Bravaccini: Up to now, the conventional tissue biomarkers (estrogen receptor [ER], PgR, human epidermal growth factor receptor 2 [HER2], and Ki-67) are always evaluated in the clinical practice to define BC patients’ prognosis and to predict the response to therapy in TNBC subtype. Despite the experimental limitations due to the lack of reproducibility of PgR and Ki-67 evaluation intra and inter-laboratories, some authors showed the prognostic value of Ki-67 in TNBC, using a cut-off point of 60%, depending on the patients’ age. The current guidelines recommend germline BRCA mutation evaluation to identify high-risk individuals who may benefit from genetic counseling or prophylactic procedures. Moreover, BRCA mutational profiles are evaluated also to select patients responsive to PARP inhibitors in those with HER2 negative mTNBC. Prostate-specific membrane antigen (PSMA) is present not only on tumor cells but also in endothelial cells of tumor vessels. It could be a new diagnostic and therapeutic alternative, particularly for triple-negative breast cancer that is highly PSMA positive. PSMA may have a broader potential as tumor-associated neovascular target for antiangiogenic therapy and it could become an attractive molecular target for oncological imaging and radionuclide therapy using PSMA PET/CT in TNBC. Recently, the evaluation of PD-L1 expression by using SP142 has been demonstrated to be important to select metastatic TNBC patients responsive to immune checkpoint inhibitors as first-line treatment. Mismatch repair (MMR) deficiency seems to be predictive to select patients responsive to immunotherapy even if the IHC method used to evaluate the 4 MMR proteins (MLHl, PMS2, MSH2, and MSH6) has low reproducibility due to the different IHC platforms used. Trop-2 is the target for sacituzumab govitecan but the drug seems to be active independently from Trop-2 level of expression by IHC. Standardization of the methods to detect the PD-L1, Trop-2, and the MMR proteins (MLHl, PMS2, MSH2, and MSH6) is needed. Moreover, given that they are not ideal markers, the search of new biomarkers is still ongoing in this prognostically unfavorable tumor type.

Pronzato: For the “true” TNBC – so excluding some special and rare subtypes – PD-L1 is the only biomarker clinically useful: with the aim to ensure ICI treatment to all patients who may benefit, evaluation should be made on the two platforms commercially available (since the results are not superimposable).

Bravaccini: While traditional, routinely measured biomarkers such as ERs, progesterone receptors, and HER2 still represent the best prognostic and predictive biomarkers for HR+ breast cancer, a significant proportion of patients either do not respond to endocrine therapy or develop resistance to endocrine treatment. Special attention must be given to specimen handling for an accurate ER, progesterone receptor, and HER2 biomarker assessment and appropriate management of breast cancer patients. The Oncotype DX genomic assay (Genomic Health, Inc., Redwood City, CA, USA) is a clinically validated assay that providing a breast recurrence score can be used to predict likelihood of recurrence of early stage hormone receptor positive, HER2 negative breast cancer, and it can be used therefore, in decision making with respect to systemic therapy. BRCA1/2 germline mutations are currently used to select locally advanced or metastatic ER+ HER2– BC patients to be treated with Talazoparib. The detection of molecular biomarkers and gene signatures by alternative techniques such as circulating tumor DNA (ctDNA) analyses thus complement the conventional methods, as they allow for noninvasive assessment of the disease, as well as for the study of multiple time points throughout treatment, allowing for the study of tumor evolution and treatment response. ESR1 and PI3K mutations detected in ctDNA could be used both in the adjuvant and metastatic setting to monitor BC patients and treat them with specific compounds. The search of new biomarkers is an urgency in order to be able to treat the hormone receptor positive patients with chemotherapy at a later period of time and to drive new treatment options to overcome endocrine resistance.

Pronzato: In the case of nonmetastatic cancer, the selection of patients’ candidate to the addition of chemotherapy should be based on gene expression profiling, by means of tests that have been demonstrated of clinical utility in proper prospective clinical trials. Also Ki-67 evaluation should be considered because of the emerging data concerning adjuvant CDK inhibitors In the case of metastatic cancer, ESR and PI3K mutations should be under surveillance (even in terms of liquid biopsy) with aim of possible applications of newer hormonotherapy agents (PI3K inhibitors and new SERD).

Bravaccini: Breast cancer screening is highly accurate for BC diagnosis. Despite the clinical screening permit a more effective early detection of tumors, there is a small proportion of tumors in which mammography and ultrasound of the breast are not able to distinguish between a normal nodule and an early stage tumor. BRCA 1 and 2 mutational status is currently tested in healthy individuals with familiarity for BC to establish the risk to develop BC and to insert individuals in screening programs. Together with the conventional clinical screening procedures, we need noninvasive biomarkers to be tested in liquid biopsy for the early diagnosis of BC in order to permit an easy and accurate follow-up of the patients.

Pronzato: In the case of an oncogenetic test is indicated, a panel extended beyond BRCA and including low penetrance genes should be applied.

Bravaccini: Despite the great importance of the biomarkers detectable in primary and metastatic solid tumor tissues, the dynamic heterogeneity of cancers is a critical issue that impairs their value and should be always kept in mind. I believe in routine liquid biopsy utilization but one of the open questions is what to look for in liquid biopsy. CTC and total cfDNA are the two liquid biopsy components that have been studied the most. CTCs could be used as prognostic biomarkers in patients with mTNBC for their well-established unfavorable prognostic role in this disease setting even if their predictive value needs to be further explored. Unfortunately, they are not feasible in all the laboratories, for the lack of specific instruments and personnel skills.

I think that ESR1 and PI3K mutational status if detected in the ctDNA as liquid biopsy could be useful for disease monitoring and to drive in mutated patients therapeutic strategy with anti-ESR1 and anti-PI3K compounds (such as Fulvestrant or Alpelisib, respectively). The investigation of other actionable mutations would be important.

Pronzato: Liquid biopsy should not routinely apply but may be useful in detection of PI3K mutations and patients candidate to a second-line hormonotherapy by PI3K inhibitor. Liquid biopsy has to be greatly considered for being included in newer protocols in order to monitor the evolution of metastatic disease or detect minimal residual disease after treatment of early breast cancer.

S. Bravaccini and P. Pronzato have no conflict of interest to declare.

Dr. Angelo Paradiso

Experimental Oncology Istituto tumori G paolo II IRCCS Bari

via O Flacco

65 Bari 70125

Italy

a.paradiso@oncologico.bari.it

Dr. Sara Bravaccini

IRCCS Istituto Romagnolo per lo Studio dei Tumori (IRST) “Dino Amadori”

Via Maroncelli 40

47014 Meldola (FC)

Italy

dottsarasara@yahoo.it

Dr. Paolo Pronzato

IRCCS OSPEDALE POLICLINICO San Martino

Largo Rosanna Benzi 10

16132 Genova GE

Italy

paolo.pronzato@hsanmartino.it