Daniel John Renouf

Ductal pancreatic cancer modeling and drug screening using human pluripotent stem cell- and patient-derived tumor organoids

There are few in vitro models of exocrine pancreas development and primary human pancreatic adenocarcinoma (PDAC). We establish three-dimensional culture conditions to induce the differentiation of human pluripotent stem cells into exocrine progenitor organoids that form ductal and acinar structures in culture and in vivo. Expression of mutant KRAS or TP53 in progenitor organoids induces mutation-specific phenotypes in culture and in vivo. Expression of TP53R175H induces cytosolic SOX9 localization. In patient tumors bearing TP53 mutations, SOX9 was cytoplasmic and associated with mortality. We also define culture conditions for clonal generation of tumor organoids from freshly resected PDAC. Tumor organoids maintain the differentiation status, histoarchitecture and phenotypic heterogeneity of the primary tumor and retain patient-specific physiological changes, including hypoxia, oxygen consumption, epigenetic marks and differences in sensitivity to inhibition of the histone methyltransferase EZH2. Thus, pancreatic progenitor organoids and tumor organoids can be used to model PDAC and for drug screening to identify precision therapy strategies.

Nonsmall cell lung carcinoma with neuroendocrine differentiation--an entity of no clinical or prognostic significance.

The existence of non-small cell lung carcinoma with neuroendocrine differentiation as a distinct entity and its relevance for prognostic and treatment purposes is controversial. This study assesses the frequency and biologic and prognostic significance of neuroendocrine (NE) expression of synaptophysin (SNP), chromogranin (Ch), and neural cell adhesion molecule (N-CAM) using tissue microarray (TMA) and immunohistochemistry. Six hundred nine nonsmall cell lung carcinomas (NSCLCs) were reviewed for subclassification. TMA blocks were made using duplicate 0.6-mm-diameter tissue cores and slides stained with SNP, Ch, and N-CAM. Immunoreactivity was considered if 1% or more of tumor cells were positive. Hematoxylin and eosin-stained sections were subclassified as: 243 adenocarcinoma (ACA), 272 squamous cell carcinoma (SCC), 35 large cell carcinoma, 32 non-small cell carcinoma NOS, and 6 other (carcinosarcoma, giant cell carcinoma). Positivity for either marker was identified in 13.6% of NSCLC (76/558). NSCLC showed reactivity for Ch in 0.4% of cases (2/524), for SNP in 7.5% of cases (39/521) and for N-CAM in 8.6% of cases (44/511), whereas only 0.2% of cases (1/517) showed coexpression of SNP and Ch and none of all 3 markers. The assessment of NE differentiation in NSCLC is unnecessary and expensive and is of no clinical or prognostic significance. SNP or N-CAM stains a small minority of NSCLC, whereas Ch immunoreactivity is less common. Positivity for any 2 NE markers is rare. SNP is more likely to be expressed in adenocarcinoma (P=0.01) and N-CAM in squamous-cell carcinoma (P=0.008). Otherwise there was no correlation between immunoreactivity and tumor morphology. Disease specific and overall survival is not influenced by NE differentiation and therefore non-small cell lung carcinoma with neuroendocrine differentiation should not be a subclass distinct from the other NSCLC.

Ocular Toxicity of Targeted Therapies

Molecularly targeted agents are commonly used in oncology practice, and many new targeted agents are currently being tested in clinical trials. Although these agents are thought to be more specific and less toxic then traditional cytotoxic chemotherapy, they are associated with a variety of toxicities, including ocular toxicity. Many of the molecules targeted by anticancer agents are also expressed in ocular tissues. We reviewed the literature for described ocular toxicities associated with both approved and investigational molecularly targeted agents. Ocular toxicity has been described with numerous approved targeted agents and also seems to be associated with several classes of agents currently being tested in early-phase clinical trials. We discuss the proposed pathogenesis, monitoring guidelines, and management recommendations. It is important for oncologists to be aware of the potential for ocular toxicity, with prompt recognition of symptoms that require referral to an ophthalmologist. Ongoing collaboration between oncologists and ocular disease specialists is critical as the use of molecularly targeted agents continues to expand and novel targeted drug combinations are developed.

Lessons learned from the application of whole-genome analysis to the treatment of patients with advanced cancers

Given the success of targeted agents in specific populations it is expected that some degree of molecular biomarker testing will become standard of care for many, if not all, cancers. To facilitate this, cancer centers worldwide are experimenting with targeted “panel” sequencing of selected mutations. Recent advances in genomic technology enable the generation of genome-scale data sets for individual patients. Recognizing the risk, inherent in panel sequencing, of failing to detect meaningful somatic alterations, we sought to establish processes to integrate data from whole-genome analysis (WGA) into routine cancer care. Between June 2012 and August 2014, 100 adult patients with incurable cancers consented to participate in the Personalized OncoGenomics (POG) study. Fresh tumor and blood samples were obtained and used for whole-genome and RNA sequencing. Computational approaches were used to identify candidate driver mutations, genes, and pathways. Diagnostic and drug information were then sought based on these candidate “drivers.” Reports were generated and discussed weekly in a multidisciplinary team setting. Other multidisciplinary working groups were assembled to establish guidelines on the interpretation, communication, and integration of individual genomic findings into patient care. Of 78 patients for whom WGA was possible, results were considered actionable in 55 cases. In 23 of these 55 cases, the patients received treatments motivated by WGA. Our experience indicates that a multidisciplinary team of clinicians and scientists can implement a paradigm in which WGA is integrated into the care of late stage cancer patients to inform systemic therapy decisions.

Evolution of systemic therapy for advanced pancreatic cancer

The prognosis for advanced pancreatic cancer remains poor and successful drug development in this disease continues to be a major challenge. In the last decade the approach to drug development in pancreatic cancer has included a focus on combinations of cytotoxic agents. While some promising results were seen in Phase II studies, none of the Phase III trials of cytotoxic combinations were able to demonstrate an improvement in overall survival over that seen with the single-agent gemcitabine. Newer studies have assessed the efficacy of ‘targeted’ agents that inhibit pathways thought to be important in the development, growth, invasion and metastasis of pancreatic cancer. Although some agents had promising activity in preclinical studies, none has made a major impact in the clinic. There has been some success with the addition of the EGF receptor tyrosine kinase inhibitor erlotinib to gemcitabine, which was the first combination to achieve an overall survival benefit compared with gemcitabine alone in a Phase III trial. Future directions for drug development in pancreatic cancer will mainly involve testing new targeted agents, although some cytotoxic combinations are currently in Phase III testing. There is a need to better understand the biology of the disease and incorporate this into trials in an attempt to search for predictive and prognostic markers that will aid in drug development. Control of pancreatic cancer will require combinations of targeted agents, probably individualized based on tumor genetics. We are just beginning to explore the efficacy of combining targeted agents in the clinic.

BCL-2 Expression is Prognostic for Improved Survival in Non-small Cell Lung Cancer

Objective: We used a large patient population to identify immunohistochemical biomarkers to enable improved prognostication in patients with non-small cell lung carcinoma (NSCLC). Methods: A tissue microarray was constructed using duplicate 0.6 mm cores of formalin-fixed paraffin-embedded tissue blocks from 609 patients with NSCLC. Immunohistochemical was used to detect 11 biomarkers including epidermal growth factor receptor, Her2, Her3, p53, p63, bcl-1, bcl-2, Thyroid transcription factor, carcinoembryonic antigen, chromogranin, and synaptophysin. A clinical database was generated prospectively at the time of tissue collection. Survival outcomes were obtained from a Provincial Cancer Registry database. Univariate and multivariate analyses were performed to look for a relationship between biomarker expression, smoking history, and survival. Results: Survival data for 535 cases were available. As of June 2005, 429 patients (80%) had died; of these 286 (54%) died of lung cancer, 117 (22%) died of other known causes, and for 26 (5%) the cause of death was not available. Univariate analysis revealed that bcl-2 (p = 0.007) was the only biomarker prognostic for improved overall survival (OS). bcl-2 (p = 0.021) and p63 (p = 0.025) were both found to be prognostic for improved disease-specific survival (DSS). Multivariate analysis (using age and biomarker expression) revealed that bcl-2 expression is prognostic for improved OS (p = 0.005) and DSS (p = 0.021). Conclusions: Our results suggest that bcl-2 expression is prognostic for improved OS and DSS in NSCLC. Testing for bcl-2 expression in a prospective study will help to determine its clinical relevance in prognostication.

Eligibility of Metastatic Pancreatic Cancer Patients for First-line Palliative Intent nab-paclitaxel Plus Gemcitabine Versus Folfirinox

Objectives: The PRODIGE and MPACT trials showed superiority of FOLFIRINOX and nab-paclitaxel plus gemcitabine (NG) over gemcitabine alone, respectively. However, both had strict inclusion criteria. We sought to determine the characteristics of patients with metastatic pancreatic cancer (MPC) which inform the appropriateness of first-line chemotherapy FOLFIRINOX and NG in routine practice. Materials and Methods: Patients with MPC who initiated palliative chemotherapy with gemcitabine from 2000 to 2011 at the British Columbia Cancer Agency were identified. Clinicopathologic variables and outcomes were retrospectively collected and compared among groups. Eligibility criteria for each regimen were in accordance with the respective pivotal phase III trials. Results: A total of 473 patients were included: 25% of the patients were eligible for FOLFIRINOX versus 45% for NG. Main reasons for FOLFIRINOX ineligibility were Eastern Cooperative Oncology Group (ECOG) performance status (PS)≥2 (56.5%), age older than 75 years (19.0%), and bilirubin>1.5× upper limit of normal (18.6%), whereas those for NG ineligibility were bilirubin > upper limit of normal (24.5%), ECOG PS≥3 (14.6%), and cardiac dysfunction (13.8%). Univariate analyses revealed that FOLFIRINOX and NG-eligible patients had longer median overall survival than their respective ineligible group (8.6 vs. 4.7 mo, P<0.001; 6.7 vs. 4.9 mo, P=0.008, respectively). After accounting for ECOG PS in the multivariate model, however, eligibility for either FOLFIRINOX or NG no longer predicted for better overall survival. Conclusions: The majority of patients with MPC are not candidates to either NG or FOLFIRINOX due to restrictive eligibility requirements. Specific trials addressing the unmet needs of protocol ineligible patients are warranted.

Trends in Chemotherapy Utilization for Colorectal Cancer

BACKGROUND Since 1990, significant advances have occurred in the adjuvant and metastatic treatment of colorectal cancer (CRC). Not all patients may be eligible for chemotherapy (CT), and the proportions and characteristics of patients who receive treatment are not well defined. In this study, treatment patterns of patients referred to the British Columbia Cancer Agency (BCCA) with early-stage colon cancer and metastatic CRC between 1990 and 2004 are described. PATIENTS AND METHODS This study included patients with stage II or III colon cancer or stage IV CRC at presentation referred to the BCCA during a 1-year period for 3 time cohorts: 1990, 2000, and 2004. Patients were considered treated with CT if they received > or = 1 cycle of any CT for indication of the initial referral diagnosis. RESULTS A total of 1421 patients were included: stage II/III, n = 915; stage IV, n = 506. Chemotherapy utilization increased significantly from 1990 to 2004 for adjuvant CT (1990: 29%; 2000: 45%; 2004: 52%; P < .001) and for palliative CT (1990: 35%; 2000: 51%; 2004: 63%; P < .001). The proportion of patients with stage II disease treated with adjuvant CT dramatically increased (1990: 4%; 2000: 26%; 2004: 30%; P < .001). The use of palliative CT was significantly associated with male sex (P = .025). Among older patients, only 25% received adjuvant CT, and 31% received palliative CT. CONCLUSION In this population-based study, adjuvant and palliative CT utilization increased significantly between 1990 and 2004. The majority of patients aged > 70 years did not receive CT, and women appeared less likely to receive palliative CT. Such disparities in CT utilization require further investigation.

Survival for Metastatic Colorectal Cancer in the Bevacizumab Era: A Population-based Analysis

BACKGROUND As of 2006, bevacizumab was available for the treatment of metastatic colorectal cancer (mCRC) in British Columbia (BC). This study compares survival between referred patients diagnosed with mCRC in 2003/2004 (pre-bevacizumab era) and 2006 (bevacizumab era). PATIENTS AND METHODS The BC cancer agency (BCCA) is a cancer network treating approximately 60% of patients with mCRC in BC. For this study, all patients in the BCCA diagnosed with mCRC in 2003/2004 and 2006 were included. The primary objective was to compare overall survival (OS) between the 2 cohorts. RESULTS One thousand four hundred seventeen patients were included: 969 from 2003/2004, and 448 from 2006. Between 2003/2004 and 2006, the proportion of patients treated with systemic therapy for mCRC increased (61.1% to 67.6%; P = .02). The only significant difference in treatment between the cohorts was in the proportion of patients who received bevacizumab (5.9% to 30.6%; P < .001). Median OS significantly differed between the 2 cohorts (13.8 to 17.3 months; P < .001). Median OS for patients who received systemic therapy increased (18.6-23.6 months; P = .001). Median OS for patients who did not receive systemic therapy was unchanged (6.1-5.9 months; P = .65). CONCLUSION In this population-based study, median OS for mCRC significantly increased between 2003/2004 and 2006. An increase in the proportion of patients treated with systemic therapy, and the addition of bevacizumab to chemotherapy, seem to have contributed to this improvement in survival.

Tumor budding is an independent adverse prognostic factor in pancreatic ductal adenocarcinoma.

Tumor budding is a well-established adverse prognostic factor in colorectal cancer. However, the significance and diagnostic reproducibility of budding in pancreatic carcinoma requires further study. We aimed to assess the prognostic significance of tumor budding in pancreatic ductal adenocarcinoma, determine its relationship with other clinicopathologic features, and assess interobserver variability in its diagnosis. Tumor budding was assessed in 192 archival cases of pancreatic ductal adenocarcinoma using hematoxylin and eosin (H&E) sections; tumor buds were defined as single cells or nonglandular clusters composed of <5 cells. The presence of budding was determined through assessment of all tumor-containing slides, and associations with clinicopathologic features and outcomes were analyzed. Six gastrointestinal pathologists participated in an interobserver variability study of 120 images of consecutive tumor slides stained with H&E and cytokeratin. Budding was present in 168 of 192 cases and was associated with decreased overall survival (P=0.001). On multivariable analysis, tumor budding was prognostically significantly independent of stage, grade, tumor size, nodal status, lymphovascular invasion, and perineural invasion. There was substantial agreement among pathologists in assessing the presence of tumor budding using both H&E (K=0.63) and cytokeratin (K=0.63) stains. The presence of tumor budding is an independent adverse prognostic factor in pancreatic ductal carcinoma. The assessment of budding with H&E is reliable and could be used to better risk stratify patients with pancreatic ductal adenocarcinoma.