Pelvic Lavage CEA Messenger RNA Predicts Rectal Cancer Local Recurrence

A retrospective single-center investigation of 178 patients undergoing curative-intent resection for primary rectal cancer has revealed that detecting carcinoembryonic antigen messenger RNA in pelvic washings correlates significantly with pelvic disease recurrence. Local recurrence following surgery remains a major prognostic challenge that is thought to stem from residual malignant cells situated beyond the surgical dissection plane, distinct from the biology of distant metastasis. Although the adoption of total mesorectal excision alongside modern multimodal treatments has markedly reduced recurrence frequencies within the pelvic cavity, managing pelvic relapses remains technically difficult and diminishes patient quality of life. Standard post-resection risk assessments currently rely on the circumferential resection margin measured by pathologists on fixed surgical specimens. While a margin distance of one millimeter or less reliably indicates elevated recurrence danger, this standard metric depends heavily on laboratory preparation and tissue measurement techniques across pathology departments. Moreover, fixed histological margin evaluations cannot capture free or shed cancer cells dispersed into the pelvis during surgical manipulation, leaving the wider pelvic cavity unmonitored.

To evaluate whether molecular detection can capture shed malignant cells that evade pathological margin assessments, investigators analyzed pelvic lavage washings collected from 178 consecutive surgical cases treated between April 2013 and November 2023. Curative-intent intervention was strictly defined as achieving macroscopic tumor clearance with microscopically negative margins, classified as an R0 resection of the primary growth. Patients presenting with synchronous peritoneal dissemination, concurrent multiple colorectal malignancies, or double primary cancers were excluded from the analysis, and written informed consent was documented across the cohort. The patient collective had a median age of 69 years, spanning 30 to 93 years, with 116 male and 62 female participants. Rectal lesions were anatomically distributed across the upper rectum in 56 patients, the middle rectum in 40 patients, and the lower rectum in 82 patients. Pathological depth staging identified T1 tumors in 25 individuals, T2 lesions in 41 cases, T3 invasion in 88 individuals, and T4 extension in 24 cases. Lymph node metastasis was confirmed pathologically in 68 patients and absent in 110 patients, yielding overall disease distributions of 49 stage I, 60 stage II, 59 stage III, and 10 stage IV cases. Among those with stage IV disease, five presented liver metastases and five presented lung metastases, with complete metastatic resections achieved in eight individuals while two underwent primary tumor resection without removal of metastatic deposits. Surgical access included 13 open procedures, 99 laparoscopic resections, and 66 robot-assisted operations, while 17 patients received neoadjuvant chemoradiotherapy, seven completed neoadjuvant chemotherapy, and 37 underwent lateral pelvic lymph node dissections.

The fluid collection methodology followed a standardized intraoperative sequence designed to isolate shed cellular material while strictly avoiding contamination from internal bowel flora. After surgeons achieved complete distal mobilization of the rectum but before making any enterotomy into the bowel wall, 100 milliliters of normal saline was introduced into the pelvic basin, agitated gently, and subsequently aspirated into sterile collection containers. Delaying the enterotomy until after fluid retrieval ensured that intraluminal contents and mucosal tissue remained fully sequestered. The collected aspirate was centrifuged at 2000 revolutions per minute for 10 minutes to isolate the cellular fraction. Following supernatant removal, the isolated pellet underwent five successive washing steps with heparinized normal saline. Technicians then resuspended the pellet in 600 microliters of extraction lysis buffer before freezing the samples at minus 80 degrees Celsius. Total RNA was subsequently isolated using commercial purification columns, followed by reverse transcription into complementary DNA with commercial transcriptase reagents.

Quantitative real-time reverse transcription polymerase chain reaction assays were performed using specific primer sets and fluorescent hybridization probes on a LightCycler platform, employing glyceraldehyde-3-phosphate dehydrogenase as an endogenous reference standard. Receiver operating characteristic curve analysis of the expression data identified an optimal cutoff threshold of 0.26 relative expression units via the Youden index. This mathematical cutoff yielded a baseline area under the curve of 0.648, with a 95 percent confidence interval spanning 0.450 to 0.846, alongside a diagnostic sensitivity of 37.5 percent and a diagnostic specificity of 97.7 percent. Internal bootstrap validation over 1000 resampling iterations confirmed statistical consistency, showing a mean area under the curve of 0.646 with 2.5th to 97.5th percentiles between 0.441 and 0.851. Measured molecular levels ranged from 0 to 2.77 across the cohort, with seven individuals, representing 3.9 percent of the total patient population, testing positive above the 0.26 threshold. Among these seven positive cases, six were male, three had tumors located in the middle rectum and three in the lower rectum, five presented T3 tumor depth, and five exhibited lymph node metastasis. Pathological staging among the marker-positive subset showed two stage II cases and five stage III cases, with no stage I or stage IV representations. Operative approaches among this subgroup included three open, three laparoscopic, and one robotic procedure, while only one positive patient had undergone prior neoadjuvant chemoradiotherapy and none had received neoadjuvant chemotherapy.

Circumferential resection margin status was concurrently verified by a dedicated institutional pathologist examining formalin-fixed, paraffin-embedded surgical specimens, with margin positivity designated as tumor cells extending within one millimeter of the surgical resection edge. Overall, 11 of the 178 surgical cases, or 6.2 percent, demonstrated positive margin involvement. The median age among margin-positive individuals was 66 years, with eight male and three female patients. Pathological evaluation of these margin-compromised specimens demonstrated that lesions were situated in the lower rectum in eight cases, the middle rectum in two cases, and the upper rectum in one patient. Furthermore, all 11 margin-positive tumors exhibited advanced invasion depths, comprising seven T3 lesions and four T4 growths, while lymph node metastasis was identified in six patients. Stage distribution among these 11 individuals showed four stage II, six stage III, and one stage IV diagnosis, with surgical management accomplished via open laparotomy in two patients, laparoscopic surgery in six patients, and robot-assisted resection in three patients. Neoadjuvant chemoradiotherapy had been administered preoperatively to two of these patients, with nine receiving no neoadjuvant therapy.

During a median clinical follow-up period of 59.1 months, ranging from 0.7 to 137.9 months, local recurrence occurred in eight of the 178 enrolled patients, representing an overall pelvic recurrence incidence of 4.5 percent. Recurrence was formally established when tumor regrowth appeared below the sacral promontory or at the colorectal anastomosis on scheduled surveillance computed tomography, serum testing, and clinical evaluations, excluding isolated lateral lymph node progression. Local recurrence developed in 36.4 percent of patients harboring positive resection margins, accounting for four recurrences out of 11 individuals, and in 42.9 percent of patients testing positive for pelvic lavage carcinoembryonic antigen messenger RNA, representing three recurrences out of seven individuals. Notably, all three individuals in the entire study who were simultaneously positive for both molecular pelvic washings and histological margins developed local pelvic recurrence, representing a 100 percent recurrence rate. In contrast, among the 163 patients who were negative for both circumferential margin involvement and lavage messenger RNA, local recurrence appeared in only four cases, establishing a baseline recurrence rate of 2.5 percent.

Contingency analyses using Fisher’s exact test revealed specific clinicopathological features associated with intraoperative molecular positivity. Detection of the messenger RNA marker was significantly linked to deeper pathological invasion, specifically pT3 and pT4 stages, with all seven positive cases occurring in advanced depth categories compared to 105 of 171 marker-negative cases, yielding a p-value of 0.047. Marker positivity was also strongly associated with pathological margin involvement, showing positive margins in three of the seven marker-positive cases compared to eight of 171 marker-negative individuals, with a p-value below 0.001. Surgical modality demonstrated another significant association, as three of the seven positive tests occurred among the 13 patients undergoing open surgery compared to four positives among 165 minimally invasive procedures, generating a p-value of 0.009. Conversely, no statistically significant associations emerged between messenger RNA status and patient age, patient sex, anatomical tumor location, regional lymph node status, or whether lateral pelvic lymph node dissection had been performed.

Statistical evaluation of clinical factors influencing pelvic recurrence demonstrated that advanced tumor depth, molecular washings, and microscopic margin compromise were all individually tied to failure of local control. Specifically, all eight local recurrences developed in patients with pT3 or pT4 disease, whereas zero recurrences occurred among the 66 patients with pT1 or pT2 disease, producing a p-value of 0.027. Pelvic lavage messenger RNA positivity demonstrated a significant association with recurrence, present in three of eight recurring patients versus four of 170 non-recurring patients, yielding a p-value of 0.002. Similarly, positive resection margins were documented in four of eight recurrent cases compared to seven of 170 non-recurrent cases, generating a p-value below 0.001. Simultaneous positivity for both biomarkers was found in three recurrent patients and zero non-recurrent patients, also reaching a p-value below 0.001. Kaplan–Meier survival estimates corroborated these patterns, demonstrating significantly diminished local recurrence-free survival distributions for individuals exhibiting molecular wash positivity as well as those exhibiting positive surgical margins, with both log-rank tests achieving significance below 0.001.

Multivariable proportional hazards modeling confirmed that molecular lavage findings provide prognostic signals independent of standard circumferential margin status. When entered into a two-variable Cox regression model, circumferential margin positivity retained a strong hazard ratio of 11.09, with a 95 percent confidence interval spanning 2.06 to 59.68 and a p-value of 0.005. Concurrently, intraoperative messenger RNA positivity maintained an independent hazard ratio of 5.99, with a 95 percent confidence interval from 1.06 to 33.81 and a p-value of 0.043. Because real-time quantitative reverse transcription polymerase chain reaction produces objective, standardized numerical results, investigators observed that lavage assessments may capture detached neoplastic cells freed into the pelvic basin during tissue dissection that escape standard histological specimen examination. This complementary dynamic may explain why patients with dual positivity demonstrated complete local failure during surveillance, suggesting that molecular washings reflect distinct oncological phenomena from static margin measurements.

Despite these informative observations, the investigators noted several methodological limitations that mandate cautious interpretation. The retrospective, single-institution design restricts immediate generalizability to broader surgical populations. More critically, the occurrence of only eight local recurrences and seven molecularly positive cases resulted in an events-per-variable ratio of four in the multivariable model, widening the confidence intervals, increasing vulnerability to statistical overfitting, and framing the numerical hazard ratios as hypothesis-generating rather than definitive proof of prognostic independence. Furthermore, the significant association between molecular positivity and open surgery suggests that selection bias cannot be ruled out, as open laparotomy was typically chosen for advanced, bulky, or technically demanding anatomies. The modest sensitivity of 37.5 percent and area under the curve of 0.648 preclude using this marker as an isolated screening tool, and the 0.26 cutoff requires external validation. The authors concluded that while molecular washings cannot justify escalating adjuvant chemotherapy alone, intraoperative testing may ultimately support individualized surveillance strategies, such as intensified pelvic magnetic resonance imaging, while motivating prospective multi-institutional trials and the future evaluation of complementary markers such as cytokeratin 20.

Subject of Research: Intraoperative pelvic lavage CEA messenger RNA detection and rectal cancer local recurrence

Article Title: Carcinoembryonic Antigen Messenger RNA in Pelvic Lavage Fluid Predicts Local Recurrence After Curative‐Intent Resection of Rectal Cancer

Article References: Asai, H., Yamakawa, Y., Shiga, K., Uehara, S., Kato, A., Suzuki, T., Ushigome, H., Takahashi, H., Matsuo, Y., & Takiguchi, S. (2026). Carcinoembryonic Antigen Messenger RNA in Pelvic Lavage Fluid Predicts Local Recurrence After Curative‐Intent Resection of Rectal Cancer. Annals of Gastroenterological Surgery, 10(6), 1762-1769. https://doi.org/10.1002/ags3.70257

Image Credits: AI Generated

DOI: 10.1002/ags3.70257

Keywords: rectal cancer, carcinoembryonic antigen, pelvic lavage, messenger RNA, local recurrence, circumferential resection margin, oncology, surgical oncology, Carcinoembryonic, Antigen, Messenger, Pelvic