John L. McCall

Risk of Dissemination With Biopsy of Colorectal Liver Metastases

AbstractPURPOSE: Liver surgeons usually recommend against biopsy of colorectal liver metastases because of the risk of local dissemination. To date, only case reports describing this problem have been published. This study is an attempt to quantify the risk of biopsy-related dissemination. nMETHODS: A multicenter, retrospective review was undertaken of cases of colorectal liver metastases presenting for surgery that had undergone a preoperative biopsy. nRESULTS: Two hundred thirty-one cases of colorectal liver metastases presenting for surgery were identified. Forty-three cases had undergone a preoperative biopsy (18.6 percent). Seven patients had evidence of dissemination related to the biopsy, giving a risk of dissemination of 16 percent (95 percent confidence interval, 7–30 percent). The risk of dissemination was not related to the type of biopsy. Within the follow-up period (median, 21 months), 3 of the 7 cases with evidence of dissemination and 11 of the 35 without dissemination were alive without disease. Twenty-five percent of the 36 cases without dissemination were resected, whereas 6 of the 7 cases with dissemination were resected. nCONCLUSIONS: There is a significant risk of local dissemination with biopsy of colorectal liver metastases. In this series this was independent of the type of biopsy. There was no demonstrated effect on resectability or survival, but numbers were small, and the median follow-up was short.

Analysis of potential markers for detection of submicroscopic lymph node metastases in breast cancer.

SummaryWe have developed sensitive assays for cytokeratin (K) 8, 16, 19, stromelysin 3 (ST3), MUC1 and maspin mRNAs using reverse transcription polymerase chain reaction (RT-PCR) and used these to assess lymph node status in patients undergoing surgery for breast cancer. In addition the RT-PCR assays were tested against lymph nodes from non-cancer patients to determine their specificity. Despite high sensitivity RT-PCR assays for K8, K16, K19, ST3 and maspin were not found to be useful as markers of submicroscopic disease as transcripts of these genes were detected in the great majority of control lymph nodes tested. Expression of MUC1 was also not found to be useful as it was both insensitive and non-specific. The importance of assessing potential markers against an adequately sized control population is demonstrated, as failure to do so can lead to erroneous conclusions.

Maintaining confidentiality of interim data to enhance trial integrity and credibility

Background For clinical trials of interventions that could affect mortality or major morbidity, Data Monitoring Committees have an important role in safeguarding patient interests and enhancing trial integrity and credibility. In trials overseen by an independent DMC it is widely recognized that interim data should remain confidential to the DMC and to the statistical group preparing reports. However, we have found that the principle of confidentiality is not always followed in practice, particularly where the interim data include complete results on a short-term outcome measure. Purpose To discuss the reasoning and evidence supporting the principle of confidentiality of interim data with emphasis on the setting where the interim data include complete results on a short-term outcome. Methods We review the reasons why wider access to interim data can increase the risk of false positive or false negative conclusions and discuss the types of harm which can occur. We provide illustrations and insights from recent experiences and discuss the level of consensus in the research community. Results The arguments in favor of early release of interim data include the need to provide reliable data in a timely manner to patients and physicians, the potential to increase the enthusiasm of trial investigators, and to restore equipoise. However interim data, even where these include complete results on a short-term outcome measure, provide an unreliable and biased assessment of the overall benefit-to-risk profile of the trial treatments. Pre-judgment based on over-interpretation of such interim data can affect recruitment, treatment delivery, and follow-up, risking the ability of the trial to achieve its goals. Conclusions In order to preserve the integrity of a trial and safeguard the interests of patients, interim data, including complete data on short-term outcomes, should remain confidential to the DMC and the statistical group responsible for preparing interim reports until the trial has achieved its primary objectives. Clinical Trials 2008; 5: 157—167. http://ctj.sagepub.com

Analysis of IGF2 gene imprinting in breast and colorectal cancer by allele specific-PCR

The insulin‐like growth factor II (IGF2) gene is imprinted with the paternal allele expressed and the maternal one silent. Loss of imprinting (LOI) of IGF2 has been suggested to play a role in the development of tumours, but the reported incidence of IGF2 LOI in tumours shows considerable variation, which may stem from different methodologies employed. In particular, partial digestion of reverse transcriptase‐polymerase chain reaction (RT‐PCR) products by restriction enzymes can lead to inaccurate measurements. To overcome the problem of partial enzymatic digestion, a novel method termed allele specific‐polymerase chain reaction (AS‐PCR) has recently been reported, which provides a significant advance over enzymatic digestion. A second problem with measurements of biallelic IGF2 transcription is that the co‐amplification of contaminating genomic DNA during the RT‐PCR step can lead to an overestimation of the frequency of biallelic IGF2 expression. To investigate the extent of this problem, total RNA from breast and colorectal cancer was analysed using two methods. The first method involved a first‐round PCR using cDNA generated with primers spanning exons 8 and 9 (exon connection), followed by a second round of AS‐PCR using primers from within exon 9. The second method used only AS‐PCR with primers from within exon 9. The result was that the exon‐connection approach was more accurate, thereby highlighting a significant problem in imprinting analyses where genomic DNA contamination cannot be completely ruled out. Copyright

Keratin 20 is a specific marker of submicroscopic lymph node metastases in colorectal cancer: validation by K-RAS mutations

Lymph node status has major prognostic importance in colorectal cancer and greater precision in the diagnosis of lymph node metastases should provide better prognostic and therapeutic guidance. Keratin 20 (K20) gene expression has been used as a marker of lymph node metastases, but the evidence for this remains circumstantial. This study has therefore sought to determine K20 specificity and to correlate K20 expression with mutant K‐RAS expression, in order to provide direct evidence that K20 expression in lymph nodes of colorectal cancer patients genuinely reflects metastatic disease. Specificity of K20 expression was established against a range of tissue types and 289 lymph nodes from 41 non‐cancer control patients. K20 expression was restricted to gastrointestinal epithelia and was only present in one of the 289 control lymph nodes, giving a calculated specificity of 97.6% (95% confidence limits: 87.1–99.9%). Forty‐two tumour samples were analysed for the presence of K‐RAS codon 12 gene mutations using a RT‐PCR mutant allele‐specific amplification (MASA) technique. Thirteen tumours (31%) had codon 12 mutations detected by MASA and these were further analysed to determine the exact nature of the mutation. MASA was then used to screen the lymph nodes from these patients for the presence of the tumour‐specific K‐RAS transcript and the results were compared with K20 RT‐PCR and histopathology from the same samples. Whilst K‐RAS MASA was not as sensitive as K20 RT‐PCR, there was substantial agreement between the assays. There were no K20‐negative lymph nodes which were found to be K‐RAS MASA‐positive, whereas seven nodes in four patients were K20‐positive and K‐RAS‐negative, in keeping with the differences in assay sensitivity. These results further validate K20 as a marker by providing greater certainty that what is being detected represents occult metastatic disease. Copyright

Determination of two major metabolites of the novel anti-tumour agent 5,6-dimethylxanthenone-4-acetic acid in hepatic microsomal incubations by high-performance liquid chromatography with fluorescence detection

High-performance liquid chromatographic methods have been developed and validated for the glucuronidated and oxidative metabolites of the novel anti-tumour agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA), produced in human liver microsomal incubations. Calibration curves for DMXAA acyl glucuronide (DMXAA-Glu) and 6-hydroxymethyl-5-methylxanthenone-4-acetic acid (6-OH-MXAA) were constructed over the concentration ranges of 0.25 to 20 and 0.5 to 40 μM, respectively. Assay performance was determined by intra- and inter-day accuracy and precision of quality control (QC) samples. The difference between the theoretical and measured concentration, and the coefficient of variation, were less than 15% at low QC concentrations, and less than 10% at medium and high QC concentrations for both analytes. The methods presented good accuracy, precision and sensitivity for use in kinetic studies of the glucuronidated and oxidative metabolites of DMXAA in human liver microsomes.

Analysis of Intragraft Gene and Protein Expression of the Costimulatory Molecules, CD80, CD86 and CD154, in Orthotopic Liver Transplant Recipients

CD40‐CD154 and/or CD28‐CD80/86 costimulatory blockade induces long‐term allograft survival in numerous animal models. Studies examining the expression of costimulatory molecules during acute cellular rejection (ACR) have been limited to renal and cardiac allografts.

Eosinophilic cholangitis: a case of 'malignant masquerade'.

BACKGROUNDnA variety of causes of inflammatory bile duct stricture can masquerade as hilar cholangiocarcinoma. Eosinophilic cholangitis is a further example.nnnCASE OUTLINEnA 50-year-old woman with epigastric pain and deranged liver function was found to have a stricture of the common hepatic duct on ERCP with an associated mass on MRl.The lesion was excised with reconstruction of the right and left hepatic ducts, and the patient recovered well. Histopathological examination of the resected gallbladder and bile duct showed diffuse inflammation with a predominant eosinophil infiltrate.The presence of Candida albicans in the bile duct lumen can probably be attributed to the preoperative biliary stent.There was a modest postoperative rise in peripheral eosinophil count.nnnDISCUSSIONnA literature search reveals only six previous cases of eosinophilic cholangitis, but similar infiltrates have also been seen in occasional cholecystectomy specimens. As the present patient did not have gallstones, the aetiology remains unclear. Peripheral eosinophilia is an unreliable clue to the diagnosis, which is usually likely to escape detection until the biliary stricture has been resected.

Systematic review and meta-analysis of outcomes after liver resection in patients with hepatocellular carcinoma (HCC) with and without bile duct thrombus

INTRODUCTIONnThis meta-analysis aimed to compare perioperative and survival outcomes in patients who underwent hepatectomy with and without Bile Duct Tumour Thrombus (BDTT).nnnMETHODSnA comprehensive search of Cochrane Library, PubMed, MEDLINE and EMBASE was performed to identify relevant articles. The perioperative, postoperative and long term outcomes were compared.nnnRESULTSnEleven studies including 6051 patients met the inclusion criteria. The perioperative outcomes were comparable between the 2 groups. The BDTT group had higher proportion poorly differentiated tumours (OR = 1.87, X(2) = 10.00, df = 6, p = 0.002, I(2) = 40%), Lymphovascular invasion (LVI) (OR = 4.85, X(2) = 28.21, df = 9, p = <0.001, I(2) = 68%) and Macrovascular invasion (MVI) (OR = 5.41, X(2) = 8.73, df = 9, p = <0.001, I(2) = 0%). There was no difference in 1 and 3 year survival, however 5-yr survival was poorer in the BDTT group (OR = 0.37, X(2) = 37.04, df = 7, p = <0.001, I(2) = 81%). The mean difference (MD) in overall survival in the BDTT group was -20 months [-32.31, -7.06], p = 0.002, I(2) = 95%.nnnCONCLUSIONnPatients with HCC with BDTT had more advanced stage HCC with adverse histological features including higher rates of MVI, LVI and poor differentiation. Hepatectomy in this group of patients offers similar survival at 3 years but inferior long-term survival and should be considered when feasible.

Intragraft gene and protein expression in rat liver allografts treated with costimulatory blockade alone or in combination with CyA.

BACKGROUNDnCostimulatory blockade has been shown to prevent acute rejection (AR) and promote long-term graft survival in a number of animal models including nonhuman primates. The effect of concomitant administration of conventional immunosuppressives on long-term liver allograft survival and intragraft expression of immune mediators has not previously been examined.nnnMATERIALS AND METHODSnA high-responding Dark Agouti to Lewis orthotopic liver transplant (LEW OLT) model was used to compare anti-CD154 alone, or in combination with cyclosporin (CyA) on allograft survival. Donor-specific reactivity was assessed by mixed lymphocyte reaction (MLR) and allogeneic skin grafts. Surviving rats were euthanized on day 150 and intragraft gene (CD80, 86, 152, 154, IFN-gamma, IL-2, IL-6, IL-10, IL-13, TNF-alpha, TGF-beta, IL-7, Fas-ligand, Granzyme B, bax, and bcl(2)) and protein (CD4, CD8, ED1, CD154, CD80, CD86) expression was measured.nnnRESULTSnUntreated control recipients had a median survival time of 5 days. Recipients treated with anti-CD154 survived to beyond 150 days with no evidence of AR. Concomitant administration of CyA did not alter the long-term survival. There was no difference in the serum aspartate aminotransferase between treatment groups or a change over time. All treated recipients showed a reduction in donor-specific MLR at day 40 and 60 but had persistence of donor reactivity to skin grafts at day 100. Histologically, liver architecture was well preserved despite the presence of a nondestructive mononuclear cell infiltrate. Analysis of intragraft gene expression revealed an inverse relationship between the duration of anti-CD154 therapy and the gene expression of costimulatory molecules and Th1 cytokine transcripts. The pro-apoptotic gene, bax, was increased in recipients treated with anti-CD154, but not CyA, compared with normal liver.nnnCONCLUSIONSnThese data demonstrate that anti-CD154 therapy either alone or in combination with CyA allows for the long-term survival of liver allografts in the rat despite there being a difference in the intragraft gene and protein profile.