Optimization of Breast Biopsy and Mastectomy Sample Collection Procedures for Biobanking, Personalized Medicine, and Research Applications

The scope aims to improve cancer sample collection for biobanking so that it will enable better personalized treatment through high quality RNA sequencing and organoid culture from Filipino breast cancer tissues. Recent advances include using patient-derived organoids and RNA sequencing to predict treatment response in breast cancer in a recent paper, particularly for cases with lymphovascular invasion linked to drug resistance. We use ultrasound guided biopsies, RNA sequencing tumor percentage estimation and 3D organoid cultures to analyze breast cancer tissues precisely.

Main challenges include obtaining high quality RNA from small biopsy samples, quality issues for organoid preservation. So after starting with breast cancer, we'll explore further gene expression in a larger Filipino cohort and use organoids to test therapies for the following:thyroid, ovarian, pancreatic, and rare cancers like phyllodes tumors. To begin, set up a 14 gauge core needle biopsy gun under ultrasound guidance in an outpatient clinic, operating room, or other aseptic environment.

For each core, collect a breast sample approximately two centimeters in length from the center of the tumor and one centimeter away for adjacent normal tissue. Collect two types of specimens, normal tissue and tumor tissue, from each patient. Hand each sample to the research assistant for placement into a Petri dish containing PBS solution.

Sort the nine tumor tissue samples into six cores for RNA analysis and three cores for biobanking. Cut each core lengthwise or crosswise using a sterile blade. Place the three normal tissue cores in Petri dishes.

Cut the cores in half and allocate them for analysis. Distribute samples among the tubes according to priority. Place half of the cores into designated two milliliter cryo vials with 1.25 milliliters of RNA stabilization solution.

Fix the other halves in specimen vials containing 10%neutral buffered formalin. Complete and submit the surgical pathology forms with the fixed samples to the pathology department. After mastectomy, place the sample into a clean specimen bag after documentation and transport immediately to the Department of Laboratories pathology without formalin to reduce cold ischemic time.

Obtain a minimum one cubic centimeter tumor tissue block during grossing and cut into two halves. Evenly trisect the fresh half block for RNA sequencing, biobanking and organoid culture. Immediately place the tissue for RNA sequencing in five milliliter pre-chilled and pre-labeled cryo vials pre-filled with 2.5 milliliters of RNA stabilization solution.

For biobanking, use five milliliter cryo vials with the same solution. For organoid culture, use 15 milliliter conical vials, pre-filled with five milliliters of tissue storage solution and 0.01 milliliters of primocin. Pull one third of all tumor blocks into one conical tube.

Obtain a minimum of one cubic centimeter normal tissue block and slice it in half for RNA biobanking and routine histopathology. Transfer the tissue block into five milliliter pre-chilled and pre-labeled cryo vials pre-filled with 2.5 milliliters of RNA stabilization solution for RNA sequencing and biobanking. Then seal all cryo vials with parafilm.

Store at four degrees Celsius for 24 hours. Then transfer to minus 20 degrees Celsius for two to four hours. And finally, store at minus 80 degrees Celsius.

Seal the cryo vial caps with parafilm. Place the sealed cryo vials in a test tube rack and cover with absorbent and bubble wrap. Place the wrapped rack in a resealable bag with a temperature logger.

Position gel packs on the bottom of a polystyrene box. Then place the bag over the gel packs. Put another layer of gel packs on top of the bag.

Make sure the tubes are sandwiched between gel packs to ensure the specimens will be kept cold during transport. Put the closed polystyrene box into a cardboard shipping box and include the manifest in a Ziploc bag. Seal and label the box for shipping.

Before courier handoff, note the number of vials and the departure time from the biobank. Two samples with varying RIN values and concentration failed in the library preparation step during sequencing and were excluded in the final analysis. All samples had great sequencing quality scores where more than 90%of base calls had an accuracy of at least 99.9%Bright-field imaging showed successful organoid formation from stage three breast cancer mastectomy tissues after neoadjuvant therapy with abundant spheroid-like structures visible at low and high magnification.