Breast Cancer Prognosis: A Genetic Code for Personalized Therapy


 Background: Breast cancer is distinguished in three different subtypes, based on clinical and molecular parameters: Luminal - HER2 - Basal type. Luminal carcinomas (which represent about 65% of the total) are distinguished by a particular heterogeneity of biological behaviour with disease recovery in about 40-50% and death in about two-thirds of these patients at 5 years from diagnosis, despite initial anatomopathological pictures of apparent low aggressiveness. Precisely, more biomolecular parameters are desirable for this diagnostic category which starting from an estimated disease recovery can guide therapeutic choices in a more articulated way that is modelled on the actual needs.Method: The aim of this work is to build a panel of genes that can be used to personalize therapy and consequently reduce mortality. Our kit is patented and includes 33 genes that best characterize neoplastic heterogeneity and sensitivity to drugs. The study involved the total transcriptome (RNA) sequencing of 40 patient samples carried out in the two-year period 1994/1995 with the aim of identifying a group of genes expressed differentially among patients with good prognosis and those with poor prognosis. Results: Total RNA extraction from formalin-fixed, paraffin-embedded samples and then Library preparation for RNA-Seq was achieved. The study highlighted some genes: CXCL13 life gene, IFITM10 death gene always present regardless of molecular subtype, and DSCAM-AS1 gene, specific for Luminal A subtype, that if present, let the patient avoid standard PBI and neoadjuvant therapy. Conclusion: The goal is to implement a different surgical and adjuvant personalized therapy for every single patient.

a particular heterogeneity of biological behavior, with recovery of disease in approximately 40-50% and death in approximately two-thirds of these patients 5 years after diagnosis, despite initial anatomopathological pictures of apparent low aggressiveness [2][3][4][5][6][7][8][9]. Precisely for this diagnostic category, biomolecular parameters derived from the genome/transcriptome that are capable of orienting the therapeutic choices in a more precise and personalized way on the patient's actual therapeutic needs are desirable [10,11]. Currently, the clinical (T, nodal status) and biopathological (hormonal status) parameters obtained from membrane receptor expression provide prognostic information and an indication of any adjuvant systemic chemoradiotherapy treatment [12][13][14]. However, adjuvant therapy reduces the risk of recurrence by only 25-30% [13]. These data are probably due to: 1. Clinicopathological parameters of stratification of the risk of disease recovery are not sufficiently adequate for the patient's prognostic framework.

2.
Adjuvant therapies are not specific enough toward the cells responsible for disease recovery [15,16].
Knowing the details of the mutations of every single tumor allows us to predict the biological behavior of that neoplasm and to adequately stratify the risk. Genetic tests, such as Mammaprint and Oncotype DX, EndoPredict [17,18], assist clinicians in choosing the most suitable adjuvant treatment by analyzing the expression profile of genes involved in the metastasis process (St Gallen 2017). These tests are very expensive and often have to be sent to foreign laboratories. The genetic profile is of the utmost importance in the evaluation of parameters already known as the expression of hormonal receptors and HER2; these are currently determined with immunohistochemistry (IHC) or FISH methods, which provide information about the morphological expression of the receptor but not about their functional state. In any case, knowing that the receptor is expressed at the membrane level is not sufficient information to guarantee the effectiveness of the drug addressed to it because that protein may not be functionally active.
Therefore, it is necessary to ascertain the functional activation of the gene responsible for the synthesis of the protein to guarantee its functionality, more than its presence. Moreover, from the gene expression profile, additional information that specifically correlates the expression of some genes to the response to individual therapies can be obtained; for example, in HER2-positive   patients, the high expression of IGF1R correlates with resistance to   Herceptin, as well as the hyperexpression of CCNE1; instead, ER+   patients with high PDGFRA expression are resistant to tamoxifen treatment [14,19,20]. The use of gene profiling tests offers the opportunity for a more adequate risk stratification, an improvement   In practice, for samples with a number of neoplastic cells ≥ 30% of the total, it was not necessary to proceed with macrodissection.
In the first phase of the experimentation, so with the 33 starting samples, the enrichment of the sample was not necessary, but 2/4 sections of the FFPE fabric of 10 µm thickness were cut in sequence on microtome and placed in 1.5 ml safe look tubes ready to be extracted. Total RNA, after appropriate quantitative and qualitative evaluation, was used for the preparation of cDNA for sequencing purposes. The library used is Illumina's TruSeq Total RNA. In the second phase of the experimentation, 2 pilot samples were used to validate the panel of genes obtained with the first phase; they were homogeneous both as biopathological characteristics of the lesion (early breast cancer; tumor diameter; lymph node status; state of HER2 and so on) and as the age of the patients. In the end, for each patient, we obtained 2 tubes of tumor tissue and 2 tubes of healthy tissue ready to be extracted. The extraction kit used to obtain total RNA was the same as that used in phase I of the trial. Total RNA, after appropriate quantitative and qualitative evaluation, was used for the preparation of cDNA for sequencing purposes. The library used was Illumina's TruSeq RNA Access, suitable for processing RNA extracted from paraffin samples. Total RNA was extracted from sections of paraffin tissue using the "Tissue Preparation Reagents" kit -Sividon Diagnostic, from the Pathological Anatomy Section of the Santa Maria Della Misericordia Hospital. The chosen extraction method was previously "validated" on 2 samples with characteristics of "age of the sample", "type of tissue" and "origin" The statistical significance that will be used in the two packages is shown below: An analysis of the network of genes significantly differentially expressed in the two groups was also conducted using GeneMania   4. in HERB2+ patients, we found other genes, among which PAX8-AS1 was responsible for stem cell proliferation.   In the postoperative phase, targeted therapy allows a better stratification of adjuvant therapies based on the amplification of the genes that regulate, for example, resistance to tamoxifen and/ or to trastuzumab.

Declaration Section
We give our consent for the publication of identifiable details, which can include case history and/or details within the text ("Material") to be published in the above journal and article. The datasets generated and/or analyzed during the current study are not publicly available due to privacy reasons but are available from the corresponding author on reasonable request.

Ethics Approval and Consent to Participate
The study protocol was approved by official authorization of

Competing Interests
All authors declare that they have no competing interests.

Funding
This study was supported by the Italian League for the fight against cancer -LILT (Ministero della Salute, 5 x l000 year 2014).

Authors' Contributions
AR designed the study, reviewed and analyzed the clinical data and did the critical revision of the manuscript; LF reviewed and analyzed the clinical data and did the critical revision of the manuscript; SZ data manager; PC reviewed and analyzed the clinical data and did the critical revision of the manuscript; FS enrolled the patients; CP performed the NGS, analyzed the data and drafted the manuscript; AS performed the NGS, analyzed the data and drafted the manuscript; FB reviewed the manuscript. All authors read and approved the manuscript's final version.