The Falciani Foundation promotes clinical research to improve timely diagnosis and appropriate treatment of cancer, which is responsible for the death of more than 8

million people in developed countries alone.

LIQUID BIOPSY IN BREAST CANCER: TOPICALITY AND FUTURE PROSPECTS

B. Damascelli MD*, V.Tichà MD*, Elena Repetti MD°, Tshering Dorji MD^* Oncological Interventional Radiology, Emodinamica Columbus, Milan, Italy° Molecular Genetics Section, ImpactLab Group Laboratory, Milan, Italy^ Pathological Anatomy Section, ImpactLab Group Laboratory, Milan, Italy

The term liquid biopsy currently refers to the identification of mutations present in tumor-derived DNA, which can be isolated in plasma or from circulating tumor cells in the blood of cancer patients. The advantages of this diagnostic approach are significant because it is not invasive, it can be repeated over time, and it is able to capture the molecular heterogeneity of a tumor better than an invasive tissue biopsy which reflects the genome profile of only one area of the disease.

Liquid biopsy is normally performed on peripheral blood samples. Circulating tumor cells (CTC) are identified and circulating fragmented DNA (cfDNA), consisting of DNA derived from normal cells and to a minimal extent from tumor cells (ctDNA), is extracted. The amounts of ctDNA are not related to tumor size but to release mechanisms. CTC are rare in blood, where one tumor cell is found for each 600,000 nucleated cells, obtained from a 5-10 ml blood sample.

Various solutions have been proposed to overcome the scarcity of genetic material obtained from blood. Some of these are highly technological, but inevitably costly and complex in application, such as inserting devices capable of capturing circulating tumor cells into the venous circulation and leaving them for a certain time. Others are more simple, such as selective blood sampling from the body areas affected by the tumor.

Percutaneous catheterization from brachial orfemoral veins under fluoroscopy. Virtuallyreaches every body district. It can take thename of topographic if more than one sample iscollected simultaneously, including theperipheral one for comparison. It is minimallyinvasive, ambulatory, repeatable, does not havecontraindications, does not require preparation.

Selective venous biopsy increases the likelihood of capturing both ctDNA and CTC. In addition, the sample, which can be obtained from any venous district, allows topographical analysis of cancer distribution with any differences in genome characteristics. This method, implemented by the Falciani Foundation, can be applied in critical cases with negative results on peripheral sampling or as a first resort thanks to the greater sensitivity and specificity of the results.

Liquid biopsy in breast cancer is readily illustrated by the following description of some case studies

In breast cancer, extraction of fragmented DNA from plasma (liquid biopsy) and tumorcell counts are used for genome analysis, tumor burden estimation and prognostication.

About 6% to 10% of breast cancer patients have stage 4 disease right from the initialdiagnosis. It is estimated that 20 to 30% of women with breast cancer will developmetastatic disease.

Failure of systemic treatment is due to the difficulty in coding the heterogeneity ofthe genetic profile of cancer, which is unstable and changes over the course of itsevolution.

Genome analysis by liquid biopsy and circulating tumor cell counts are critical piecesof information, obtained in real time, which guide the use of targeted drugs,monitoring their effects.

BREAST COLON RECTUM PROSTATE

UNFAVORABLE ≥5 CTC ≥3 CTC ≥5 CTC

FAVORABLE 0-4 CTC 0-2 CTC 0-4 CTC

Prognostic indications according to the liquid biopsy CTC count for metastatic breast, colorectal and prostate cancer

53-year-old woman with BI-RADS 4 microcalcifications in the left breast. Peripheral liquid biopsy was negative.Selective liquid biopsy from the left subclavian vein was positive for ERBB4 gene mutation. Biopsy diagnosis ofductal carcinoma in situ, surgically confirmed. Liquid biopsy at 14 months was negative for circulatingfragmented DNA.

51-year-old woman with lesion in the left breast found to be triple negative infiltrating carcinoma onpercutaneous biopsy. Selective liquid biopsy from the superior vena cava was positive for TP53 gene mutation, whichis among the somatic mutations of triple negative breast cancer. Circulating tumor cell count of 7 in the left subclavianvein sample.

Liquid Biopsy

Gene TP53 c.916C>T p.(Arg306*) esone 8 1%VF variant non sense. Classification: Likely Pathogenic. In the other investigated genes no pathogenetic mutations relevant to the clinic were found. No benign variants are indicated in the report.Six months follow-up after chemotherapy.FISH for tumor circulating cells (FISH-CTC). Result: In the 8ml of analyzed blood a single cell with polysomy of the chromosomes 3,7 and 17 was highlighted.

55-year-old woman with previous radical mastectomy and subsequent finding of lungmetastases treated with chemotherapy and surgery. Liquid biopsy was positive for PTEN genemutation, which is found in 7% of breast cancers and has a role in negative regulation of theP13K pathway. At the onset of brain metastases liquid biopsy from the right jugular vein waspositive for DNMT3A gene mutation, which has a negative prognostic role in breast cancer.

2008: mastectomy for invasive carcinoma, G3, 60% ER-positive, PgR-negative, 10% Ki 67, HER2 3+, epirubicin andTaxotere CHT, Herceptin, hormone therapy.

2017: Lung metastases

2018: Brain metastases (stereotactic RT)

2014: Wide local excision of right breast for invasive dutal carcinoma, pT3 pN0 M0, G3, 95% ER-positive, 20% PgR-positive, 40% Ki-67, cerbB2 score 0, EC CHT, RT, letrozole.

2018: Right supraclavicular metastasis, metastasis in D8, RT, VET CHT.

2019: Retrosternal metastasis, left acetabulum, CEA 39.3 on 13.09.2019, 3.5 on 9.10.2019.

23/01/2019 05/06/2019

59-year-old woman

Polisomia CEP 3Her2/Neu

Evolution of metabolic disease and quantitative trend of circulating tumor DNA and CTC count.

First PET21.01.19

Second PET22.05.19

Third PET05.09.19

DNA 1,87ng/uL DNA 2,12ng/uL

The findings reported in the literature for liquid biopsy confirm that circulating tumor cellcounts at presentation have a predictive value for progression-free survival and for overallsurvival, while quantification of specific mutations in ctDNA correlates with the tumor burden.The criticality of liquid biopsy, basically in the scarcity of circulating tumor DNA and the rareoccurrence of circulating tumor cells, can be improved by direct enrichment methods or byselective venous sampling from the areas of interest.

Dear plastic surgeons,It can happen that a complex reconstruction must be undertaken without any certainty that no minimal residual disease persists, even some time after exhaustive treatment for breast cancer.No imaging modality is capable of providing really early information on residual disease.

Liquid biopsy, by its very nature, offers this potential, which is consolidated day after day by the unstoppable progress of the genetic laboratory.

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