasive BC, occurring in about 30%-35% of all cases, and are mostly found in TNBC tumors. 35,36TP53 is activated in DNA damage response and is involved in cell-cycle arrest and apoptosis. 37Therefore, the TP53 mutation is a marker for resistance to chemotherapy and radiotherapy and, consequently, a poor prognosis of the disease. 38,39| MATERIALS AND METHODS

| Tumor specimens
Tumor tissues included n = 306 mBCs from the archive of the institute of pathology of Hannover Medical School MHH.Samples with one metastatic lesions to brain, liver, lung, or bone marrow, with available archive paraffin blocks in the time period between 2001 and 2022 were entered into the study.Furthermore, n = 231 cases of an already published bone metastases cohort were added. 40,41In total, n = 521 cases (521/537, 97.0%) with BC metastases yielded enough DNA of sufficient quality for further studies.From these cases 389 metastatic bone lesions, 49 metastases to the brain, 64 to the liver, and 19 to the lungs were analyzed.For every patient one metastatic tissue taken at the time point of metastatic diagnosis was analyzed.
Expression of estrogen receptor (ER) and progesterone receptor (PR) as well as HER2 status was determined according to established guidelines 42 using immunohistochemistry and in-situ hybridization in case of equivocal HER2 results (described previously 43 ).
Because many samples were sent to us from other hospitals for an external histopathological assessment we often had only limited clinical data from these patients (e.g., no hormone receptor status or HER2 status).The characteristics of the study population included in this retrospective molecular analysis for the final statistical analysis are reported in Table 1.The local ethic committee (MHH, Hannover) approved this study.

| DNA extraction
Depending on tumor size 6-8 sections (10 μm) were taken from tumor-bearing paraffin blocks.Genomic DNA was extracted from formalin-fixed paraffin embedded (FFPE) specimens with the Maxwell ® RSC DNA FFPE KIT on a Maxwell ® RSC instrument (Promega, Madison, WI, USA) according to the manufacturer's recommendations.DNA quantification was performed using the Qubit 2.0 Fluorometer with dsDNA high sensitivity Assay kit (ThermoFisher Scientific, Dreieich, Germany).

| Next generation sequencing
Mutational analysis of ERBB2, ESR1, PIK3CA, and TP53 was carried out by next generation sequencing (NGS) using a customized NGS panel, which covered the complete protein-coding sequences.Library preparation was performed with Ion AmpliSeq™ Library Kit 2.0 (Thermo Fisher Scientific, Waltham, MA, USA).For quantification of prepared libraries, the Ion Library TaqMan™ Quantitation Kit (Thermo Fisher Scientific) was used.Sequencing was performed on an Ion S5 instrument (Thermo Fisher Scientific).Mean mapped reads per sample was 842 985 (range 27 429 to 13 844 604) (Table S1).Evaluation of sequencing data and variant annotation was performed with the ANNOVAR software and database tools (http://annovar.openbioinformatics.org/en/latest/). 44riants with unknown significance were predicted as deleterious, when they were considered as pathogenic in the following in silico prediction tools: MutationTaster, SIFT, and PolyPhen-2.
DNA is amplified in a total volume of 25 μL using 0.5 units Platinum-Taq, 1.5 μM MgCl 2 , 0.2 μM of each dNTP, and 0.2 μM of each AKT1 primer (Table S2).The PCR conditions were as follows: initial denaturation step at 95 C for 5 min, followed by 40 cycles of denaturation at 95 C for 30 s, annealing at 60 C for 45 s, and elongation at 72 C for 30 s, concluding with a final elongation step at 72 C for 5 min.The PCR products were checked by polyacrylamide gel electrophoresis before sequencing.

| Statistics
Statistical analyses were performed with GraphPad Prism software Version 5.00 (GraphPad Software, San Diego, CA, USA).The two-sided Fisher's exact test, one-way ANOVA with Bonferroni's multiple comparison test, and the Chi square test were used for contingency analyses.Results were considered as statistically significant if p ≤ 0.05.
T A B L E 1 Characteristics of the study cohort.Unless otherwise stated, the values are given in the format n (%), with n, number of patients.HER2 positive cases were either immunohistochemically 3+ or 2+ and HER2 gene amplified according to in situ-hybridization.TP53 mutations were mostly present in the DNA binding domain (Figure 2).

| Frequency of ESR1 and TP53 mutations differ depending on the metastatic site
Depending on the organ site affected by metastases the genetic alterations differed significantly (Table 3 and Figure 4).only one out of 49 cases (2.0%).TP53 mutations were mostly observed in brain metastases (51.0%), with a significant difference compared to bone (16.5%) and liver metastases (32.8%).
Each intrinsic subtype has different preferential sites of metastatic involvement. 19For this reason, we have specified the results again for the luminal subtype (HR-positive and HER2-negative, see

| DISCUSSION
About 20%-30% of BC patients will develop distant metastases, preferentially in bones, liver, lung, and brain. 1,2,10,11[19] Luminal BCs commonly metastases to the bone, whereas HER2-positive BC is associated with a higher frequency of liver metastases. 21R2-positive and TNBC often metastasizes to the central nervous system, liver, and lungs. 20,21This is in line with the results presented in our study (Table 1).Further, these subtypes vary in the abundance of genetic alterations. 22,23By that, genetic alterations may influence the localizations of metastases.
In the current study, n = 521 BC metastases of the four major metastatic sites for progressive BC (bone, brain, liver, and lung) were investigated for the prevalence of AKT1, ERBB2, ESR1, PIK3CA, and TP53 mutations via next generation sequencing and pyrosequencing.
Somatic mutations were present in 64% of cases.Genes were mainly affected by missense mutations, only in TP53 truncating mutations were also frequent, which is in line with previous studies. 39,45,46With 35% and 22% PIK3CA and TP53 were the most frequently mutated genes under study.This is in line with previous studies. 22,23,47ESR1 mutation appears to be a secondary aberration acquired by BC during clonal evolution of metastatic disease and may occur during endocrine treatment with aromatase inhibitors. 4,48,49[52][53] Tumors with different intrinsic subtypes vary in the frequency of genetic alterations (Figure 3).The prevalence of genetic alterations depends on the intrinsic subtype of BC. 22,23 TP53 mutations being most frequent in triple negative subtype and least frequent in the HR-positive/HER2-negative subtype ( p < 0.0001), which is in line with previous studies. 35,45In HR-positive/HER2-negative mBC, TP53 mutations were associated with a poor outcome. 29In contrast  S1).
Depending on the organ site affected by metastases the genetic alterations differed significantly (Figure 4).With about 30%, the highest percentage of ESR1 mutated samples was observed in liver and lung metastases.Whereas metastatic lesions to the brain showed ESR1 mutation in only one out of 49 cases (2.0%, p < 0.0001).In contrast, TP53 mutations were mostly observed in brain metastases (51.0%).Metastases outside of the brain (bone, liver, and lung) revealed a lower proportion of TP53 mutated samples than metastases involving the brain.Overall, mutation frequencies were consistent with independent previous studies. 19,22,29,54,55In contrast to other studies, we performed our analysis not on plasma samples but on metastatic biopsies. 5,19,26Only few previous studies performed analysis with exploited DNA from biopsies, but mostly without site differentiation. 22,28,29Bertucci et al. differentiated metastases in their analysis but did not include metastases from the bone and brain. 29In our study, we can provide an analysis of the mutational profile of BCs and the affected metastatic site.
Each intrinsic subtype has different preferential sites of metastatic involvement, so we specified the analysis for the luminal BC (HR-positive and HER2-negative) only. 19ESR1 and PIK3CA mutations were enriched in liver metastases ( p = 0.0186 each).Additionally, AKT1 p.E17K mutations were enriched in liver metastases with borderline significance ( p = 0.0583) and occurred less frequently in bone metastases ( p = 0.0333).With 36.4% TP53 mutations are enriched in metastases spread to the brain and occur less frequently in bone metastases (11.6%,Table 3).
The findings of this study have to be seen in light of some limitations, starting with the retrospective approach of molecular analyses.We evaluated available cases from the archive of the institute of pathology.As a result, only limited clinical information was available (e.g., no treatment data and a substantial subset of cases with an unclassified intrinsic subtype).The relatively high number of unclassified tumor is caused by the fact that a proportion of cases stem from the era when re-determination of receptor expression in metastatic breast cancer was not yet required by the German guidelines.A better-defined cohort could help to consolidate the results and may allow statistical analyses of HER-positive BC and TNBC metastases.Further, we did not analyzed the primary BC; consequently it is unclear if the mutations found in the metastases where already present in the primary BC and determined the metastatic site.We only performed a small gene panel for the molecular analysis, focusing on the most frequently mutated genes in BC.However, further gene mutations could play a role in the difference of metastatic sites.Additionally, the small number of lung metastases might be insufficient for statistical analysis.
However, there is a lack of studies in the literature using metastatic biopsies and differentiating between metastatic sites, because of this our study has its eligibility.
Within this study, we were able to find significant differences of mutational status in mBC depending on the affected organ.

F
I G U R E 1 Mutation frequencies in advanced BC. Bar charts showing the frequencies of genetic alterations in candidate genes.BC, breast cancer; ins, insertion; del, deletion.F I G U R E 2 Distribution of mutations within the functional domains.

PIK3CA
and ESR1 mutations occurred less frequently in TNBC T A B L E 3 (Continued) 0.0002, respectively p = 0.0003).ESR1 mutations were found exclusively in HR-positive BC ( p < 0.0001, Table Organotropism of metastatic cancer spread may be influenced by the mutational profile of individual BCs.Differences could also be shown when only luminal BCs were analyzed.Understanding the molecular mechanisms of metastatic organotropism is essential for biomarkerbased prediction and prognosis.The detected ESR1 mutations confer resistance against antiestrogen therapy.However, the tumor cells are potentially sensitive for a treatment with fulvestrant.PIK3CA mutated tumors are potentially treatable with alpelisib or other PIK3 inhibitors which are currently tested in clinical studies.AKT1 and ERBB2 mutations represent a possible target for capivasertib and neratinib.Therefore, we strongly recommend AKT1, ERBB2, ESR1, and PIK3CA sequencing in patients that suffer from bone, lung, and liver metastases. Genomic characteristics in advanced BC. Immunohistochemical subgroups, and genetic alterations identified in n = 424 mBCs (n = 97 metastases with an unclassified subtype were excluded).The type of the alterations and histology are color-coded according to the legend.BC, breast cancer.
The highest percentage of ESR1 mutated samples was observed in liver and lung metastases (31.3% and 26.3%, Table3), with significantly less mutations in brain metastases compared with bone and liver.Bone metastases revealed a mutated ESR1 gene in 52 out of 389 cases (13.4%).By contrast, metastatic lesions to the brain showed ESR1 mutation inF I G U R E 3 T A B L E 2 Genomic characteristics in different subtypes.The values are given in the format n (%, of all investigated cases for this subtype), with n, number of patients.χ 2 -Test was used for statistical analysis.Significant values are highlighted in bold.T A B L E 3 Genomic characteristics according to localization in different subtypes.The values are given in the format n (%), with n, number of patients.The Fisher's Exact test was used for statistical analysis.Significant values are highlighted in bold.

Table 3 )
Mutations of ESR1 and PIK3CA are significantly enriched in liver metastases of luminal BC.ESR1