Christine E. Sheehan

Identification of new ALK and RET gene fusions from colorectal and lung cancer biopsies

Applying a next-generation sequencing assay targeting 145 cancer-relevant genes in 40 colorectal cancer and 24 non–small cell lung cancer formalin-fixed paraffin-embedded tissue specimens identified at least one clinically relevant genomic alteration in 59% of the samples and revealed two gene fusions, C2orf44-ALK in a colorectal cancer sample and KIF5B-RET in a lung adenocarcinoma. Further screening of 561 lung adenocarcinomas identified 11 additional tumors with KIF5B-RET gene fusions (2.0%; 95% CI 0.8–3.1%). Cells expressing oncogenic KIF5B-RET are sensitive to multi-kinase inhibitors that inhibit RET.

Xenogeneic extracellular matrix grafts elicit a TH2-restricted immune response1

Background. Porcine small intestinal submucosa (SIS) is an acellular, naturally derived extracellular matrix (ECM) that has been used for tissue remodeling and repair in numerous xenotransplantations. Although a vigorous immune response to xenogeneic extracellular matrix biomaterials is expected, to date there has been evidence for only normal tissue regeneration without any accompanying rejection. The purpose of this study was to determine the reason for a lack of rejection. Methods. Mice were implanted s.c. with xenogeneic tissue, syngeneic tissue, or SIS, and the graft site analyzed histologically for rejection or acceptance. Additionally, graft site cytokine levels were determined by reverse transcriptase polymerase chain reaction and SIS-specific serum antibody isotype levels were determined by ELISA. Results. Xenogeneically implanted mice showed an acute inflammatory response followed by chronic inflammation and ultimately graft necrosis, consistent with rejection. Syngeneically or SIS implanted mice, however, showed an acute inflammatory response that diminished such that the graft ultimately became indistinguishable from native tissue, observations that are consistent with graft acceptance. Graft site cytokine analysis showed an increase in interleukin-4 and an absence of interferon-&ggr;. In addition, mice implanted with SIS produced a SIS-specific antibody response that was restricted to the IgG1 isotype. Reimplantation of SIS into mice led to a secondary anti-SIS antibody response that was still restricted to IgG1. Similar results were observed with porcine submucosa derived from urinary bladder. To determine if the observed immune responses were T cell dependent, T cell KO mice were implanted with SIS. These mice expressed neither interleukin-4 at the implant site nor anti-SIS-specific serum antibodies but they did accept the SIS graft. Conclusions. Porcine extracellular matrix elicits an immune response that is predominately Th2-like, consistent with a remodeling reaction rather than rejection.

HER-2/neu gene amplification by FISH predicts poor survival in Barrett's esophagus-associated adenocarcinoma

The HER-2/neu oncogene is localized to chromosome 17q and shares significant homology with the epidermal growth factor receptor. HER-2/neu protein overexpression has been associated with poor prognosis in a variety of tumors, but its significance in Barretts esophagus-associated adenocarcinoma (BEAd) is unknown. Therefore, the aim of this study was to evaluate the prevalence and prognostic value of HER-2/neu gene amplification by fluorescence in situ hybridization (FISH) in 63 cases of BEAd. Routinely processed tissue sections from resection specimens of 63 patients with BEAd (M/F ratio, 10:1; mean age, 63 years) were assayed for HER-2/neu gene amplification by FISH using the Ventana unique sequence probe (Ventana Medical Systems, Inc, Tuscon, AZ). FISH results were correlated with the pathological features of the tumors and with patient survival. Clinical follow-up data were available for 54 patients (mean follow-up, 31 months [range, 1 to 152 months]). The HER-2/ neu gene was amplified in 12 of 63 (19%) cases. The presence of HER-2/neu gene amplification showed a trend toward a correlation with depth of tumor invasion (P = .07), lymph node metastasis (P = .13), and pathological stage (P = .14), but did not correlate with any of the other pathological features, such as degree of differentiation or tumor size. On both univariate and multivariate analysis, HER-2/neu gene amplification was associated with shortened survival (P = .03). HER-2/neu oncogene amplification, as determined by FISH, correlates with shortened patient survival and independently predicts poor outcome in patients with BEAd.

Expression of Nuclear Factor-κB and IκBα Proteins in Prostatic Adenocarcinomas: Correlation of Nuclear Factor-κB Immunoreactivity with Disease Recurrence

Purpose: The nuclear transcription factor nuclear factor-κB (NFκB) and its inhibitor, IκB, regulate the transcription of various genes involved in cell proliferation, adhesion, and survival. The NFκB transcription factor complex plays a role in cancer development and progression through its influence on apoptosis. More recently, NFκB has been shown to be activated in human and androgen-independent prostate cancer cells. To our knowledge, this is the first study demonstrating the prognostic significance of NFκB immunoreactivity in prostate adenocarcinomas (PACs). Experimental Design: Using prostatectomy specimens, we performed immunohistochemical staining for NFκB and IκBα (Santa Cruz Biotechnology) on formalin-fixed, paraffin-embedded sections obtained from 136 patients with PAC. Cytoplasmic and nuclear immunoreactivity was scored for intensity and distribution, and results were correlated with preoperative serum prostate-specific antigen, tumor grade, stage, DNA ploidy (Feulgen spectroscopy), and biochemical disease recurrence. Results: Forty-nine percent of PACs overexpressed cytoplasmic NFκB, and 63% showed decreased IκB expression. Cytoplasmic NFκB overexpression correlated with advanced tumor stage (P = 0.048), aneuploidy (P = 0.022), and biochemical disease recurrence (P = 0.001). When we compared the means for the NFκB-positive and -negative subgroups, NFκB overexpression correlated with preoperative serum prostate-specific antigen (P = 0.04) and DNA index (P = 0.05). Fifteen percent of PACs expressed nuclear NFκB, which correlated with high tumor grade (P = 0.001) and advanced stage (P = 0.05). Decreased IκBα expression correlated with high tumor grade (P = 0.015). On multivariate analysis, tumor stage (P = 0.043) and NFκB overexpression (P = 0.006) were independent predictors of biochemical recurrence. Conclusion: These results support a role for NFκB pathway proteins in the tumorigenesis of PACs. The findings are also consistent with reported experimental studies suggesting a new strategy of combined chemotherapy and specific NFκB blockade in decreasing the rate of disease relapse.PURPOSE The nuclear transcription factor nuclear factor-kappa B (NF kappa B) and its inhibitor, I kappa B, regulate the transcription of various genes involved in cell proliferation, adhesion, and survival. The NF kappa B transcription factor complex plays a role in cancer development and progression through its influence on apoptosis. More recently, NF kappa B has been shown to be activated in human and androgen-independent prostate cancer cells. To our knowledge, this is the first study demonstrating the prognostic significance of NF kappa B immunoreactivity in prostate adenocarcinomas (PACs). EXPERIMENTAL DESIGN Using prostatectomy specimens, we performed immunohistochemical staining for NF kappa B and I kappa B alpha (Santa Cruz Biotechnology) on formalin-fixed, paraffin-embedded sections obtained from 136 patients with PAC. Cytoplasmic and nuclear immunoreactivity was scored for intensity and distribution, and results were correlated with preoperative serum prostate-specific antigen, tumor grade, stage, DNA ploidy (Feulgen spectroscopy), and biochemical disease recurrence. RESULTS Forty-nine percent of PACs overexpressed cytoplasmic NF kappa B, and 63% showed decreased I kappa B expression. Cytoplasmic NF kappa B overexpression correlated with advanced tumor stage (P = 0.048), aneuploidy (P = 0.022), and biochemical disease recurrence (P = 0.001). When we compared the means for the NF kappa B-positive and -negative subgroups, NF kappa B overexpression correlated with preoperative serum prostate-specific antigen (P = 0.04) and DNA index (P = 0.05). Fifteen percent of PACs expressed nuclear NF kappa B, which correlated with high tumor grade (P = 0.001) and advanced stage (P = 0.05). Decreased I kappa B alpha expression correlated with high tumor grade (P = 0.015). On multivariate analysis, tumor stage (P = 0.043) and NF kappa B overexpression (P = 0.006) were independent predictors of biochemical recurrence. CONCLUSION These results support a role for NF kappa B pathway proteins in the tumorigenesis of PACs. The findings are also consistent with reported experimental studies suggesting a new strategy of combined chemotherapy and specific NF kappa B blockade in decreasing the rate of disease relapse.

New Routes to Targeted Therapy of Intrahepatic Cholangiocarcinomas Revealed by Next-Generation Sequencing

BACKGROUND Intrahepatic cholangiocarcinoma (ICC) is a subtype of primary liver cancer that is rarely curable by surgery and is rapidly increasing in incidence. Relapsed ICC has a poor prognosis, and current systemic nontargeted therapies are commonly extrapolated from those used in other gastrointestinal malignancies. We hypothesized that genomic profiling of clinical ICC samples would identify genomic alterations that are linked to targeted therapies and that could facilitate a personalized approach to therapy. METHODS DNA sequencing of hybridization-captured libraries was performed for 3,320 exons of 182 cancer-related genes and 36 introns of 14 genes frequently rearranged in cancer. Sample DNA was isolated from 40 μm of 28 formalin-fixed paraffin-embedded ICC specimens and sequenced to high coverage. RESULTS The most commonly observed alterations were within ARID1A (36%), IDH1/2 (36%), and TP53 (36%) as well as amplification of MCL1 (21%). Twenty cases (71%) harbored at least one potentially actionable alteration, including FGFR2 (14%), KRAS (11%), PTEN (11%), CDKN2A (7%), CDK6 (7%), ERBB3 (7%), MET (7%), NRAS (7%), BRCA1 (4%), BRCA2 (4%), NF1 (4%), PIK3CA (4%), PTCH1 (4%), and TSC1 (4%). Four (14%) of the ICC cases featured novel gene fusions involving the tyrosine kinases FGFR2 and NTRK1 (FGFR2-KIAA1598, FGFR2-BICC1, FGFR2-TACC3, and RABGAP1L-NTRK1). CONCLUSION Two thirds of patients in this study harbored genomic alterations that are associated with targeted therapies and that have the potential to personalize therapy selection for to individual patients.

HER-2/neu gene amplification status in prostate cancer by fluorescence in situ hybridization

HER-2/neu expression has been established as a prognostic factor in breast and other cancers. In prostate cancer (PC), a similar predictive role has been hindered by variable immunohistochemical (IHC) results. The authors studied DNA amplification of the HER-2/neu gene on 4-microm sections obtained from 62 formalin-fixed, paraffin-embedded PCs by fluorescence in situ hybridization (FISH). The results were compared with HER-2/neu protein expression as determined by IHC and correlated by logistic regression analysis with Gleason tumor grade, DNA ploidy, serum prostate specific antigen (PSA), and pathological stage. The HER-2/neu gene was localized using the Oncor (Gaithersburg, MD) digoxigenin-labeled unique sequence probe. Amplified PCs had at least 20 malignant cells, with 5 or more copies of the sequence. Amplification of HER-2/neu correlated with Gleason score (P = .0001). The mean Gleason score of unamplified tumors was 5.7 and that of amplified tumors was 7.5. Nondiploid tumors had a significantly greater rate of HER-2/neu amplification compared with diploid tumors (P = .0003). Of the 62 cases evaluated by IHC and FISH, 18 cases (29%) were overexpressed by IHC, and 27 cases (44%) were amplified by FISH. A trend for similar HER-2/neu status in each PC by the two methods did not reach statistical significance (P = .23). HER-2/neu amplification by FISH was associated with advanced pathological stage; however, this relationship reached only near-statistical significance (P = .06). There was no correlation of HER-2/neu amplification by FISH with patient age or preoperative serum PSA levels. The authors conclude that HER-2/neu gene amplification status can be determined by FISH on archival prostate cancer specimens, significantly correlates with high tumor grade and nondiploid DNA content, and is more frequently encountered in tumors with advanced pathological stage. Also, FISH is more sensitive than IHC for detection of abnormalities in the HER-2/neu gene, and further studies should be undertaken to determine whether a FISH-based HER-2/neu detection method may prove of importance in the prediction of prognosis and planning of therapy in prostate cancer patients.

Prostate-specific membrane antigen expression in the neovasculature of gastric and colorectal cancers

Prostate-specific membrane antigen (PSMA), a type II transmembrane metallo-peptidase highly overexpressed in prostate cancer cells, has been studied as a targeting molecule in prostate cancer. Recently, PSMA has also been found to be expressed in the neovasculature of multiple nonprostatic solid tumors. Because of its unique expression pattern limited to tumor-associated endothelial cells, PSMA may also be an interesting molecule for vascular targeting. In this study, PSMA expression was determined by immunohistochemistry in 119 cases of primary gastric adenocarcinoma, 130 cases of primary colorectal adenocarcinoma, and 24 metastasis of colorectal adenocarcinoma. Expression data were correlated with clinicopathologic information. PSMA expression was detected in tumor-associated neovasculature of 79 (66%) of 119 gastric and 110 (85%) of 130 colorectal carcinomas. Furthermore, the neovasculatures of 16 (84%) of 19 liver and 4 (80%) of 5 nodal metastases from colorectal carcinomas were prostate-specific membrane antigen positive. There was a trend for high-grade tumors to higher PSMA expression (Spearman r = 0.18, P = .046) in colorectal cancers. No association between PSMA expression and overall- or disease-free survival was observed in gastric or colorectal cancers. This study provides the first in-depth look at PSMA expression in gastric and colorectal cancer. Because of its highly tumor-restricted expression and its accessibility to targeted therapy, PSMA represents a promising therapeutic and diagnostic target in colorectal and gastric cancer.

PTEN expression in normal skin, acquired melanocytic nevi, and cutaneous melanoma

BACKGROUND Although various studies have shown mutations of the tumor suppressor gene, PTEN/MMAC1, in primary, metastatic, and cultured cutaneous melanoma specimens, little is known about the pattern of PTEN protein expression in early melanocytic tumor progression. OBJECTIVE To further investigate the role of PTEN in melanocytic tumor development. METHODS We assessed the level and distribution of PTEN in normal skin, 39 acquired melanocytic nevi, and 30 primary cutaneous melanomas, including lentigo malignas, by immunostaining. RESULTS We found high levels of PTEN expression in cutaneous muscles, nerves, and muscular arteries, and moderate-to-high amounts of PTEN in the epidermis, follicular epithelium, and sebaceous and eccrine glands. PTEN staining in cutaneous lymphatics, dermal and periadnexal adventitial fibroblasts, and chondrocytes were variably absent. Junctional melanocytes and chondrocytes frequently exhibited preferential nuclear staining. We found uniformly strong PTEN expression in the cytoplasm of almost all benign and dysplastic nevi. However, there was some evidence of nuclear PTEN loss even in the benign melanocytic proliferations. In addition, out of 30 primary cutaneous melanomas and lentigo malignas, we detected diffuse expression of PTEN in 11 (37%) tumors, widespread loss of PTEN in 11 (37%) tumors and mixed PTEN expression in 8 (27%) lesions. In the primary cutaneous melanomas, PTEN was largely localized to the cytoplasm. CONCLUSIONS The presence of PTEN in benign melanocytic tumors and the absence of PTEN in a significant proportion of primary cutaneous melanomas support a role for PTEN loss in the pathogenesis of melanoma.

Advanced urothelial carcinoma: next-generation sequencing reveals diverse genomic alterations and targets of therapy

Although urothelial carcinoma (UC) of the urinary bladder generally portends a favorable prognosis, metastatic tumors often follow an aggressive clinical course. DNA was extracted from 40 μm of formalin-fixed, paraffin-embedded (FFPE) sections from 35 stage IV UCs that had relapsed and progressed after primary surgery and conventional chemotherapy. Next-generation sequencing (NGS) was performed on hybridization-captured, adaptor ligation-based libraries for 3320 exons of 182 cancer-related genes plus 37 introns from 14 genes frequently rearranged in cancer to at an average sequencing depth of 1164 × and evaluated for all classes of genomic alterations (GAs). Actionable GAs were defined as those impacting the selection of targeted anticancer therapies on the market or in registered clinical trials. A total of 139 GAs were identified, with an average of 4.0 GAs per tumor (range 0–10), of which 78 (56%) were considered actionable, with an average of 2.2 per tumor (range 0–7). Twenty-nine (83%) cases harbored at least one actionable GA including: PIK3CA (9 cases; 26%); CDKN2A/B (8 cases; 23%); CCND1 (5 cases; 14%); FGFR1 (5 cases; 14%); CCND3 (4 cases; 11%); FGFR3 (4 cases; 11%); MCL1 (4 cases; 11%); MDM2 (4 cases; 11%); EGFR (2 cases, 6%); ERBB2 (HER2/neu) (2 cases, 6%); NF1 (2 cases, 6%) and TSC1 (2 cases, 6%). Notable additional alterations included TP53 (19 cases, 54%) and RB1 (6 cases; 17%). Genes involved in chromatin modification were altered by nonsense mutation, splice site mutation or frameshift indel in a mutually exclusive manner in nearly half of all cases including KDM6A (10 cases; 29%) and ARID1A (7 cases; 20%). Comprehensive NGS of 35 UCs of the bladder revealed a diverse spectrum of actionable GAs in 83% of cases, which has the potential to inform treatment decisions for patients with relapsed and metastatic disease.

Natural anti-galactose α1,3 galactose antibodies delay, but do not prevent the acceptance of extracellular matrix xenografts

Naturally occurring antibodies to the galactose alpha1,3 galactose (alpha gal) epitope expressed on xenogeneic grafts are a major barrier to organ transplantation in humans. Porcine small intestinal submucosa (SIS) expresses the alpha gal epitope and is currently being used as a bioscaffold for tissue remodeling. To examine in detail the potential role of the alpha gal epitope in immune recognition of this acellular, avascular biomaterial, we have used mice which have a genetic disruption in the alpha1,3 galactosyltransferase gene (alpha gal(-/-)mice) and thus express natural anti-alpha gal antibodies in a manner similar to humans. It was found that alpha gal(-/-)mice produced IgM anti-alpha gal antibodies in addition to IgG1 SIS-specific antibodies, which did not bind to the alpha gal epitope. Histological examination of implant sites demonstrated an early inflammatory response that consisted predominantly of neutrophils in both alpha gal(+/+) and alpha gal(-/-)mice. However, while alpha gal(+/+)mice completely remodeled SIS implants by day 25, alpha gal(-/-)mice still exhibited some visible SIS together with inflammatory cellular infiltrates at this time point. Nevertheless, by day 35, the implant site in alpha gal(-/-)mice appeared to be entirely remodeled although a few acute inflammatory cells were still present. Immunization of alpha gal(-/-)mice with sheep erythrocytes to enhance anti-alpha gal antibody levels led to a more robust early inflammatory response following implantation but did not change the ultimate fate of the graft. We conclude that, in contrast to xenotransplantation of whole organs, naturally-occurring anti-alpha gal antibodies do not influence the ability of xenogeneic extracellular matrices to serve as bioscaffolds for tissue remodeling.