Lincoln D. Nadauld

Oncogenic transformation of diverse gastrointestinal tissues in primary organoid culture

The application of primary organoid cultures containing epithelial and mesenchymal elements to cancer modeling holds promise for combining the accurate multilineage differentiation and physiology of in vivo systems with the facile in vitro manipulation of transformed cell lines. Here we used a single air-liquid interface culture method without modification to engineer oncogenic mutations into primary epithelial and mesenchymal organoids from mouse colon, stomach and pancreas. Pancreatic and gastric organoids exhibited dysplasia as a result of expression of Kras carrying the G12D mutation (KrasG12D), p53 loss or both and readily generated adenocarcinoma after in vivo transplantation. In contrast, primary colon organoids required combinatorial Apc, p53, KrasG12D and Smad4 mutations for progressive transformation to invasive adenocarcinoma-like histology in vitro and tumorigenicity in vivo, recapitulating multi-hit models of colorectal cancer (CRC), as compared to the more promiscuous transformation of small intestinal organoids. Colon organoid culture functionally validated the microRNA miR-483 as a dominant driver oncogene at the IGF2 (insulin-like growth factor-2) 11p15.5 CRC amplicon, inducing dysplasia in vitro and tumorigenicity in vivo. These studies demonstrate the general utility of a highly tractable primary organoid system for cancer modeling and driver oncogene validation in diverse gastrointestinal tissues.

Metastatic tumor evolution and organoid modeling implicate TGFBR2 as a cancer driver in diffuse gastric cancer

BackgroundGastric cancer is the second-leading cause of global cancer deaths, with metastatic disease representing the primary cause of mortality. To identify candidate drivers involved in oncogenesis and tumor evolution, we conduct an extensive genome sequencing analysis of metastatic progression in a diffuse gastric cancer. This involves a comparison between a primary tumor from a hereditary diffuse gastric cancer syndrome proband and its recurrence as an ovarian metastasis.ResultsBoth the primary tumor and ovarian metastasis have common biallelic loss-of-function of both the CDH1 and TP53 tumor suppressors, indicating a common genetic origin. While the primary tumor exhibits amplification of the Fibroblast growth factor receptor 2 (FGFR2) gene, the metastasis notably lacks FGFR2 amplification but rather possesses unique biallelic alterations of Transforming growth factor-beta receptor 2 (TGFBR2), indicating the divergent in vivo evolution of a TGFBR2-mutant metastatic clonal population in this patient. As TGFBR2 mutations have not previously been functionally validated in gastric cancer, we modeled the metastatic potential of TGFBR2 loss in a murine three-dimensional primary gastric organoid culture. The Tgfbr2 shRNA knockdown within Cdh1-/-; Tp53-/- organoids generates invasion in vitro and robust metastatic tumorigenicity in vivo, confirming Tgfbr2 metastasis suppressor activity.ConclusionsWe document the metastatic differentiation and genetic heterogeneity of diffuse gastric cancer and reveal the potential metastatic role of TGFBR2 loss-of-function. In support of this study, we apply a murine primary organoid culture method capable of recapitulating in vivo metastatic gastric cancer. Overall, we describe an integrated approach to identify and functionally validate putative cancer drivers involved in metastasis.

Molecular Profiling of Gastric Cancer: Toward Personalized Cancer Medicine

Gastric cancer is the fourth most common cancer worldwide and the second leading cause of global cancer deaths. The prognosis of patients diagnosed with gastric cancer continues to be dismal, despite improving surgical and adjuvant treatment approaches, with a 5-year overall survival less than 25%. The high disease incidence and poor outcomes continue to make gastric cancer a topic of active clinical and basic scientific research. Notwithstanding these efforts, relatively little is known about the molecular changes that contribute to the development of this deadly disease and how they may influence outcomes and therapeutic results. Gastric cancer can be considered a multifactorial disease because many inherited and environmental factors play a role in its carcinogenesis, including the genetic background of the host, infectious agents such as Helicobacter pylori, and dietary habits. Gastric cancer is a heterogeneous disorder that can be divided into at least the following two main histologic types on the basis of the Lauren classification: intestinal and diffuse types. These two types have distinct morphologic, clinical, and epidemiologic features and may also represent a different molecular mechanism of tumor development and progression. The identification of germline E-cadherin (CDH1) mutations in families with hereditary diffuse gastric cancer provided early insight into the initial changes that promote gastric tumorigenesis and established CDH1 as a bona fide tumor suppressor. Subsequent studies indicated that, although germline CDH1 mutations are common in hereditary gastric cancer of the diffuse, but not intestinal, type, somatic mutations in CDH1 are frequently identified in sporadic gastric cancer of both major histologic subtypes. In addition, somatic epigenetic alterations, such as CDH1 promoter hypermethylation, are present in diffuseand intestinal-type tumors and result in gene silencing. This epigenetic silencing of CDH1 gene expression represents the “second hit” in approximately 50% of hereditary diffuse gastric cancers, whereas the prevalence of CDH1 promoter hypermethylation in sporadic gastric cancers ranges in estimates from 11% to 46% depending on the histologic subtype. Relatively small sample sizes and a focus on hereditary cancers or a single histologic subtype have hampered previous studies that evaluated the prevalence of CDH1 somatic mutations in gastric cancer overall. The current study by Corso et al evaluated a large set of familial and sporadic gastric cancers to provide a more comprehensive analysis of the prevalence of CDH1 somatic mutations and their correlation with patient survival. The authors analyzed 246 patients with sporadic (n 174) or familial (n 72) gastric cancer for CDH1 structural changes (either mutation or loss of heterozygosity) or epigenetic alterations (CDH1 promoter hypermethylation). The results indicated that, overall, 31% of gastric cancers carried CDH1 alterations, two thirds of which were epigenetic alterations and one third of which were structural alterations. Patients with CDH1 structural alterations displayed a poorer overall survival than in patients with either no changes or epigenetic changes only. In addition, the authors report that CDH1 alterations (both structural and epigenetic) were found in 27.5% of intestinal-type and 38.4% of diffuse-type gastric cancers. Although these alterations were not associated with a worse overall survival in patients with diffusetype tumors, there was a strong association between CDH1 structural alterations and poorer overall survival in patients with intestinaltype tumors. The major finding by Corso et al that nearly one third of gastric cancers harbor somatic CDH1 alterations and those with structural alterations carry a poorer prognosis raises the question of whether somatic CDH1 alterations should influence the clinical management of gastric cancer. Although the findings in this study significantly clarify the extent of somatic CDH1 alterations in gastric cancer, there continues to be insufficient evidence to warrant regular CDH1alteration analysis as a standard prognostic indicator or for predictive use in therapeutic decision making. However, the incorporation of this and other molecular features to more specifically characterize gastric cancers should certainly be part of the design of prospective studies of surgical and adjuvant therapy for this disease. The role of epigenetic changes and tumor-suppressor silencing through promoter hypermethylation in human cancers has raised the specter of the use of demethylating agents such as 5-azacytadine as a therapeutic intervention. Large-scale changes in DNA methylation have been noted in the gastric cancer genome, and the CpG island methylator phenotype may predict a poor outcome independent from the tumor stage. A large number of genes that are suppressed by hypermethylation have been reported in gastric cancer in addition to CDH1, including the DNA mismatch repair genes, CDKN2A (p16), and MGMT (O6-methylguanine DNA methyltransferase). Whether therapeutic demethylation of some or all of these genes in gastric cancer may lead to tumor responses has yet to be tested. The use of demethylating agents has also been considered a potential chemoprevention strategy for carriers of the germline CDH1 mutation to slow or reverse the epigenetic second hit that inactivates CDH1 expression. JOURNAL OF CLINICAL ONCOLOGY E D I T O R I A L VOLUME 31 NUMBER 7 MARCH 1 2013

Quantitative and Sensitive Detection of Cancer Genome Amplifications from Formalin Fixed Paraffin Embedded Tumors with Droplet Digital PCR

For the analysis of cancer, there is great interest in rapid and accurate detection of cancer genome amplifications containing oncogenes that are potential therapeutic targets. The vast majority of cancer tissue samples are formalin fixed and paraffin embedded (FFPE) which enables histopathological examination and long term archiving. However, FFPE cancer genomic DNA is oftentimes degraded and generally a poor substrate for many molecular biology assays. To overcome the issues of poor DNA quality from FFPE samples and detect oncogenic copy number amplifications with high accuracy and sensitivity, we developed a novel approach. Our assay requires nanogram amounts of genomic DNA, thus facilitating study of small amounts of clinical samples. Using droplet digital PCR (ddPCR), we can determine the relative copy number of specific genomic loci even in the presence of intermingled normal tissue. We used a control dilution series to determine the limits of detection for the ddPCR assay and report its improved sensitivity on minimal amounts of DNA compared to standard real-time PCR. To develop this approach, we designed an assay for the fibroblast growth factor receptor 2 gene (FGFR2) that is amplified in a gastric and breast cancers as well as others. We successfully utilized ddPCR to ascertain FGFR2 amplifications from FFPE-preserved gastrointestinal adenocarcinomas.

A Retrospective Analysis of Precision Medicine Outcomes in Patients With Advanced Cancer Reveals Improved Progression-Free Survival Without Increased Health Care Costs

Purpose: The advent of genomic diagnostic technologies such as next-generation sequencing has recently enabled the use of genomic information to guide targeted treatment in patients with cancer, an approach known as precision medicine. However, clinical outcomes, including survival and the cost of health care associated with precision cancer medicine, have been challenging to measure and remain largely unreported. Patients and Methods: We conducted a matched cohort study of 72 patients with metastatic cancer of diverse subtypes in the setting of a large, integrated health care delivery system. We analyzed the outcomes of 36 patients who received genomic testing and targeted therapy (precision cancer medicine) between July 1, 2013, and January 31, 2015, compared with 36 historical control patients who received standard chemotherapy (n = 29) or best supportive care (n = 7). Results: The average progression-free survival was 22.9 weeks for the precision medicine group and 12.0 weeks for the control group (P = .002) with a hazard ratio of 0.47 (95% CI, 0.29 to 0.75) when matching on age, sex, histologic diagnosis, and previous lines of treatment. In a subset analysis of patients who received all care within the Intermountain Healthcare system (n = 44), per patient charges per week were

Pyoderma Gangrenosum With the Use of Sunitinib

4,665 in the precision treatment group and

Linked read sequencing resolves complex genomic rearrangements in gastric cancer metastases

5,000 in the control group (P = .126). Conclusion: These findings suggest that precision cancer medicine may improve survival for patients with refractory cancer without increasing health care costs. Although the results of this study warrant further validation, this precision medicine approach may be a viable option for patients with advanced cancer.

Family History As a Positive Prognostic Factor in Gastric Cancer

In December 2008, a previously healthy 47-year-old man presented with bilateral leg pain and swelling. Laboratory evaluation was notable for an elevated serum creatinine. Ultrasound of the legs confirmed the presence of bilateral femoral vein deep venous thrombosis with extension into the inferior vena cava. Computed tomography and magnetic resonance imaging of the abdomen revealed an exophytic mass of the right kidney that measured 5.7 5.3 cm. Right radical nephrectomy was performed and final pathology confirmed renal cell carcinoma of clear-cell histology with extension into the renal vein and inferior vena cava (pT3c). The patient underwent additional work-up that revealed several lesions in the right liver lobe and gluteus muscle that were consistent with metastatic disease. In February 2009, the patient began receiving sunitinib 50 mg/d with an administration schedule of 4 weeks on and 2 weeks off. Initially, treatment was well tolerated and the patient demonstrated a good response as evidenced by stability of tumor growth and no indication of clinical progression. However, approximately 3 months after beginning treatment, the patient developed a small, erythematous papule on the right lateral lower leg. The lesion evolved into a pustular and violaceous plaque that additionally developed into a full-thickness ulcer with a purulent, undermined border (Fig 1). The edge of the wound was biopsied and demonstrated eroded epidermis with overlying hemorrhage and a neutrophil-laden crust, as well as dense perivascular and interstitial neutrophilic infiltrates that were consistent with pyoderma gangrenosum (PG; Fig 2). The patient noted that the ulcers transiently improved during the 2-week periods when he was not receiving sunitinib. When PG was diagnosed, he began receiving high-dose prednisone at 120 mg/d and experienced a dramatic resolution of his skin ulcer (Fig 3). Sunitinib was discontinued 2 days after biopsy, approximately 14 months after treatment was initiated. Subsequently, the skin ulcer resolved entirely. PG is a neutrophilic dermatosis characterized by skin lesions that typically begin as inflammatory nodules or pustules and subsequently evolve into painful necrotic ulcers. The characteristic lesions can be histologically classified as ulcerative, bullous, pustular, or vegetative. The exact cause of PG remains uncertain, but it is most commonly associated with systemic disorders such as inflammatory bowel disease, arthritis, systemic lupus erythematosus, malignancy, and immunosuppressive states. Diagnosis can be difficult because of the nonspecific nature of the histopathologic changes observed on biopsy

A quality outcomes analysis following treatment with personalized genomic cancer medicine.

BackgroundGenome rearrangements are critical oncogenic driver events in many malignancies. However, the identification and resolution of the structure of cancer genomic rearrangements remain challenging even with whole genome sequencing.MethodsTo identify oncogenic genomic rearrangements and resolve their structure, we analyzed linked read sequencing. This approach relies on a microfluidic droplet technology to produce libraries derived from single, high molecular weight DNA molecules, 50 kb in size or greater. After sequencing, the barcoded sequence reads provide long range genomic information, identify individual high molecular weight DNA molecules, determine the haplotype context of genetic variants that occur across contiguous megabase-length segments of the genome and delineate the structure of complex rearrangements. We applied linked read sequencing of whole genomes to the analysis of a set of synchronous metastatic diffuse gastric cancers that occurred in the same individual.ResultsWhen comparing metastatic sites, our analysis implicated a complex somatic rearrangement that was present in the metastatic tumor. The oncogenic event associated with the identified complex rearrangement resulted in an amplification of the known cancer driver gene FGFR2. With further investigation using these linked read data, the FGFR2 copy number alteration was determined to be a deletion-inversion motif that underwent tandem duplication, with unique breakpoints in each metastasis. Using a three-dimensional organoid tissue model, we functionally validated the metastatic potential of an FGFR2 amplification in gastric cancer.ConclusionsOur study demonstrates that linked read sequencing is useful in characterizing oncogenic rearrangements in cancer metastasis.

Abstract 3165: Metastatic tumor evolution delineates clinically important oncogenic drivers in diffuse gastric cancer.

Conflicting reports have created controversy with respect to the effects of a positive family history on the survival of patients who are diagnosed with gastric cancer. Previously, Yatsuya et al reported that a positive family history of gastric cancer was negatively associated with patient survival. Other studies have reported that family history either reduces or has no effect on the risk of death from gastric cancer. In the article that accompanies this editorial, Han et al report on a study that seeks to clarify the relationship between a positive family history and survival of gastric cancer in a large, retrospective review of surgical cases from Korea. The authors retrospectively evaluated 1,273 Korean patients with gastric cancer for the presence of firstor second-degree relatives with any cancer. Results were reported for percentage of patients with first-degree relatives with any cancer (38.6%) or cancer type, including gastric cancer (20.6%). The authors reported a statistically significant improvement in disease-free survival, recurrence-free survival, and overall survival at 5 years for patients with a first-degree family history of gastric cancer compared with those without a positive family history of gastric cancer. These findings are similar to reports in other cancer types (ie, breast and colon) in which a positive family history is associated with improved prognosis. In addition, these results are most consistent with those previously reported by Yatsuya et al wherein a family history of gastric cancer was associated with improved patient survival. This study confirms that a positive family history of gastric cancer is clinically significant and prognostically favorable. These conclusions carry important implications for the families of individuals who are diagnosed with gastric cancer because it affects the mode of screening in first-degree family members, particularly in Asia where individuals with a first-degree family history of gastric cancer undergo more strict screening programs. This study also serves as a reminder of the importance of obtaining a detailed family history, which continues to be something of a lost art. Indeed, a history of cancer in firstor second-degree relatives has important implications for patients and for their family members who would potentially be candidates for more aggressive screening measures, particularly in cases of familial colon, breast, gastric, and other cancers. Despite the new insights about the significance of family history in gastric cancer that are provided by this study, we are left to wonder about the mechanism by which a positive history affects prognosis. Several potential explanations exist, including the possibility that individuals with a family history of gastric cancer share a genetic predisposition for their improved survival. For example, the presence of microsatellite instability (MSI) is a hallmark of all patients with hereditary nonpolyposis colon cancer and is present in 15% of sporadic colon cancers. Furthermore, MSI in colorectal cancer is positively associated with patient survival. MSI also serves as a negative predictive marker for response to treatment in patients with colorectal cancer. The role of MSI has been investigated in gastric cancer and is identified in 13% to 44% of sporadic tumors. Although a trend toward improved survival in MSI-high gastric cancer has been noted in several studies, these observations have not consistently reached statistical significance, and thus it is unclear whether the presence of MSI in gastric tumors serves as a prognostic or predictive indicator as it does in colon cancer. Additional studies are required to definitively determine whether the superior prognosis that has been observed in patients with a positive family history of gastric cancer is attributable to underlying genetic differences such as MSI or other genetic characteristics yet to be identified. The risk of developing gastric cancer is influenced by both genetic and environmental factors. Gastric adenocarcinoma is observed as part of inherited cancer syndromes that include Li-Fraumeni, hereditary nonpolyposis colon cancer, familial adenomatous polyposis, Peutz-Jeghers, and hereditary diffuse gastric cancer. Hereditary diffuse gastric cancer is an inherited gastric cancer predisposition syndrome that is associated with germline transmission of a CDH1 mutation and confers an approximate 75% lifetime risk of developing diffuse-type gastric cancer. Women with CDH1 germline mutations also carry an approximate 40% lifetime risk of developing lobular breast cancer. In contrast, environmental factors such as Helicobacter pylori (H pylori) infection, which confers an approximate five-fold increased risk of developing gastric cancer (primarily intestinal type), significantly affect gastric cancer risk. However, the role of H pylori infection in gastric cancer does not seem to be strictly environmental. Some evidence suggests that genetic factors contribute to an individual’s risk of developing gastric cancer after infection with H pylori. It will be important to determine whether a family history of H pylori infection and gastric cancer influences patient survival in a similar fashion. Additional explanations for the observed differences in prognosis for patients with a family history of gastric adenocarcinoma compared with those without such a history include behavioral differences and variable adherence to screening. A history of tobacco or alcohol use, as noted by the authors, is associated with a poorer prognosis in patients with gastric cancer. The study by Han et al identified a higher rate of smoking cessation and alcoholic consumption in patients with a firstdegree family history, which suggests that this health-related behavioral difference might influence the reported results. In contrast, the rate of adherence to screening recommendations was not observed to be different among those with a first-degree family history of gastric cancer (although this observation did not achieve statistical significance), which suggests that differential adherence to screening cannot account for the observed survival benefit. Editorials