Over the course of the PLCO trial, over 2.9 million biospecimens were collected. The majority of these specimens were collected on the intervention arm participants during their visits to the screening centers. Serum was collected every year from T0 to T5 except T3, and an additional remainder vial exists from the serum that was used for the cancer screens (PSA for men, CA-125 for women). The serum samples were allowed to clot for 60 minutes prior to centrifugation. Plasma was collected at T0, T3, T4 and T5 in both sodium heparin vials and liquid EDTA vials. Buffy coat was selected at T0, T3 and T5, along with a T4 buffy coat/red blood cell vial. 12 vials of whole blood were collected. 80% of participants had their whole blood drawn at T3, though those randomized early had it drawn at T5 (17%) or both T3 and T5 (2.7%). Lastly, red blood cell vials were collected at T0, T3 and T5.

Between 2000 and 2003, participants in the control arm of the trial were sent a buccal cell collection kit.

Between 2017 and 2018, a subset of participants in both arms of the trial were sent a buccal cell collection kit. The supernatant was also preserved from this collection.

85% of the participants in the intervention arm had at least one collection, while compliance in the control arm was expectedly lower at 66%. 24% of PLCO participants were eligible for the second buccal collection and provided a sample.

All tubes, except for whole blood samples, were centrifuged for either 15 minutes at 1200xg or 6 minutes at 3900xg. Buccal samples and specimens collected within the T0 – T3 timeframe, except for whole blood, were stored at -70° C. Whole blood and specimens collected at T4/T5 were stored at -157° C.

Number of Biospecimen Vials Collected per Person (Blood Derived - Intervention Arm Only)

Material	T0	T1	T2	T3	T4	T5
Serum 2.0 mL	4	2	2		2	2
Serum (Zinc Free, No Anticoagulant) 2.0 mL	1
Serum (Remainder From Cancer Screen) 1.5 mL	1	1	1	1	1	1
Plasma (Sodium Heparin) 2.0 mL	2			2
Plasma (Liquid EDTA) 1.0 mL [T3]; 3.6 mL [T4]; 2.0 mL [T5]				4	1	4
Buffy Coat 2.0 mL	1			2		2
Buffy Coat/RBC 3.6 mL					1
Whole Blood 1.8 mL				12
Red Blood Cell 2.0 mL	1			1		1

Number of Biospecimen Vials Collected per Person (First Buccal Collection - Control Arm Only)

Material	2000-2003
Buccal Cell 1.5 mL	2

Number of Biospecimen Vials Collected per Person (Second Buccal Collection – Both Arms)

Material	2017-2018
Buccal Cell 1.5 mL	2
Supernatant 4.5 mL	2

PLCO has created tissue microarrays (TMAs) for prostate, lung, colorectal, ovarian and extra-ovarian cancers, female and male breast cancers, bladder cancer, and colorectal adenoma. The TMAs are formalin-fixed paraffin embedded samples. Early selections were sent to UCLA for processing, while those selected in 2012 and beyond were sent to the Pathology/Histotechnology Laboratory (PHL) in Frederick, MD. In addition to the cores on the TMAs, other cores were collected for nucleic acid extraction. Generally, up to three blocks that were most representative of the cancer were selected, as well as up to three blocks of matched normal tissue for the TMA cores; the DNA cores were only produced from the cancerous tissue. Each block had one 4-µm slide cut for H&E staining and up to 15 cores were taken. The core size was 0.6 or 1.0 mm, depending on the tissue type. There are approximately 216 cores from 24 - 48 subjects on a TMA.

Tissue Microarrays

Site	Year(s) of Selection	Comments	TMA Core Diameter (mm)
Prostate	Fall 2007	Selected participants had prostatectomy. Participants were either in the CGEMS study, had blood/buccal material available, or were in the PCP study.	0.6
Lung	Fall 2009	Targeted participants were either never smokers, interval cancers, small aggressive lung cancers, or African American. Those identified for selection had evidence of a biopsy or surgery obtaining tissue.	0.6
Colorectal	Spring 2006; Fall 2013	All eligible participants were targeted for selection. Cases are split into pre-treatment and post-treatment tissue (Spring 2006). Newly eligible participants were targeted for selection (Fall 2013).	1.0
Ovarian	Summer 2006; Fall 2010	Subjects with either Path T or an oophorectomy were identified for selection. Cancer cases included invasive ovarian, fallopian tube, and primary peritoneals.	1.0
Adenoma	Fall 2006	Selected groups include: ACF subjects, African Americans, and participants with recurrent adenomas, incident adenomas, and prevalent adenomas. Only large adenomas were selected.	0.6
Breast	Fall 2010; Fall 2012	Participants from the breast CGEMS study and invasive cases were preferentially selected. Only tumors of size 0.5 cm or greater were selected (Fall 2010). DCIS cases selected only. No TMA cores created - membrane slides available for nucleic acid extraction only (Fall 2012).	1.0
Male Breast	Fall 2010	All confirmed and reported cases were selected. Tumor size was not taken into account.	1.0
Bladder	Fall 2012	All available invasive and in situ cases of morphologies 8010, 8020, 8050, 8120, 8130, and 8980 were selected.	1.0

For more information regarding EEMS and the application process, please visit the PLCO EEMS Application page .