Ben George

Combined Effects of p53, p21, and pRb Expression in the Progression of Bladder Transitional Cell Carcinoma

PURPOSE To determine the combined effects of p53, p21, and pRb alterations in predicting the progression of bladder transitional cell carcinoma. PATIENTS AND METHODS p53, p21, and pRb expression was examined immunohistochemically on archival radical cystectomy samples from 164 patients with invasive or high-grade recurrent superficial transitional cell carcinoma (TCC; lymph node-negative, 117 patients; lymph node-positive, 47 patients). Median follow-up was 8.6 years. Based on percentage of nuclear reactivity, p53 was considered as wild-type (0% to 10%) or altered (>10%); p21 was scored as wild-type (>10%) or altered (<10%); and pRb status was considered wild-type (1% to 50%) or altered (0% or >50%). RESULTS As individual determinants, the p53, p21, and pRb status were independent predictors of time to recurrence (P<.001, P<.001, and P<.001, respectively), and overall survival (P<.001, P=.002, and P=.001, respectively). By examining these determinants in combination, patients were categorized as group I (no alteration in any determinant, 47 patients), group II (any one determinant altered, 51 patients), group III (any two determinants altered, 42 patients), and group IV (all three determinants altered, 24 patients). The 5-year recurrence rates in these groups were 23%, 32%, 57%, and 93%, respectively (log-rank P<.001), and the 5-year survival rates were 70%, 58%, 33%, and 8%, respectively (log-rank P<.001). After stratifying by stage, the number of altered proteins remained significantly associated with time to recurrence and overall survival. CONCLUSION This study suggests that alterations in p53, p21, and pRb act in cooperative or synergistic ways to promote bladder cancer progression. Examining these determinants in combination provides additional information above the use of a single determinant alone.

p53 gene and protein status: the role of p53 alterations in predicting outcome in patients with bladder cancer.

PURPOSE The p53 gene status (mutation) and protein alterations (nuclear accumulation detectable by immunohistochemistry; p53 protein status) are associated with bladder cancer progression. Substantial discordance is documented between the p53 protein and gene status, yet no studies have examined the relationship between the gene-protein status and clinical outcome. This study evaluated the clinical relationship of the p53 gene and protein statuses. MATERIALS AND METHODS The complete coding region of the p53 gene was queried using DNA from paraffin-embedded tissues and employing a p53 gene-sequencing chip. We compared p53 gene status, mutation site, and protein status with time to recurrence. RESULTS The p53 gene and protein statuses show significant concordance, yet 35% of cases showed discordance. Exon 5 mutations demonstrated a wild-type protein status in 18 of 22 samples. Both the p53 gene and protein statuses were significantly associated with stage and clinical outcome. Specific mutation sites were associated with clinical outcome; tumors with exon 5 mutations showed the same outcome as those with the wild-type gene. Combining the p53 gene and protein statuses stratifies patients into three distinct groups, based on recurrence-free intervals: patients showing the best outcome (wild-type gene and unaltered protein), an intermediate outcome (either a mutated gene or an altered protein) and the worst outcome (a mutated gene and an altered protein). CONCLUSION We show that evaluation of both the p53 gene and protein statuses provides information in assessing the clinical recurrence risk in bladder cancer and that the specific mutation site may be important in assessing recurrence risk. These findings may substantially impact the assessment of p53 alterations and the management of bladder cancer.

Neoadjuvant FOLFIRINOX for Borderline Resectable Pancreas Cancer: A New Treatment Paradigm?

BACKGROUND Borderline resectable pancreatic cancer is best treated by multimodality therapy. FOLFIRINOX (5-fluorouracil, oxaliplatin, irinotecan, and leucovorin) tripled the response rate and significantly increased median survival for patients with advanced pancreatic cancer and shows promise for neoadjuvant use. Toxicity concerns prompted a careful analysis of our initial FOLFIRINOX experience. METHODS All patients diagnosed with borderline resectable, biopsy-proven pancreatic adenocarcinoma treated with neoadjuvant FOLFIRINOX between July 2010 and December 2012 were reviewed. Primary outcome was surgical resectability. Secondary outcomes were treatment-related toxicities and survival. RESULTS FOLFIRINOX followed by gemcitabine- or capecitabine-based chemoradiation was initiated in 18 patients. The most common grade 3 or 4 toxicities during chemotherapy were gastrointestinal, including nausea/emesis (n = 5), weight loss (n = 3) and diarrhea (n = 2), and hematologic (n = 2; neutropenia); five patients (36%) required a total of six admissions. Neoadjuvant therapy was completed in 15 of 18 patients (83%), and 12 (67%) underwent pancreatectomy (10 Whipple, 2 total pancreatectomy) including portal vein resection/reconstruction in 10 (83%). Disease progression precluded surgery in 6 of the 18 patients (33%). All 12 resected patients had negative (R0) margins. Only 2 of 12 (17%) were node positive (median node count: 26.5 [range: 15-39]). There were no in-hospital or 30-day mortalities and no clinical pancreatic leaks or reoperations. Of the 12 patients who completed all intended therapy, 7 (58.3%) are alive, including 5 who have no evidence of disease (median months from diagnosis: 22 months [range: 18-35 months). The six patients who did not complete all planned therapy are deceased (months from diagnosis: 6.9-17.5 months). CONCLUSION FOLFIRINOX followed by chemoradiation as neoadjuvant therapy for borderline resectable pancreatic adenocarcinoma is safe, and our initial experience suggests favorable resection rates compared with previous reports in this high-risk patient population.

Hyperphosphorylation of pRb: a mechanism for RB tumour suppressor pathway inactivation in bladder cancer.

Loss of heterozygosity, mutations or deletions of the RB1 gene usually result in loss of pRb expression, which has been regarded as an indicator of loss of pRb function in human tumours. It has previously been shown that in addition to loss of pRb expression, aberrantly high (pRb2+) pRb expression also indicates loss of pRb function in bladder tumours compared with moderate (normal, pRb1+) pRb expression. The aim of this study was to elucidate the mechanism by which pRb is functionally inactivated in bladder tumours expressing aberrantly high levels of pRb. Constitutive phosphorylation was therefore investigated as a mechanism of pRb inactivation in bladder tumours. Of 28 bladder tumours examined, western blotting demonstrated pRb hyperphosphorylation in 5/7 (71%) pRb2+ bladder tumours compared with only 4/11 (36%) pRb1+ tumours (p = 0.002). All cases with undetectable pRb showed moderate to high p16 expression and none showed cyclin D1 expression by immunohistochemistry. All pRb1+ tumours with underphosphorylated pRb showed p16 but not cyclin D1 expression. All pRb2+ tumours with hyperphosphorylated pRb showed loss of p16 expression and/or cyclin D1 overexpression. Thus, elevated pRb expression was associated with pRb hyperphosphorylation, which, in turn, was associated with loss of p16 expression and/or increased cyclin D1 expression. In order to analyse this association in vitro, T24 cells, which express high levels of pRb, were transfected with p16 cDNA. Transfection with p16 cDNA resulted in a marked decrease in pRb phosphorylation, decreased cell proliferation, and a change in expression of pRb from high to moderate phenotype as assessed by immunohistochemistry. This paper gives the biological basis for constitutive alteration of pRb function in human tumours in the presence of an intact, expressed pRb protein; the mechanism of pRb inactivation is through hyperphosphorylation, which results from loss of p16 expression and/or cyclin D1 overexpression. Immunohistochemical expression of pRb appears to be a reliable indicator of pRb function. Copyright

The use of genetic programming in the analysis of quantitative gene expression profiles for identification of nodal status in bladder cancer.

BackgroundPrevious studies on bladder cancer have shown nodal involvement to be an independent indicator of prognosis and survival. This study aimed at developing an objective method for detection of nodal metastasis from molecular profiles of primary urothelial carcinoma tissues.MethodsThe study included primary bladder tumor tissues from 60 patients across different stages and 5 control tissues of normal urothelium. The entire cohort was divided into training and validation sets comprised of node positive and node negative subjects. Quantitative expression profiling was performed for a panel of 70 genes using standardized competitive RT-PCR and the expression values of the training set samples were run through an iterative machine learning process called genetic programming that employed an N-fold cross validation technique to generate classifier rules of limited complexity. These were then used in a voting algorithm to classify the validation set samples into those associated with or without nodal metastasis.ResultsThe generated classifier rules using 70 genes demonstrated 81% accuracy on the validation set when compared to the pathological nodal status. The rules showed a strong predilection for ICAM1, MAP2K6 and KDR resulting in gene expression motifs that cumulatively suggested a pattern ICAM1>MAP2K6>KDR for node positive cases. Additionally, the motifs showed CDK8 to be lower relative to ICAM1, and ANXA5 to be relatively high by itself in node positive tumors. Rules generated using only ICAM1, MAP2K6 and KDR were comparably robust, with a single representative rule producing an accuracy of 90% when used by itself on the validation set, suggesting a crucial role for these genes in nodal metastasis.ConclusionOur study demonstrates the use of standardized quantitative gene expression values from primary bladder tumor tissues as inputs in a genetic programming system to generate classifier rules for determining the nodal status. Our method also suggests the involvement of ICAM1, MAP2K6, KDR, CDK8 and ANXA5 in unique mathematical combinations in the progression towards nodal positivity. Further studies are needed to identify more class-specific signatures and confirm the role of these genes in the evolution of nodal metastasis in bladder cancer.

Survival of patients with resectable pancreatic cancer who received neoadjuvant therapy

BACKGROUND Enthusiasm for neoadjuvant therapy is growing from the emerging consensus that pancreatic cancer is a systemic disease at the time of diagnosis. Those who remain in favor of upfront surgery often cite the lack of reported data to support alternative treatment sequencing. We therefore report the results of all patients treated outside of a clinical trial under the direction of a multidisciplinary pancreatic cancer working group. METHODS We reviewed all patients with resectable pancreatic cancer treated with neoadjuvant therapy (NeoTx) from 2009 to 2013; we excluded those patients treated on prospective clinical trials as they will be the subject of subsequent reports. Data regarding demographics, NeoTx, operative outcomes, pathology, and survival data were abstracted from a prospective database. RESULTS NeoTx was initiated in 69 patients; median age was 65 years (interquartile range [IQR]: 11) and median carbohydrate antigen 19-9 at diagnosis was 96.5 (IQR 210). NeoTx consisted of chemotherapy alone (n = 10, 14%), chemotherapy and radiation (chemoradiation, n = 53, 77%), or both (n = 6, 9%). Median carbohydrate antigen 19-9 after NeoTx was 39 (IQR 104) corresponding to a median decrease of 60%. Operative resection was completed in 60 (87%) of the 69 patients. At restaging after NeoTx, 5 (7%) of 69 patients were not considered candidates for surgery because of the development of metastatic disease (n = 4) or an inadequate performance status (n = 1). At the time of surgery, 4 (6%) of 64 patients had metastatic disease found at laparoscopy. Of the 60 patients who underwent surgical resection, a complete pathologic response was observed in 2 (3%) patients; 20 (33%) had positive lymph nodes, and the median number of positive lymph nodes was 2 (IQR 3). R0 resections were achieved in 58 (97%) of the 60 patients. Additional postoperative adjuvant therapy was administered to 37 (62%) of the 60 patients. Median survival of all 69 patients was 31.5 months; 44.9 months for the 60 patients who completed all NeoTx and resection compared with 8.1 months for the 9 patients who were not resected (log rank P < .001). CONCLUSION NeoTx for resectable pancreatic cancer was associated with a median overall survival of 32 months; something not reported for patients treated with surgery first if based on intent-to-treat analysis. Treatment sequencing may provide an oncologic benefit beyond that of the selection bias afforded surgery after a period of induction therapy.

Predictive and prognostic markers in colorectal cancer.

Colorectal cancer (CRC) has a complex pathogenesis involving multiple sequential steps with accumulation of genetic alterations including mutations, gene amplification, and epigenetic changes. Treatment of CRC has undergone a paradigm shift over the past decade due in part to a better understanding of the biology of the disease and development of newer drugs including biologic agents. In the era of personalized medicine, it is attractive to investigate the molecular pathways leading to colorectal cancer tumorigenesis, thus raising the possibility of identifying novel therapeutic targets. It has intuitive appeal to hypothesize that biomarkers that have prognostic and/or predictive value are those that are intimately connected to the pathogenesis of CRC. In this article, we focus on prognostic and predictive markers in CRC that have a substantial body of data in support of their potential role in routine clinical practice.

Arterial resection at the time of pancreatectomy for cancer

BACKGROUND Tumor-induced arterial abutment/encasement has been traditionally a contraindication to surgery in patients with localized pancreatic cancer (PC). One recent meta-analysis reported greater mortality rates in this setting. We report herein a series of planned arterial resections in carefully selected patients who responded favorably to combined modality therapy for localized PC. METHODS We reviewed all patients with PC and arterial encasement treated between May 2011 and September 2013; all patients received an extensive course of neoadjuvant therapy before surgery. RESULTS Of 15 patients taken to surgery, 2 had peritoneal disease at laparoscopy, and therefore, laparotomy was not performed. Pancreatectomy (pancreaticoduodenectomy, 3; distal, 8; central pancreatectomy, 1; total, 1) was performed in the remaining 13, 10 of whom required arterial resection. The most common operation was an Appleby procedure. Of 10 patients who underwent combined pancreatectomy and arterial resection, their median age was 62 years (range, 33-75), median operative time was 7.5 hours, and median blood loss was 725 mL. Complications occurred in 3 of 15 patients with no perioperative mortality. Median duration of hospital stay was 9 days (range, 5-19). An R0 resection was achieved in 11 (85%) of 13 patients. At a median follow-up of 21 months, 8 of these 13 resected patients (62%) are alive without disease. CONCLUSION Planned arterial resection at the time of pancreatectomy can be performed with acceptable morbidity and mortality; patient selection and induction therapy are likely critically important variables that seem to impact patient outcome. Those patients with stable or responding disease after induction therapy represent the subset of patients with potentially favorable tumor biology in whom extended resections may enhance survival duration.

Sensitivity and reproducibility of standardized-competitive RT-PCR for transcript quantification and its comparison with real time RT-PCR

BackgroundProbe based detection assays form the mainstay of transcript quantification. Problems with these assays include varying hybridization efficiencies of the probes used for transcript quantification and the expense involved. We examined the ability of a standardized competitive RT-PCR (StaRT PCR) assay to quantify transcripts of 4 cell cycle associated genes (RB, E2F1, CDKN2A and PCNA) in two cell lines (T24 & LD419) and compared its efficacy with the established Taqman real time quantitative RT-PCR assay. We also assessed the sensitivity, reproducibility and consistency of StaRT PCR. StaRT PCR assay is based on the incorporation of competitive templates (CT) in precisely standardized quantities along with the native template (NT) in a PCR reaction. This enables transcript quantification by comparing the NT and CT band intensities at the end of the PCR amplification. The CT serves as an ideal internal control. The transcript numbers are expressed as copies per million transcripts of a control gene such as β-actin (ACTB).ResultsThe NT and CT were amplified at remarkably similar rates throughout the StaRT PCR amplification cycles, and the coefficient of variation was least (<3.8%) when the NT/CT ratio was kept as close to 1:1 as possible. The variability between the rates of amplification in different tubes subjected to the same StaRT PCR reaction was very low and within the range of experimental noise. Further, StaRT PCR was sensitive enough to detect variations as low as 10% in endogenous actin transcript quantity (p < 0.01 by the paired students t-test). StaRT PCR correlated well with Taqman real time RT-PCR assay in terms of transcript quantification efficacy (p < 0.01 for all 4 genes by the Spearman Rank correlation method) and the ability to discriminate between cell types and confluence patterns.ConclusionStaRT PCR is thus a reliable and sensitive technique that can be applied to medium-high throughput quantitative transcript measurement. Further, it correlates well with Taqman real time PCR in terms of quantitative and discriminatory ability. This label-free, inexpensive technique may provide the ability to generate prognostically important molecular signatures unique to individual tumors and may enable identification of novel therapeutic targets.

Systematic review of outcomes of patients undergoing resection for colorectal liver metastases in the setting of extra hepatic disease

BACKGROUND Surgical resection for patients with colorectal liver metastases (CRLM) can offer patients a significant survival benefit. We hypothesised that patients with CRLM and extra hepatic disease (EHD) undergoing metastasectomy had comparable survival and describe outcomes based on the distribution of metastatic disease. METHODS A systematic search using a predefined registered protocol was undertaken between January 2003 and June 2012. Primary exposure was hepatic resection for CRLM and primary outcome measure was overall survival. Meta-regression techniques were used to analyse differences between patients with and without extra hepatic disease. FINDINGS From a pool of 4996 articles, 50 were retained for data extraction (3481 CRLM patients with EHD). The median survival (MS) was 30.5 (range, 9-98) months which was achieved with an operative mortality rate of 0-4.2%. The 3-year and 5-year overall survival (OS) were 42.4% (range, 20.6-77%) and 28% (range, 0-61%) respectively. Patients with EHD of the lungs had a MS of 45 (range, 39-98) months versus lymph nodes (portal and para-aortic) 26 (range, 21-48) months versus peritoneum 29 (range, 18-32) months. The MS also varied by the amount of liver disease - 42.2months (<two lesions) versus 39.6months (two lesions) versus 28months (⩾three lesions). INTERPRETATION In the evolving landscape of multimodality therapy, selective hepatic resection for CRLM patients with EHD is feasible with potential impact on survival. Patients with minimal liver disease and EHD in the lung achieve the best outcome.