Abstract
Background/Aim: Detecting free tumor cells in the peritoneal lavage fluid of gastric cancer patients permits to assess a more accurate prognosis, predict peritoneal recurrence and select cases for a more aggressive treatment. Currently, cytology and molecular biology comprise the two most popular methods of detection that are under constant study by researchers. Materials and Methods: We burrowed into the available literature comparing cytological with molecular detection of free intraperitoneal gastric cancer cells. PubMed, Science Direct, Scopus and Google Scholar were the search engines investigated. Results: As of 2017, 51 dedicated studies have been published. Messenger RNA of carcinoembryonic antigen was the genetic target most frequently described. The genetic technique is usually superior to cytology in sensitivity (38-100% vs. 12.3-67% respectively), whereas cytological examination tends to show a slight pre-eminence in specificity (approximately 100%). Conclusion: So far, given the imperfection of each method, employment of both cytology and molecular examination seem to be mandatory.
- Gastric cancer
- peritoneal lavage
- peritoneal carcinomatosis
- peritoneal recurrence
- cytopathology
- molecular detection
- RT-PCR
- review
Currently, despite the amelioration and standardization of surgery techniques and multi-modal therapy, the prognosis of gastric cancer (GC), especially that of advanced GC (AGC) with serosal invasion (T3 or T4 cancers), remains very poor with a 5-year overall survival (OS) of less than 35% (1). Peritoneal dissemination is the most common route of metastasis followed by AGC and leads to peritoneal recurrence (PR) which is the most frequent cause of death (up to 60% of cases within 2 years) even if curative resection is performed (2). Hence, in the case of AGC, detecting intraperitoneal free cancer cells is of paramount importance because it is significantly related to the prediction of peritoneal metastasis (PM) and patients' prognosis (1, 2). Accordingly, since 1998 the Japanese Gastric Cancer Association (JGCA) recommends to perform peritoneal lavage cytology (PLC) to detect free floating malignant cells within the peritoneal cavity; in addition, in 2010, a positive PLC was classified as metastatic disease also in the 7th edition of the American Joint Committee on Cancer (AJCC) tumor node metastasis (TNM) staging system for GC (3). However, despite its high specificity, conventional PLC shows a questionable sensitivity (11.1 to 80%): in fact, cytology-negative cases often develop PR and meet with worse prognosis (1, 2). In an effort to enhance sensitivity, in the last decades researches have focused on the detection of several epithelial cell-related targets using molecular biology methods. Herein, we offer a meticulous review of the knowledge and progress so far achieved in terms of diagnostic and prognostic results through cytological and genetic examinations of peritoneal lavage (PL) in patients affected with AGC.
Materials and Methods
We systematically reviewed the world literature dealing with the detection of free intraperitoneal cancer cells and comparing cytopathological with molecular examination of peritoneal lavage fluid obtained from patients with GC and AGC. The investigation was carried out through four popular search engines (PubMed, Science Direct, Scopus and Google Scholar). GC, AGC, PC, PM, PLC, genetic detection, molecular diagnosis and Real time reverse chained transcriptase-polymerase chain reaction (RT-PCR) were the key words utilized for searching. Only works comparing the two aforementioned diagnostic techniques (both conventional PLC and molecular analysis) were included in the review.
Results
As of 2017, we found 51 studies dealing with PLC and molecular biological detection of free malignant cells in the peritoneum of GC patients (2, 4-54). Table I summarizes the principle features of the studies included in the review.
PLC. PL was collected by introducing, stirring and aspirating from the abdominal cavity an aliquot of saline solution ranging from 50 ml (9, 27) to 200 ml (24, 41). All the patients did not receive any neoadjuvant treatment and PLs were performed at the beginning of laparotomic gastrectomy (most works), laparoscopy surgery (conducted with curative or staging purpose) (35-40, 43), or paracentesis (only one case, 48). Conventional Papanicolaou staining was adopted in the vast majority of cases, followed by ordinary hematoxylin and eosin (H&E) coloration (9, 11) and Giemsa stain (17, 28). Immunocytochemistry (ICC) was described only occasionally (10, 31, 40, 53). Sensitivity (12.3% to 67%) and specificity (94% to 100%) of PLC were clearly expressed as a percentage ratio only in 12 and 8 studies respectively (Table I).
Molecular detection of intraperitoneal free cancer cells. As for the molecular method, the mature messenger ribonucleic acid (messenger RNA, mRNA) of carcinoembryonic antigen (CEA) has been the target most commonly studied (41 articles) (2, 4-43) followed by 11 studies dealing with cytokeratin 20 (CK-20) mRNA (18, 25, 29-32, 36-39, 42, 49), 10 of which examined CEA mRNA concomitantly (18, 25, 29-32, 36-39, 49). Besides these, 23 additional mRNAs of other molecules have been occasionally investigated (11, 20-22, 28, 31, 36, 39, 44-54). Concerning the type of molecular biological technique, qualitative RT-PCR has been the one most frequently adopted (27 studies), followed by quantitative RT-PCR (Q-RT-PCR) (24 studies). Recently, other ultra-rapid non-PCR tests, such as transcription-reverse transcription concerted reaction (TCR), and PCR-tests, such as reverse transcription loop-mediated isothermal amplification (RT-LAMP), have been successfully employed for genetic analysis (33, 34, 43, 54). Sensitivity and specificity of molecular examination were clearly expressed as a percentage ratio in 23 and 16 studies respectively (Table I). Sensitivity and specificity of CEA mRNA were respectively 38-100% and 7.3-100%, whereas for CK-20 mRNA they were 25-64% and 80.3 to 94% (Table I).
Discussion
Major routes of metastatic spread in GC are direct infiltration of contiguous structures, hematogenous metastasis to the liver, regional lymph node metastasis, intraperitoneal dissemination, mesogastric pathway and intragastric exfoliation (29, 55). Of these, peritoneal dissemination is reported to be the most frequent pattern of metastasis and recurrence (32-54%) in AGC (42). PM from GC results from a 2 step-process: the former is the exfoliation of free cancer cells from the serosal surface of the primary tumor into the peritoneal cavity, the latter is the attachment of cancer cells to a preferable intraperitoneal site (such as omentum, mesenterium and Douglas pouch) with subsequent growth and invasion of the abdominal cavity (2, 29, 42). Furthermore, PM is also recognized as the most important independent prognostic factor for GC PR (29, 30). In fact, due to the development of PM even after R0 tumor resections, prognosis of AGC patients remain poor: to date, except for some individual experiences, no systemic or intraperitoneal treatment proved to effect a complete cure of AGC related-PM (56, 57). For this motive, the cytological examination of peritoneal lavage fluid has been adopted in clinical practice since 1999 by JGCA to detect free tumor cells floating in the abdominal cavity and predict PR (3, 24, 41). However, despite an excellent specificity approximately of 100%, conventional PLC through Papanicolaou or other classical stainings lack sensitivity (11.1 to 80%) since PR is often observed in PLC-negative patients as well as in non-AGC cancers (that is not invading the serosal layer) (30, Table I). Such a disappointing constraint has been in part referred to the technical personal skills of the cytologist but, mostly, it has been reported that the manipulation of the tumor as well as surgical maneuvers (especially when the surgeons open the gastric wall or the lymphovascular vessels) can cause tumor spillage from gastric lumen to peritoneal cavity ensuing PM-PR (2, 58-64); in this sense, of interest, gastric lavage as well as PL might be helpful preventive methods to minimize the risk of spillage of GC cells and PR (2). To increase the sensitivity and reliability of cancer cell detection in PL, in the last two decades researchers employed ICC and molecular biological methods to detect epithelial cell-related targets (40). Compared to standard cytology, ICC with antibodies has been described improving the detection rate by 5% to 15%; nevertheless, PM can still occur in PLC-ICC negative cases (36, 40). Molecular detection with qualitative or quantitative RT-PCR have identified potent molecular markers of various target genes such as transcripts of CEA, CK-20, MMP-7, heparanase and many other molecules (Table I). In most cases, if not all, molecular results (especially those on CEA mRNA) have proved to be superior to cytological ones in sensitivity and prognostic prediction for survival (10, 14, 27, 34-36, 39); in particular, GC patients expressing molecular positive tests, have been identified to benefit from more aggressive adjuvant treatment including intraperitoneal chemotherapy with paclitaxel (43). Concerning specificity, on the other hand, both methods have often achieved 100% (4, 34) and PLC not infrequently was superior (14, 25, 39, 48). Currently however, yet promising, the detection molecular methods are not applied for routine use everywhere: sometimes, in fact, they provided controversial results (such as false-positive cases with mRNAs released by lymphocytes and sane mesothelial cells and other false-positives results due to isolated tumor cells -that is clinically insignificant cells without metastatic potential- and not to micrometastases -the utter active metastatic cells-) and, most of all, they are time-consuming, labor-intensive and expensive (1, 32, 24, 40). With this respect, testing novel ultra-rapid molecular methods (such as OSNA, TCR and LAMP) (33, 34, 40, 43, 54) with innovative targets isolated not only from PLs but also from blood or tissue specimens of AGC patients (65, 66) could surmount some temporal and costly limitations of the current genetic techniques. Furthermore, they could provide an opportunity to perform reliable tailor-made surgery for GC as a common procedure in general hospitals (54).
Main features of the 51 studies dealing with cytological and molecular detection of free intraperitoneal tumor cells in gastric cancer patients.
Conclusion
Detecting free tumor cells in peritoneal lavage fluid of AGC patients is of paramount importance in order to predict PR, assess a more accurate prognosis and select cases for more intensive treatment. So far, given the imperfection of each method, employment of both cytology and molecular method seem to be mandatory for achieving this aim.
Footnotes
This article is freely accessible online.
Conflicts of Interest
The Authors declare no conflicts of interest.
- Received December 15, 2017.
- Revision received January 27, 2018.
- Accepted January 29, 2018.
- Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
