PTEN and P-4E-BP1 might be associated with postoperative recurrence of rectal cancer patients undergoing concurrent radiochemotherapy

The clinical data of 1599 rectal cancer patients who received radiotherapy with concurrent 5-fluorouracil (5-FU) based chemotherapy and total mesorectal excision (TME) surgery were collected from January 2012 through May 2020, from Tianjin Union Medical Center, Nankai University. Clinical staging was classified by endosonography, computed tomography (CT) scan, and magnetic resonance imaging (MRI). The inclusion criteria were as follows; (1) pathologically diagnosed rectal adenocarcinoma; (2) cT3, cT4 or N + according to American Joint Committee on Cancer (AJCC) criteria (7th edition); (3) no evidence of distant metastasis; and (4) Karnofsky performance status over 70. To perform the study, 109 recurrent patients were selected, and another 109 patients were randomly selected from 1490 non recurrent ones. Sixty-nine patients with incomplete pathologic data or follow up data were excluded, reducing the total number of patients analyzed to 149, which were divided into a local recurrence group (LR, n = 73) and a nonrecurrence group (NR, n = 76).

All patients underwent radiotherapy with conventional fractionation of 1.8 Gy up to a total dose of 45 Gy in 25 fractions. For patients undergoing preoperative radiotherapy, an additional 5.4 Gy in 3 fractions was delivered as a boost to the gross tumor volume (GTV). The clinical target volume (CTV) was defined as the GTV and regional lymphatics including the mesorectal, presacral, internal iliac, and distant common iliac lymph nodes. To cover the CTV properly, the borders of the pelvic fields were as follows: (1) superior: the sacral promontory at the L5-S1 junction level, (2) inferior: 3 to 5 cm distal to the GTV, (3) lateral: 1.5 cm lateral to the widest bony margin of the true pelvic walls, (4) anterior: posterior border of the symphysis pubis, and (5) posterior: 1.5 cm behind the anterior bony sacral margin. The concurrent chemotherapy regimens were as follows: (1) CAPEOX, two cycles of L-OHP (130 mg/m² day1) and capecitabine (825 mg/m² twice daily day1-14 ) given at the first, and fourth weeks of radiotherapy; (2) CAPE, oral capecitabine (825 mg/m² twice daily day1-5) given at every week of radiotherapy; (3) mFOLFOX6, L-OHP (85 mg/m² day1), calcium levofolinate (200 mg/m² day1) and 5-Fu (400 mg/m² bolus, 2400 mg/m² civ 46 h) given at the first, third and fifth weeks of radiotherapy; and (4) Tegafur (20 mg/kg day1-3) given at the first, and fourth weeks of radiotherapy. Patients receiving neoadjuvant therapy underwent planned TME surgery 4–6 weeks after the end of radiotherapy, and patients receiving adjuvant therapy underwent TME surgery 4–8 weeks before radiotherapy.

The patients were seen every 3 months during the first 2 years after surgery and every 6 months thereafter. Follow-up evaluations included physical examination, complete blood count, biochemical profile, abdominopelvic CT (or MRI), chest radiography, and proctoscopy (every 1 year).

Tissue samples were fixed in buffered 4% formalin, paraffin embedded, and used for TMA construction. Hematoxylin-eosin–stained histologic sections from resection specimens were used to mark representative tumor areas. A 1.0-mm tissue core was punched out from the tumor area of each specimen and transferred to a TMA recipient block using a TM-1 tissue arrayer (Bilcool Inc, Beijing, China). Three TMAs contained 149 tumor spots consisting of 73 rectal cancers of the local recurrence group and 76 rectal cancers of the nonrecurrence group.

The 4-µm–thick sections from TMA were deparaffinized and rehydrated using xylene and ethanol, and immersed in a 3% hydrogen peroxide solution for 10 min to block endogenous peroxidase. The sections were then boiled for 30 min in 10 mM/L citrate buffer solution (pH 6.0) for antigen retrieval. Slides were incubated with 5% skim milk for 20 min and then with PTEN antibody (1:1000, #9559, Cell Signaling Technology, Danvers, MA, USA), SIRT1 antibody (1:2000, ab110304, Abcam Biotechnology, Cambridge, MA, UK), p-4E-BP1 antibody (1:1000, #2855, Cell Signaling Technology, Danvers, MA, US) and pS6 antibody (1:1000, #5364, Cell Signaling Technology, Danvers, MA, USA) at 4 °C for 16 h, and visualized using the SP-9000 Polymer Detection System (Golden Bridge International Corp, Wharton, TX, USA) following the manufacturer’s instructions. Subsequently, the slides were incubated with 3, 3′-diaminobenzidine tetrahydrochloride-H₂O₂ solution for visualization, and counterstained with hematoxylin. All slices were observed under an Olympus DS-Ri2 microscope (Olympus, Tokyo, Japan), and PTEN, SIRT1, p-4E-BP1, and pS6 staining was defined as yellow or brown particles in either the nucleus or the cytoplasm. Immunostained slides were evaluated by two experienced pathologists who were blinded to all clinical parameters. The staining results were evaluated using a semiquantitative method: the percentage of stained cells × the staining intensity. This percentage was scored as 0 for < 5%, 1 for 5–25%, 2 for 26–50%, 3 for 51–75%, and 4 for 76–100%, and the staining intensity was then scored as 0 for no staining, 1 for light yellow, 2 for yellow brown, and 3 for brown. A score less than 4 is defined as negative expression, and a score greater than or equal to 4 is defined as positive expression [17].