Patient case: 61-year-old male with Ampullary carcinoma. Genetic variant: KRAS. Other conditions: None.

Clinical trial: Circulating Tumour DNA (ctDNA) Rectal Cancer and the Relationship to Extramural Venous Invasion

Summary: In almost one third of patients with rectal cancer, cancer spreads through the bowel wall into nearby veins which can be seen on MRI scans and is known as mrEMVI. It has been observed that this is linked with worse survival due to the spread of cancer to the liver and other organs. The investigators think that this may occur by spread of cancer cells in the blood stream. Circulating cell-free tumour DNA (ctDNA) can be detected in the blood of patients with both metastatic and primary carcinoma (but not healthy individuals).
  This trial does not interfere with the patient's routine treatment pathway. Participants will be chosen if they demonstrate mrEMVI on their baseline MRI scan. 20 patients who become mrEMVI positive (following pre-operative therapy) and 20 patients who are mrEMVI negative will have blood samples taken before and during surgery and a comparison will be made between the two groups. If the investigators can find a link between the cancer cells seen in blood to mrEMVI they may be able to use this test to help guide treatment in the future. This study will serve as the feasibility of a larger, comparative study.

Detailed description: Vascular or venous invasion in patients with rectal cancer has been considered a significant prognostic factor for over half a century. Interest in understanding the mechanisms that govern tumour spread from the rectum to distant sites such as the liver has driven the study of lymphatic and vascular invasion by malignant cells. Venous invasion has traditionally been identified on histological analysis of resection specimens but there remains variability between pathologists. Inconsistencies in the histological methods used in identifying venous or vascular invasion have led to difficulties in interpreting the actual incidence or prevalence; the literature reports prevalence ranging between 9-61%. None-the-less EMVI is considered an independent prognostic factor and as such has been recognised by the Royal College of Pathologists in the UK by including it in the minimum dataset for reporting colorectal cancers.
  More recently, the increasing accuracy of high-resolution magnetic resonance imaging (MRI) has meant that prognostic factors such as extra mural venous invasion can be confidently identified on imaging. The literature has shown that EMVI can be well identified using MRI. mrEMVI has also been shown to be an independent prognostic factor for disease recurrence following chemoradiotherapy. Furthermore MRI can identify additional cases of EMVI when compared to routine histopathology and the degree of improvement of mrEMVI on post-CRT scans is associated with improved disease-free survival.
  Circulating cell-free tumour DNA (ctDNA) can be detected in the blood of patients with both metastatic and primary carcinoma. By virtue of the presence of EMVI, it is likely that tumour cells and DNA fragments are more likely to be shed, and a higher blood circulating % mutation frequency will be seen as compared to patients who are mrEMVI negative.
  These tumour fragments may then act as microemboli to other sites and potentially increase the risk of developing metastases. Furthermore intra-operative manipulation of the tumour itself may lead to greater release of tumour cells into the mesenteric circulation suggested by wedge shaped defects in the liver after surgical resection due to portal vein thrombi.
  There are several ways in which to identify cell free tumour DNA. Studies using polymerase chain reaction (PCR- a way to concentrate the amount of ctDNA to become a meaningful measurable amount which can then be used to assess for aberrations) also showed ctDNA to be associated with increased risk of disease recurrence and worse survival outcomes. There is no standard technique and confounding factors are present in each step of the analysis of ctDNA, which can lead to sampling errors and inconsistencies. This lead to development of several techniques to try to increase the isolation of the DNA such as fast spin columns so that ctDNA only binds to a silica gel and then eluted by using water or a buffer system. Polymerase chain reaction can be used to quantify the tumour ctDNA. Other techniques include double spinning and the use of phenol-chloroform, amplification methods or rapid spinning to retrieve small DNA fragments. Early studies measured and quantified the total amount of circulating DNA and found that higher amounts of overall cell free DNA was associated with tumour. ctDNA from tumour cells can be distinguished from normal ctDNA by examining its profile in relation to size and fragmentation however debate arose on whether longer or shorter fragments were associated with tumour resulting in unclear conclusions. Circulating tumour DNA has been hypothesized as arising from tumour, normal tissue or tumour-associated stromal cells, however, tumour derived ctDNA tended to be more fragmented and exhibit a greater variation in size although overall were larger than ctDNA arising from normal tissue.
  Investigation into whether there is a spike in the intra-operative phase would be of interest. A further study shows that recurrent disease is related to ctDNA increases and is higher in patients with metastatic disease and furthermore those patients with higher ctDNA levels and metastases have a poorer prognosis. The challenge with this type of analysis is that the cell-free DNA may or may not be directly related to tumour and so more recently focus has been turned on tumour specific cell free tumour DNA as defined by known mutations such as kras and p53 (tumour p53 is termed tp53).
  In colorectal cancer the majority of patients have non-hypermutated tumours on histology. Of the hyper-mutated tumours TP53 and KRAS make up 60% and 43% of mutations respectively. The investigators will screen blood-based ctDNA for p53 and KRAS. When the investigators take the blood they will get a certain amount of circulating DNA which could be tumour or normal and then they analyse this to see if it has the mutations and if it does then the investigators can call it ctDNA (circulating tumour DNA). If there is no mutation this does not necessarily exclude ctDNA because there is a sensitivity and specificity associated with the analysis methods of the mutations (i.e KRAS and tp53) and not all tumours will have the KRAS or p53 mutations. In some reports from colorectal cancer the pick-up rate for these mutations in blood correlates to 100% of those tumours found to have the same mutation on histology. Furthermore plasma or serum sampling may be more sensitive and so in a cohort of 100 patients the pick-up rate of tumours may be up to 70% for one or the other mutations (kras and TP53). In other cancers such as lung cancer, the investigators pick up 65% of mutation in the blood, therefore on average the investigators can expect to detect 0.65 x 60 = 39% of patients to have detectable mutations in the blood. For this study it is likely then that the pick-up rate will lie between 39 - 70%. In this group, the investigators will be able to compare the ctDNA profile between EMVI positive and negative tumours after neoadjuvant long course chemoradiotherapy and determine if the ctDNA profile changes intraoperatively.

Eligibility: Inclusion Criteria:
  -  High-risk patients with biopsy proven rectal adenocarcinoma who will undergo long-course chemoradiotherapy (CRT) and who are potentially eligible for curative surgery
  -  Patients who have persistently (i.e. in baseline and post-treatment MRI) mrEMVI positive (with superior rectal vein invasion) or mrEMVI negative tumours.
  -  Patients aged over 18 years
  -  Negative (on CT) for metastatic disease
  Exclusion Criteria:
  -  Under 18 years of age
  -  Patients with any metastatic disease
  -  Patients with a synchronous second malignancy

Relevance to patient case: not relevant