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

Summary

This article presents a detailed protocol for collecting breast cancer samples, designed to optimize their utility for biobanking, personalized medicine, and other research applications. The workflow for sample collection, including breast biopsy and mastectomy procedures, is described in detail, focusing on minimizing tissue damage and maximizing sample quality.

Abstract

Breast cancer is the most prevalent cancer affecting women, particularly in Asia, with the highest incidence in the Philippines. Despite ongoing efforts, the understanding of breast cancer biology remains inadequate, compounded by the lack of effective screening programs and timely treatments. Addressing these issues, biobanks and other repositories for biological specimens have gained global momentum. The University of the Philippines- Philippine General Hospital (UP-PGH) has established a cancer biobank, demonstrating the viability of a government-supported initiative for cancer research in the Philippines. The study involves a prospective observation of 100 Filipino breast cancer patients at various disease stages, leveraging the expertise of UP-PGH, Philippine Genome Center (PGC), and University of the Philippines Diliman - Institute of Biology (UPD-IB). It integrates the collection, processing, and storage of biospecimens alongside comprehensive patient data. Procedures include RNA extraction, library preparation, and sequencing. Additionally, a subset of patients participates in an in vitro three-dimensional (3D) cell culture experiment for chemotherapy pre-screening. The aggregated data aims to uncover therapeutic strategies that can lead to personalized treatments where standard clinical approaches are inadequate. Key techniques include ultrasound-guided core needle biopsy, meticulous normal breast tissue collection, and precise tumor percentage determination to ensure high-quality RNA extraction, sequencing, and 3D cell culture. The study also addresses challenges encountered in prior biobanking endeavors. Ultimately, this research aims to contribute significantly to breast cancer research, positioning itself as a model for collaborative research, biobank establishment, and the advancement of precision medicine in developing countries, such as the Philippines.

Introduction

Breast cancer stands as the most prevalent form of cancer among Filipino women. In Asia, the Philippines exhibits one of the highest breast cancer mortality rates and lowest mortality-to-incidence ratio¹. The rates may have been greatly affected by the absence of screening programs and timely and effective treatment for breast cancer. In the country, breast cancer is typically diagnosed in later stages since most cancer patients only seek consultation at advanced cancer stages. In the Philippines, where a high incidence rate of breast cancer is observed, 53% of breast cancer cases were diagnosed in Stages III and IV, and only 2-3% were diagnosed in Stage I^1,2,3. Aside from promoting screening uptake and patient education on breast cancer, breast cancer-related research and development should be explored further to decrease breast cancer incidence in the country.

The establishment of biobanks across the globe allowed the acceleration of medical advances. Biobanks are used for the storage and preservation of scientifically valuable biological specimens for future research and development⁴. Since then, biobanks have been used for translational research⁵ and improving personalized medical approaches⁶. In Asia, biobanks are mostly concentrated in East Asia, especially in China, Japan, and Korea⁴. In low- to middle-income countries in Asia, such as the Philippines, the concept of biobanking is still emerging. Biobanks in the Philippines are mostly owned by private healthcare institutions, where the health and exposure status of the broader Philippine demographics may not be well-represented.

The establishment of the cancer biobank at the University of the Philippines Manila (UPM) Philippine General Hospital (PGH) has proven the feasibility of establishing a government-supported cancer biobank that can advance cancer-related studies in the Philippines⁷. Building upon this foundation through a collaborative effort that bridges medical expertise, research innovation, and healthcare policy, we endeavor to create a positive shift in the landscape of breast cancer care and research in the Philippines. With that, there is an imperative need to have a cancer phenome biobanking workflow and an expanded approach that streamlines the collection of extensive and stratified patient-specific data on a national scale. This study describes the protocol for breast biopsy and mastectomy sample collection procedures, focusing on their integration into the biobanking framework. Anticipating not only advancing our understanding of breast cancer pathogenesis within the context of the Filipino population but also facilitating the development of precision treatments that align with the unique genetic, environmental, and clinical attributes of affected individuals.

Protocol

This is a prospective, observational study with a collection of de-identified biospecimens (tumor and normal breast tissue) and clinicopathologic, treatment, and outcome data from 100 Filipino breast cancer patients of various stages. This is a collaborative work between the Philippine General Hospital (PGH), the Philippine Genome Center (PGC), and the Institute of Biology (IB) of the University of the Philippines Diliman, each with designated tasks. The protocol was registered in the Research Grants Administration Office (RGAO-2020-0933), and reviewed and approved by the University of the Philippines Manila Research Ethics Board (UPMREB) for implementation under ethics clearance number UPMREB 2020-822-01. All laboratories used adhere to the biosafety guidelines of each institution. Recruiting patients from PGH ensured the variance and representation in terms of clinicopathologic factors, ethnicity, and environmental exposures. A standard operating procedure (SOP) developed and validated in previous projects was utilized as the framework for the acquisition, collection, transportation, and other logistical arrangements related to the samples.

NOTE: See Figure 1 - Diagrammatic workflow of the protocol for the summary of the protocol workflow and the roles of each institution involved in the study.

1. Pre-breast sample collection procedure

1. Identify and screen the eligible patients for study inclusion during a clinic consult or preoperative conference.
   1. Inclusion criteria for cancer patients
      1. Include Filipino, female, aged 19-75 years old diagnosed with Stage 1, 2, and 3 breast cancer, and Filipino females aged 19-70 years old diagnosed with Stage 3B and 4 breast cancer.
      2. Include clinically suspected or pathologically diagnosed cases of invasive breast cancer of any stage.
      3. Include patients who will undergo surgical treatment (modified radical mastectomy or radical mastectomy) either upfront (Clinical Stage I or II) or post-neoadjuvant chemotherapy (Stage IIIB, IIIC). Stage IIIA patients may undergo either upfront surgery or neoadjuvant therapy as per the physician's recommendations. For Stage IV patients, those willing to undergo core needle biopsy of the breast tumor and adjacent normal tissue. Stage IV breast cancer patients will not undergo surgical treatment.
      4. Include Stage III breast cancer patients who have undergone neoadjuvant therapy prior to recruitment, and there is a complete medical and clinical treatment response record.
      5. Include patients who can understand and sign informed consent.
   2. Exclusion criteria for cancer patients
      1. Exclude patients who have prior or concurrent malignancy, including prior diagnosis of breast cancer.
      2. Exclude patients with bilateral breast cancer.
      3. Exclude patients who underwent prior neoadjuvant radiotherapy or hormonal therapy.
      4. Exclude patients who underwent prior core or incisional biopsy where there is no consent to repeat biopsy procedure for those patients who will undergo neoadjuvant chemotherapy (since no pre-chemotherapy fresh tumor specimen can be obtained)
      5. Exclude patients who underwent prior excisional biopsy with no residual tumor.
      6. Exclude patients with comorbidities precluding surgical management or neoadjuvant chemotherapy.
   3. Withdrawal criteria
      1. Inform participants that they can withdraw anytime for safety or other concerns without affecting their medical care. This non-interventional study will not impact the choice of treatment for participants.
2. Secure consent from the patient and fill out the data collection form.
   NOTE: The assigned fellow-in-charge (FIC) or the consultant, together with the university research associate (URA), secures the consent.
3. Obtain and verify the patient's schedule for the core needle biopsy procedure after confirming their participation in the study.
4. Conduct procedures in compliance with the guidelines set and approved by the Ethics Board Committee and Biosafety Committee of the institution.

2. Breast biopsy sample collection procedure

NOTE: See Figure 2 for the detailed workflow of the core needlebreast biopsy procedure for stage III and IV patients, showing the distribution of samples for each procedure.

1. Pre-label tubes before specimen collection as GB, meaning Genomics - Breast (the study the specimen is under), xxx which is the participant number and can range from 001 to 100, or more, and the letters A, C and D. The following are the letter codes to be used in labeling the tubes:
   A - means core needle biopsy, tumor tissue, for sending to PGC
   C - means core needle biopsy, tumor tissue, for biobanking
   ​D - means core needle biopsy, normal tissue
   1. Assign the samples submitted for sequencing with the laboratory's label: "BC" and two two-digit sample numbers (e.g., BC01, BC21). Each sample has a corresponding "GB" code which correlates to the code assigned during specimen collection.
2. Obtain up to nine tissue cores using a 14 G core needle biopsy gun under ultrasound guidance, in an outpatient clinic, operating room, or other aseptic environment.
   1. For each core, obtain a breast sample approximately 2 cm in length at the center of the tumor and 1 cm away from the tumor for adjacent normal tissue. Collect two types of specimens from each participant: tumor tissue and normal tissue.
   2. Hand each sample to the URA for placement into a petri dish containing phosphate-buffered saline (PBS) solution.
      NOTE: In this study, core needle biopsies were performed only on patients with Stage III and IV breast cancer. For Stage III patients, core needle biopsies provided chemotherapy-naïve tumor samples. For Stage IV patients, core needle biopsies served as the primary tumor sample, as mastectomy was not performed. For patients with Stage I and II breast cancer, no core needle biopsies were performed. Instead, tumor and adjacent normal tissues were obtained from mastectomy specimens.
3. Cut the biopsy specimens in half to distribute evenly between the study and histopathological analysis.
   1. For tumor tissue, obtain up to 9 cores per study patient using a 14-G needle.
   2. Separate the tumor cores obtained into 2 groups in the Petri dish: samples for RNA analysis (6 cores) and samples for biobanking (3 cores).
   3. Cut each core into 2 lengthwise, but in cases where it is difficult to do this, cut the cores into 2 crosswise. Use a sterile blade to cut the cores. Send half of the tissue core for RNA analysis or store for biobanking. Send the other half of the tissue core to Pathology for routine histopathological analysis.
   4. For normal tissue, obtain up to 3 cores per study patient using a 14 G needle. Use a different needle from the one used to obtain tumor tissues to avoid contamination. In instances where this is not possible, obtain normal breast tissues first before getting tumor tissues.
   5. Place each normal tissue obtained in a Petri dish similar to what was done with the tumor tissues, and cut the cores in half. Distribute the samples for the study and to the Pathology Department for the histopathological analysis.
4. After sample collection, distribute the samples among the tubes, following a manner and order of priority.
   1. Place half of the cores in the designated 2 mL cryovial pre-filled with 1.25 mL of RNA stabilization solution (all cores for RNA analysis placed together in 1 vial and all cores for biobanking in a separate vial, maintain samples in cold temperature at all times) and place the corresponding halves of these cores in ordinary specimen vial/tube and fixed with 10% neutral buffered formalin (10% NBF), for histopathological analysis. Ensure that the two corresponding half-groups, the one going to the designated center (e.g., samples sent for RNA analysis), and the corresponding Pathology specimen have the same specimen code.
5. Complete the surgical pathology forms. Together with the forms, submit the Pathology specimens fixed with formalin to Pathology for routine histopathology.
6. Seal with parafilm the core needle biopsy samples soaked in tubes containing RNA stabilization solution (ideally 5:1 ratio of solution to tissue, so if 0.25 cm tissue, 1.25 mL of RNA stabilization solution).
7. Store the samples at 4 °C for 24 h, and then eventually transfer it in the ultra-low freezer, with a temperature of -80 °C.
   NOTE: A guide on storage will be discussed in Section 4 (Storage, packing and transport of specimens).

3. Breast mastectomy sample collection procedure

1. Follow the pre-breast sample collection procedure in Section 1.
   NOTE: Stage I, II, and III patients enrolled in the study will undergo mastectomy at the Operating Room (OR). For Stages I and II patients, mastectomy will be done upfront, while for Stage III patients, mastectomy will only be done upon completion of 8 cycles of neoadjuvant therapy. For stage 4, no mastectomy samples are collected. See Figure 3 for the detailed breast mastectomy sample collection protocol showing the distribution of samples per procedure.
2. Coordinate the schedule with the collaborators and the Pathology department. Do this at least 2 days prior to the target date of the procedure. In case of changes to the set schedule, inform the concerned personnel right away to avoid any conflicts in the schedule.
3. Pre-label the tubes before specimen collection as GB, meaning Genomics Breast (the study the specimen is under), xxx which is the participant number and can range from 001 to 100, or more, and the letters E and F. The following are the letter codes to be used in labeling the tubes:
   E - means mastectomy, tumor tissue, for sending to PGC, IB, and for biobanking
   ​F - means mastectomy, normal tissue
   1. Assign the samples submitted for sequencing with the laboratory's label: "BC" and two digit sample number (e.g., BC01, BC21). Each sample has a corresponding "GB" code which correlates to the code assigned during specimen collection.
4. Fill out the surgical pathology forms during the surgery, while waiting for the specimen. Send updates to the pathologist-in-charge for them to make any necessary preparations for the specimen collection.
5. Place the specimen in a clean plastic/specimen bag right after removal and documentation of the breast specimen. Ensure the mastectomy specimen is immediately brought to the Pathology department for grossing to minimize the cold ischemic time. Do not place the specimen in formalin upon submission.
   NOTE: Sample action photos during mastectomy are shown in Figure 4 (Representative images of sample collection during mastectomy procedure).
6. Obtain two types of samples from each specimen upon grossing: tumor tissue and normal tissue.
   1. Tumor tissue
      1. Obtain a minimum of one block (preferably more) measuring 1 cm × 1 cm × 1 cm of tumor tissue per specimen.
      2. Cut the obtained tissue block further into two parts, with each half block measuring 1 cm × 0.5 cm × 0.5 cm. Hand over half of the tissue block to the URA for biobanking and analysis, and then fix the other half in formalin for routine histopathology.
      3. Evenly trisect the half block of fresh tissue. This will be intended for RNA sequencing, biobanking, and organoid culture.
      4. For RNA sequencing: Immediately place in pre-chilled, pre-labeled 5 mL cryovials, pre-filled with 2.5 mL RNA stabilization solution (not exceeding 30 min, must be kept cold at all times).
      5. For biobanking: Immediately place in pre-chilled, pre-labeled, and pre-filled 5 mL cryovials with 2.5 mL RNA stabilization solution (not exceeding 30 min, must be kept cold at all times).
      6. For organoid culture: Immediately place in pre-chilled, pre-labeled, and pre-filled 15 mL conical vials with 5 mL of tissue storage solution and 0.01 mL Primocin (not exceeding 30 min, must be kept cold at all times). Pool together in one conical tube one-third of all the tumor blocks obtained from different sections of the specimen.
   2. Normal tissue
      1. Obtain a minimum of one block (preferably more) measuring 1 cm × 1 cm × 1 cm of normal breast tissue per specimen.
      2. Cut the obtained tissue block further into two parts, with each half block measuring 1 cm × 1 cm × 0.5 cm. Hand over half of the tissue block to the URA for biobanking and analysis, and then fix the other half in formalin for routine histopathology.
      3. Cut the tissue block equally into two for the study. These will be intended for RNA sequencing and biobanking.
      4. For RNA sequencing: Immediately place in pre-chilled, pre-labeled 5 mL cryovials, pre-filled with 2.5 mL cryovials with RNA stabilization solution (not exceeding 30 min, maintain at cold temperature at all times).
      5. For biobanking, immediately place in pre-chilled, pre-labeled, and pre-filled 2.5 mL cryovials with RNA stabilization solution (not exceeding 30 min, maintain at cold temperature at all times).
         NOTE: Tumor samples labeled "E" are typically obtained from three or more different sections of the specimen. To avoid confusion, tumor samples intended for RNA analysis and biobanking should be placed into separate tubes labeled as E1, E2, E3, etc., while pooled tumor samples intended for cell culture should be placed into tubes labeled simply as E. For samples labeled E1, E2, and E3, the relative position/location of each tissue sample with respect to the tumor (e.g., center, 10 o'clock position) should be recorded.
7. Seal with parafilm the cryovials containing the samples obtained from the mastectomy.
8. Store the cryovials in 4 °C for 24 h, and then eventually transfer them to the ultra-low freezer, with a temperature of -80 °C for long-term storage.
9. Pack the conical tube containing the pooled tumor blocks and prepare it for transport. These tumor blocks will be used for cell culture into organoids.

4. Storage, packing, and transport of specimens

1. Immediately seal with parafilm all cryovials containing the tumor and normal tissues and store these at 4 °C for 24 h. After 24 h, transfer the tubes to -20 °C for around 2-4 h and then eventually transfer them to -80 °C indefinitely, for long-term storage until transport.
2. Update the specimen tracker and database prior to transport of samples for RNA analysis, including the results of routine histopathology and tumor percentages, to ensure the identity of the sample.
3. Accomplish a transportation manifest for each sample, prior to transport. Ensure that the packing process is quick and the tubes containing the samples are not left at room temperature (RT) for a prolonged period.
   1. Seal all cryovial caps with parafilm to prevent leakage.
   2. Place all the tubes in a test tube rack.
   3. Set the temperature logger and place it over the rack alongside the tubes for transport.
   4. Wrap the tubes with absorbent material, then with bubble wrap, and place the items inside the resealable bag. Ensure that the temperature logger is recording temperature and that the tubes are intact and positioned upright before sealing the items inside the bag.
   5. Place the gel packs on the bottom of the polystyrene shipping box, then set the resealable bag containing the tubes on top of 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.
   6. Close the polystyrene box and put it inside a cardboard shipping box. If there is no large cardboard box available, use a larger polystyrene box. Place the completed transportation manifest inside a ziplock bag and put it inside the box. Placement of the items inside the box is shown in Figure 5.
   7. Seal the cardboard box and place the appropriate shipping label outside the box.
4. Inform the recipient of the specimen collection schedule and when the target sending of samples will be.
   1. For organoid culture, only tumor tissues derived from mastectomies are sent and utilized. Ensure these are delivered fresh, on wet ice, and within 24 h from specimen collection. Once the samples are packed and the box is sealed, quickly book a courier to transport them. Ensure that the boxes will not be left in high-temperature areas for prolonged periods, as a severe temperature drop or rise can compromise the viability of the samples.
   2. Prior to handing off to the courier, take note of the number of vials/tubes shipped and the time the courier left the Biobank. Upon arrival of the package, ensure that the recipient takes note of the following: Time of arrival and receipt at the designated center, temperature at arrival, number of vials/tubes received, and any compromised specimen
   3. For RNA analysis, check if the samples to be delivered have a corresponding histopathology result and tumor percentages from the Surgical Pathology Division, prior to sending samples for RNA analysis. Identify and select the tissue block with the highest tumor content for each patient to be submitted for RNA extraction. Transport samples only those with tumor percentages.
   4. Send the samples in groups of no more than 40 to facilitate batch processing. Choose any combination of tumor tissue from core needle biopsy or mastectomy and normal tissue from mastectomy. Review the following conditions if normal tissue from core needle biopsy is to be transported:
      -Enrolled as Stage 3, but histopathology result shows Stage 4
      -Enrolled as Stage 3, but histopathology result shows loco-regional progression of disease.
   5. Ensure that all samples are transported on wet ice. Similar to the protocol being followed when sending samples for 3D cell culture, once the specimens are packed and the box is sealed, promptly book a courier for transport. Avoid leaving the boxes in high-temperature areas for prolonged periods, as significant temperature fluctuations can compromise the viability of the samples.
   6. Prior to handing off to the courier, take note of the number of vials/tubes shipped and the time the courier left the Biobank. Note the following upon arrival of the package: time of arrival and receipt at the designated center, temperature at arrival, number of vials/tubes received, and any compromised specimen (if there is any).
5. Update the database of samples sent and input all necessary information needed prior to further processing of samples. Monitor and assess the database once a week.

Results

For the collection of samples during core needle biopsy, doing the procedure under ultrasound guidance had a huge impact on patient convenience and efficiency of workflow, as well as on the RNA integrity of the samples. If this is not done, the expected results might not be obtained, and patients must go back to the clinics to repeat the procedure, therefore prolonging sample collection and processing.

In addition to that, identification of tumor percentages per sample, which was done by the p...

Discussion

The successful implementation of this protocol relies not only on technical precision but also on the coordination of clinical and research teams to ensure consistent sample quality and data integrity. This protocol incorporates several key procedures designed to maximize diagnostic accuracy and generate high-quality biospecimens suitable for downstream molecular analyses. Key steps include ultrasound-guided core needle biopsy for obtaining both normal and tumor breast tissue, data collection on treatment response, ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
37% Formalin solution	PHILUSA Corporation	N/A	Used as a preservative of tissues for histopathological analysis
70% Isopropyl alcohol	Green Cross Inc.	N/A	Used as a disinfectant
Barcode printer	Zebra	GC420t	For printing of barcode labels to be used in labelling tubes containing the samples
Bleach	Green Cross Inc.	N/A	Used as a disinfectant; for decontamination of surgical tools
Blue ice packs	Rubbermaid	1002	For keeping samples at low temperatures during transport
Core needle biopsy 14-G needle	Bard	MN1410	An invasive tubular surgical instrument designed to be connected to a biopsy gun handpiece for the automatic extraction of a small tissue sample from an anatomical structure (a biopsy), while causing minimal surrounding tissue damage, for tissue pathological examination/testing.
Core needle breast biopsy gun	Bard	MG1522	Reusable core biopsy instrument used in collecting tissue samples during core needle biopsy
Disposable Petri Dish 90 mm x 15 mm	Phoenix Biomedical Products	LOT: 20220206	Used in core needle biopsy specimen collection when submerging tissues in PBS
Dulbecco's phosphate-buffered saline (PBS)	Thermo Fisher Scientific	21600010	A balanced salt solution used for a variety of cell culture applications, such as washing cells before dissociation, transporting cells or tissue, diluting cells for counting, and preparing reagents.
Forceps	Local Supplier	N/A
Kimtech Science KimWipes Delicate Task Wipers	Kimberly Clark Professional	KC34120	4.3 x 8.4 in. (11.0 cm x 21.3 cm); 1-ply 280 Count; for cleaning surfaces, parts, instruments, and medical offices, these wipes easily clean liquids, dust and small particles.
MACS Tissue storage solution	Miltenyi Biotec	130-100-008	Storage solution used to allow an optimized storage of fresh organ and tissue samples
Micropipette	Shanghai Lichen Instrument Technology Co., Ltd.	STEM-GC-3197-Y	for pipetting/transferring liquids in small volumes
Micropipette tips	Tarsons	521100	for pipetting/transferring liquids in small volumes
Parafilm	Parafilm M	5259-04LC PM996	For airtight and moisture-proof sealing of tubes to prevent leaks
Primocin 250 mg (5 x 1 mL)	InvivoGen	ant-pm-05	Used to protect tissues from microbial contamination
Refrigerator (4 °C)	LG	Model: GR-Y331SLZB	For storage of tissue samples and reagents
RNAlater solution	Thermo Fisher Scientific	AM7021	RNA stabilization solution used in the storage and biobanking of tissues
Sterile 15 mL conical tube	Thermo Scientific	339650	Used to make aliquots of reagents/media used in tissue storage
Sterile 5 mL Cryovials	Thermo Scientific	5000-1020	Used to make aliquots of reagents/media used in tissue storage
Sterile scalpel blade (#11)	Surgitech	N/A	For cutting tumor and normal breast tissues
Surgical scissors (18.5 cm 7 ¼" str)	Olten Instruments	BO-0125	For cutting tumor and normal breast tissues
Temperature logger	Extech Instruments	42280	For recording temperature and humidity during transport of samples
Tissue ink	Epredia	3120123	For staining the margins of excised surgical specimens for histopathological analysis
Ultra-low freezer	Haier Biomedical	DW-86L579	For indefinite storage of tumor and normal breast tissues
Urine specimen container 60mL	Sure Guard	0016UCS06PC	For storage of tissues for histopathological analysis
Ziplock Bags	Reynolds Consumer Products LLC		Used in packing the samples for transport