Hugh Mulcahy

Pathological response following long-course neoadjuvant chemoradiotherapy for locally advanced rectal cancer.

Aims : To standardize the pathological analysis of total mesorectal excision specimens of rectal cancer following neoadjuvant chemoradiotherapy for locally advanced disease (T3/T4), including tumour regression.

Tumor budding is a strong and reproducible prognostic marker in T3N0 colorectal cancer.

Background Tumor budding along the advancing front of colorectal adenocarcinoma is an early event in the metastatic process. A reproducible, prognostic budding scoring system based on outcomes in early stage colorectal cancer has not been established. Design One hundred twenty-eight T3N0M0 colorectal carcinoma patients with known outcome were identified. Tumor budding was defined as isolated tumor cells or clusters of <5 cells at the invasive tumor front. Tumor bud counts were generated in 5 regions at 200× by 2 pathologists (conventional bud count method). The median bud count per case was used to divide cases into low (median=0) and high budding (median ≥1) groups. Forty cases were reevaluated to assess reproducibility using the conventional and a novel rapid bud count method. Results Fifty-seven (45%) carcinomas had high and 71 (55%) had low budding scores. High budding was associated with an infiltrative growth pattern (P<0.0001) and lymphovascular invasion (P=0.005). Five-year cancer-specific survival was significantly poorer in high compared with low budding groups: 63% versus 91%, respectively, P<0.0001. Multivariate analysis demonstrated tumor budding to be independently prognostic (hazard ratio=4.76, P<0.001). Interobserver agreement was at least equivalent comparing the conventional to the rapid bud count methods: 87.5% agreement (κ=0.75) versus 92.5% agreement (κ=0.85), respectively. Conclusions Tumor budding is a strong, reproducible, and independent prognostic marker of outcome that is easily assessed on hematoxylin and eosin slides. This may be useful for identifying the subset of T3N0M0 patients at high risk of recurrence who may benefit from adjuvant therapy.

Prediction of esophageal varices in Patients with cirrhosis

Goals To identify predictors of esophageal varices (EV) using available clinical, laboratory, and diagnostic imaging variables. Background Patients with cirrhosis frequently undergo screening endoscopy for varices so that prophylactic therapy and/or follow up can be planned. It is unclear how often patients should be screened endoscopically for varices, and there are few data on the relationship of varices to nonendoscopic variables. Study Charts were reviewed for 247 consecutive patients with cirrhosis who underwent screening esophagogastroduodenoscopy for varices. Results A total of 184 patients (68 women) were studied. Ninety-four patients (51%) had varices; of whom, 90 had only EV (small, n = 66; large, n = 24), 13 had EV and gastric varices, and 4 had isolated gastric varices. The distribution of EV according to the Child–Turcotte–Pugh class was as follows: A, 35%; B, 60%; and C, 69%, with roughly equal prevalence of large varices (29%, 24%, and 24%, respectively) in each class. Independent predictors of large varices were thrombocytopenia (p = 0.02) and splenomegaly (p = 0.04) seen using imaging. A platelet count of less than 68,000/mm 3 had the highest discriminative value for large EV with a sensitivity of 71% and a specificity of 73%. Splenomegaly had sensitivity and specificity of 75% and 58%, respectively. Using these two variables, we placed patients into one of four groups, with a risk for large varices ranging from 4% to 34%. Conclusions The prevalence of EV in cirrhosis increases with the severity of liver disease, as expected. Thrombocytopenia and splenomegaly are independent predictors of large EV in cirrhosis. Further prospective studies might result in a discriminating algorithm to predict which patients with cirrhosis would benefit from early or regular endoscopy to detect clinically significant varices.

Circulating nucleic acids in plasma and serum as a noninvasive investigation for cancer: Time for large‐scale clinical studies?

A noninvasive blood test to detect sporadic cancer has been seen as somewhat of a holy grail by clinicians with an interest in cancer, and its delivery as a quest for many researchers. For this reason, detection of cell-free circulating DNA in the plasma and serum of cancer patients, which has genetic characteristics identical to those of the primary tumour, has resulted in substantial interest and over 200 publications in the medical literature. Interest stems not only from the fact that a blood-based diagnostic and screening test for cancer is an elegant and attractive concept in its own right but also from the fact that conventional diagnostic cancer tests tend to be imperfect.1 As an example, colorectal cancer screening presently relies on faecal occult blood testing, which is both insensitive and nonspecific. In contrast, flexible sigmoidoscopy is sensitive and specific for early distal disease but both invasive and insensitive for proximal disease. Furthermore, barium enema is relatively sensitive and specific but requires colonic preparation, radiation and a day off work, while total colonoscopy is highly sensitive and specific but also invasive and expensive. The situation appears little better for other cancers. No reliable test is available for early detection of lung cancer, with computerised tomography being the most reliable tool. In addition, although several studies indicate that mammographic screening might be a useful strategy for reducing breast cancer mortality, there remains considerable controversy regarding the value of population screening programs.2 Finally, development of conventional tumour markers, e.g., CEA, AFP and the widely used PSA, was driven largely by the introduction of new methods for quantifying small amounts of circulating proteins. However, sensitivity and specificity shortcomings with these assays remain to be overcome.3 The introduction of PCR-based technology in the late 1980s and refinements over the past 10 years have allowed us to detect and quantify extremely small amounts of nucleic acids. This has led to the identification of large numbers of novel molecular targets that may eventually become clinically useful cancer markers. Additionally, many of these markers have been detected in tumourderived nucleic acids (DNA and RNA) extracted from serum and plasma samples. However, it is over 30 years since initial studies indicated that plasma-based nucleic acids might assist in cancer diagnosis. Studies performed in the early 1970s initially showed that increased quantities of DNA could be found in the plasma of patients suffering from different malignancies,4 but it was not until the 1990s that this circulating DNA was shown to exhibit tumourrelated alterations, including decreased strand stability,5 Ras and p53 mutations, microsatellite alterations, aberrant promoter hypermethylation of several genes, rearranged immunoglobulin heavy chain DNA, mitochondrial DNA mutations and tumour-related viral DNA.6,7 Over this period, it was also shown that tumourrelated circulating DNA was not confined to any particular cancer type but appeared to be a ubiquitous finding across the cancer spectrum. Thus, mutant plasma DNA has been found in colorectal, pancreatic, biliary tree, skin, head-and-neck, lung, breast, kidney, ovarian, nasopharyngeal, liver, bladder, gastric, prostate and cervical cancers as well as in haematologic malignancies including lymphomas. The results obtained in plasma/serum DNA in many cancers are opening new research areas and indicate that plasma/ serum may eventually be a suitable source for the development of noninvasive diagnostic, prognostic and follow-up tests for cancer. The diagnostic value of plasma DNA testing appears promising in a number of cancers including melanoma,8 B-cell malignancies9 and NPC.10 However, perhaps the closest to achieving clinical significance is an assay for EBV DNA, which is closely associated with NPC in southern Asia. Using real-time PCR, EBV DNA is detectable in 95% of NPC cases compared 5% of healthy controls. In addition, following diagnosis, the test appears to be useful for determining prognosis and monitoring disease response to treatment. Overall, the absolute levels of EBV DNA at presentation are of considerable prognostic value as are levels following treatment, in so far as a high level is suggestive of the presence of residual disease. It appears likely that in the next few years estimation of EBV DNA will become a routine part of the staging procedure for NPC and will directly influence therapeutic options for this tumour. The prognostic value of plasma/serum tumor DNA has also been established for other cancers, with high levels also indicative of a poor prognosis.6,7 Surprisingly, using essentially similar methodology, cell-free mRNA can also be detected in plasma and should, at least in theory, permit plasma-based expression profiling.11 Studies with RNA markers are particularly promising due to their close association with malignancy. In this short review, we emphasise studies on the most widespread malignancies, colorectal, pancreatic, lung, breast and prostate cancers, which need a simple test that could become clinically available in the not too distant future.

Development and Independent Validation of a Prognostic Assay for Stage II Colon Cancer Using Formalin-Fixed Paraffin-Embedded Tissue

PURPOSE Current prognostic factors are poor at identifying patients at risk of disease recurrence after surgery for stage II colon cancer. Here we describe a DNA microarray-based prognostic assay using clinically relevant formalin-fixed paraffin-embedded (FFPE) samples. PATIENTS AND METHODS A gene signature was developed from a balanced set of 73 patients with recurrent disease (high risk) and 142 patients with no recurrence (low risk) within 5 years of surgery. RESULTS The 634-probe set signature identified high-risk patients with a hazard ratio (HR) of 2.62 (P < .001) during cross validation of the training set. In an independent validation set of 144 samples, the signature identified high-risk patients with an HR of 2.53 (P < .001) for recurrence and an HR of 2.21 (P = .0084) for cancer-related death. Additionally, the signature was shown to perform independently from known prognostic factors (P < .001). CONCLUSION This gene signature represents a novel prognostic biomarker for patients with stage II colon cancer that can be applied to FFPE tumor samples.

Alu Repeat Sequences Are Present in Increased Proportions Compared to a Unique Gene in Plasma/Serum DNA

Abstract: Small amounts of DNA circulate freely in plasma or serum, but the mechanism of release is not known. To determine if DNA is actively excreted from viable cells, we utilized real‐time PCR to measure the proportion of Alu repeat sequences compared to the β‐globin gene in serum and lymphocyte DNA in 27 cancer patients and 22 healthy controls. The proportion of Alu compared to β‐globin was significantly greater in serum DNA than in lymphocyte DNA both in control subjects (p= 0.003) and in cancer patients (p < 0.001). Overall, the proportion was similar in cancer and control patients (p= 0.79). Further experiments showed that the β‐globin gene was not more vulnerable to degradation by nuclease action than Alu sequences. Our results lead us to conclude that active DNA release is likely to play a significant role in the origin of circulating DNA.

Tumour Tissue Microenvironment Can Inhibit Dendritic Cell Maturation in Colorectal Cancer

Inflammatory mediators in the tumour microenvironment promote tumour growth, vascular development and enable evasion of anti-tumour immune responses, by disabling infiltrating dendritic cells. However, the constituents of the tumour microenvironment that directly influence dendritic cell maturation and function are not well characterised. Our aim was to identify tumour-associated inflammatory mediators which influence the function of dendritic cells. Tumour conditioned media obtained from cultured colorectal tumour explant tissue contained high levels of the chemokines CCL2, CXCL1, CXCL5 in addition to VEGF. Pre-treatment of monocyte derived dendritic cells with this tumour conditioned media inhibited the up-regulation of CD86, CD83, CD54 and HLA-DR in response to LPS, enhancing IL-10 while reducing IL-12p70 secretion. We examined if specific individual components of the tumour conditioned media (CCL2, CXCL1, CXCL5) could modulate dendritic cell maturation or cytokine secretion in response to LPS. VEGF was also assessed as it has a suppressive effect on dendritic cell maturation. Pre-treatment of immature dendritic cells with VEGF inhibited LPS induced upregulation of CD80 and CD54, while CXCL1 inhibited HLA-DR. Interestingly, treatment of dendritic cells with CCL2, CXCL1, CXCL5 or VEGF significantly suppressed their ability to secrete IL-12p70 in response to LPS. In addition, dendritic cells treated with a combination of CXCL1 and VEGF secreted less IL-12p70 in response to LPS compared to pre-treatment with either cytokine alone. In conclusion, tumour conditioned media strongly influences dendritic cell maturation and function.

Spatial variation of the colonic microbiota in patients with ulcerative colitis and control volunteers

Objectives The relevance of spatial composition in the microbial changes associated with UC is unclear. We coupled luminal brush samples, mucosal biopsies and laser capture microdissection with deep sequencing of the gut microbiota to develop an integrated spatial assessment of the microbial community in controls and UC. Design A total of 98 samples were sequenced to a mean depth of 31 642 reads from nine individuals, four control volunteers undergoing routine colonoscopy and five patients undergoing surgical colectomy for medically-refractory UC. Samples were retrieved at four colorectal locations, incorporating the luminal microbiota, mucus gel layer and whole mucosal biopsies. Results Interpersonal variability accounted for approximately half of the total variance. Surprisingly, within individuals, asymmetric Eigenvector map analysis demonstrated differentiation between the luminal and mucus gel microbiota, in both controls and UC, with no differentiation between colorectal regions. At a taxonomic level, differentiation was evident between both cohorts, as well as between the luminal and mucosal compartments, with a small group of taxa uniquely discriminating the luminal and mucosal microbiota in colitis. There was no correlation between regional inflammation and a breakdown in this spatial differentiation or bacterial diversity. Conclusions Our study demonstrates a conserved spatial structure to the colonic microbiota, differentiating the luminal and mucosal communities, within the context of marked interpersonal variability. While elements of this structure overlap between UC and control volunteers, there are differences between the two groups, both in terms of the overall taxonomic composition and how spatial structure is ascribable to distinct taxa.

Primary Sclerosing Cholangitis and Disease Distribution in Inflammatory Bowel Disease

BACKGROUND & AIMS The relationship between site of intestinal inflammation and primary sclerosing cholangitis (PSC) development in inflammatory bowel disease (IBD) has not been studied extensively, but may be important in understanding the pathogenesis of PSC. We aimed to determine patterns of disease distribution in IBD patients with and without PSC. METHODS We performed a 2-part study involving the following: (1) 2754 IBD patients and (2) 82 separate PSC patients attending the Irish National Liver Transplant Unit. RESULTS Fifty-nine of 2708 (2.2%) IBD patients had PSC. In ulcerative colitis patients, PSC incidence increased with increasing colonic involvement (P = .001) and was relatively rare in those without total colitis. Thirteen Crohns disease patients had PSC, none with isolated small-bowel disease had PSC (P = .03). In study 2, the majority of ulcerative colitis patients with PSC had total colitis, whereas the remainder had disease extending at least to the left colon. In addition, all 10 PSC patients with Crohns disease had colonic involvement. CONCLUSIONS An inflamed colon, but not small bowel, is important in PSC development and it is possible that bacterial translocation and subsequent portal bacteremia is important in PSC development in IBD.

Localization of nuclear cathepsin L and its association with disease progression and poor outcome in colorectal cancer

Previous in vitro studies have identified a nuclear isoform of Cathepsin L. The aim of this study was to examine if nuclear Cathepsin L exists in vivo and examine its association with clinical, pathological and patient outcome data. Cellular localization (nuclear and cytoplasmic) and expression levels v of Cathespin L in 186 colorectal cancer cases using immunohistochemistry. The molecular weight and activity of nuclear and cytoplasmic Cathepsin L in vivo and in vitro were assessed by Western blotting and ELISA, respectively. Epithelial nuclear staining percentage (p = 0.04) and intensity (p = 0.006) increased with advancing tumor stage, whereas stromal cytoplasmic staining decreased (p = 0.02). Using multivariate statistical analysis, survival was inversely associated with staining intensity in the epithelial cytoplasm (p = 0.01) and stromal nuclei (p = 0.007). In different colorectal cell lines and in vivo tumors, pro‐ and active Cathepsin L isoforms were present in both the cytoplasm and nuclear samples, with pro‐Cathepsin L at 50 kDa and active Cathepsin L at 25 kDa. Purified nuclear and cytoplasmic fractions from cell lines and tumors showed active Cathepsin L activity. The identification of nuclear Cathepsin L may play an important prognostic role in colorectal disease progression and patient outcome. Moreover, these findings suggest that altering active nuclear Cathepsin L may significantly influence disease progression.