William K. Murray

Type I natural killer T cells suppress tumors caused by p53 loss in mice.

CD1d-restricted T cells are considered to play a host protective effect in tumor immunity, yet the evidence for a role of natural killer T (NKT) cells in tumor immune surveillance has been weak and data from several tumor models has suggested that some (type II) CD1d-restricted T cells may also suppress some types of antitumor immune response. To substantiate an important role for CD1d-restricted T cells in host response to cancer, we have evaluated tumor development in p53(+/-) mice lacking either type I NKT cells (TCR Jalpha18(-/-)) or all CD1d-restricted T cells (CD1d(-/-)). Our findings support a key role for type I NKT cells in suppressing the onset of sarcomas and hematopoietic cancers caused by p53 loss but do not suggest that other CD1d-restricted T cells are critical in regulating the same tumor development.

Gene expression profiling of esophageal cancer: comparative analysis of Barrett's esophagus, adenocarcinoma, and squamous cell carcinoma.

Esophageal cancer is a particularly aggressive tumor with poor prognosis, however, our current knowledge of the genes and pathways involved in tumorigenesis of the esophagus are limited. To obtain insight into the molecular processes underlying tumorigenesis of the esophagus, we have used cDNA microarrays to compare the gene expression profiles of 128 tissue samples representing the major histological subtypes of esophageal cancer (squamous cell carcinoma and adenocarcinoma (ADC)) as well as Barretts esophagus (BE), the precursor lesion to ADC, and normal esophageal epithelium. Linear discriminant analysis and unsupervised hierarchical clustering show the separation of samples into 4 distinct groups consistent with their histological subtype. Differentially expressed genes were identified between each of the tissue types. Comparison of gene ontologies and gene expression profiles identified gene profiles specific to esophageal cancer, as well as BE. “Esophageal cancer clusters,” representing proliferation, immune response, and extracellular matrix genes were identified, as well as digestion, hydrolase, and transcription factor clusters specific to the columnar phenotype observed during BE and esophageal ADC. These clusters provide valuable insight into the molecular and functional differences between normal esophageal epithelium, BE, and the 2 histologically distinct forms of esophageal cancers. Our thorough, unbiased analysis provides a rich source of data for further studies into the molecular basis of tumorigenesis of the esophagus, as well as identification of potential biomarkers for early detection of progression.

Mutations in KIT occur at low frequency in melanomas arising from anatomical sites associated with chronic and intermittent sun exposure

In melanoma, mutations in KIT are most frequent in acral and mucosal subtypes and rarely reported in cutaneous melanomas particularly those associated with intermittent UV exposure. Conversely melanomas arising within chronic sun damaged skin are considered to harbour KIT mutations at higher rates. To characterize the frequency of KIT mutations in a representative melanoma population, 261 patients from two Australian melanoma centres were prospectively screened for mutations in exons 11, 13 and 17 of the KIT gene. A total of 257 patients had cutaneous melanoma arising from non‐acral sites and four were acral melanomas. No mucosal or ocular melanomas were analysed. KIT mutations were identified in five tumours (2% of the entire cohort) including two acral melanomas. Two of the three non‐acral melanomas with KIT mutations were associated with markers of chronic sun damage as assessed by the degree of skin elastosis. In the remaining cohort, 43% had chronically sun damaged skin. This report confirms that within an Australian population, KIT mutations are infrequent in cutaneous melanomas associated with both intermittent and chronic sun exposed skin.

Pretreatment Gene Expression Profiles Can Be Used to Predict Response to Neoadjuvant Chemoradiotherapy in Esophageal Cancer

BackgroundThe use of neoadjuvant therapy, in particular chemoradiotherapy (CRT), in the treatment of esophageal cancer (EC) remains controversial. The ability to predict treatment response in an individual EC patient would greatly aid therapeutic planning. Gene expression profiles of EC were measured and relationship to therapeutic response assessed.MethodsTumor biopsy samples taken from 46 EC patients before neoadjuvant CRT were analyzed on 10.5K cDNA microarrays. Response to treatment was assessed and correlated to gene expression patterns by using a support vector machine learning algorithm.ResultsComplete clinical response at conclusion of CRT was achieved in 6 of 21 squamous cell carcinoma (SCC) and 11 of 25 adenocarcinoma (AC) patients. CRT response was an independent prognostic factor for survival (P < .001). A range of support vector machine models incorporating 10 to 1000 genes produced a predictive performance of tumor response to CRT peaking at 87% in SCC, but a distinct positive prediction profile was unobtainable for AC. A 32-gene classifier was produced, and by means of this classifier, 10 of 21 SCC patients could be accurately identified as having disease with an incomplete response to therapy, and thus unlikely to benefit from neoadjuvant CRT.ConclusionsOur study identifies a 32-gene classifier that can be used to predict response to neoadjuvant CRT in SCC. However, because of the molecular diversity between the two histological subtypes of EC, when considering the AC and SCC samples as a single cohort, a predictive profile could not be resolved, and a negative predictive profile was observed for AC.

Pretreatment Transcriptional Profiling for Predicting Response to Neoadjuvant Chemoradiotherapy in Rectal Adenocarcinoma

Purpose: Patients presenting with locally advanced rectal cancer currently receive preoperative radiotherapy with or without chemotherapy. Although pathologic complete response is achieved for approximately 10% to 30% of patients, a proportion of patients derive no benefit from this therapy while being exposed to toxic side effects of treatment. Therefore, there is a strong need to identify patients who are unlikely to benefit from neoadjuvant therapy to help direct them toward alternate and ultimately more successful treatment options. Experimental Design: In this study, we obtained expression profiles from pretreatment biopsies for 51 rectal cancer patients. All patients underwent preoperative chemoradiotherapy, followed by resection of the tumor 6 to 8 weeks posttreatment. Gene expression and response to treatment were correlated, and a supervised learning algorithm was used to generate an original predictive classifier and validate previously published classifiers. Results: Novel predictive classifiers based on Mandards tumor regression grade, metabolic response, TNM (tumor node metastasis) downstaging, and normal tissue expression profiles were generated. Because there were only 7 patients who had minimal treatment response (>80% residual tumor), expression profiles were used to predict good tumor response and outcome. These classifiers peaked at 82% sensitivity and 89% specificity; however, classifiers with the highest sensitivity had poor specificity, and vice versa. Validation of predictive classifiers from previously published reports was attempted using this cohort; however, sensitivity and specificity ranged from 21% to 70%. Conclusions: These results show that the clinical utility of microarrays in predictive medicine is not yet within reach for rectal cancer and alternatives to microarrays should be considered for predictive studies in rectal adenocarcinoma. Clin Cancer Res; 17(9); 3039–47. ©2011 AACR.

The Lewis-Y carbohydrate antigen is expressed by many human tumors and can serve as a target for genetically redirected T cells despite the presence of soluble antigen in serum.

In this study we aimed to determine the suitability of the Lewis-Y carbohydrate antigen as a target for immunotherapy using genetically redirected T cells. Using the 3S193 monoclonal antibody and immunohistochemistry, Lewis-Y was found to be expressed on a range of tumors including 42% squamous cell lung carcinoma, 80% lung adenocarcinoma, 25% ovarian carcinoma, and 25% colorectal adenocarcinoma. Expression levels varied from low to intense on between 1% and 90% of tumor cells. Lewis-Y was also found in soluble form in sera from both normal donors and cancer patients using a newly developed enzyme-linked immunosorbent assay. Serum levels in patients was often less than 1 ng/mL, similar to normal donors, but approximately 30% of patients had soluble Lewis-Y levels exceeding 1 ng/mL and up to 9 ng/mL. Lewis-Y–specific human T cells were generated by genetic modification with a chimeric receptor encoding a single-chain humanized antibody linked to the T-cell signaling molecules, T-cell receptor-zeta, and CD28. T cells responded against the Lewis-Y antigen by cytokine secretion and cytolysis in response to tumor cells. Importantly, the T-cell response was not inhibited by patient serum containing soluble Lewis-Y. This study demonstrates that Lewis-Y is expressed on a large number of tumors and Lewis-Y–specific T cells can retain antitumor function in the presence of patient serum, indicating that this antigen is a suitable target for this form of therapy.

Perforin-mediated suppression of B-cell lymphoma

In the present study, we have examined the effect of perforin (pfp) deficiency in 4 models of mouse B-cell lymphomagenesis. We have examined pfp loss on the background of either Mlh1 tumor suppressor allele loss or oncogene expression [Ig heavy chain (Eμ)-v-Abl, Eμ-myc, and vav-bcl2]. Pfp was shown to act as a suppressor of B-cell malignancies characteristically driven by v-Abl or bcl-2, whereas Mlh loss cooperated in accelerating spontaneous B-cell lymphomas characteristic of pfp loss. No protective role for pfp was observed in the more aggressive Eμ-myc model of B-cell lymphoma. These transgenic models have allowed us to distinguish the role of pfp in surveillance of B-cell lymphomagenesis, as opposed to its loss simply driving the onset of a spontaneous lymphoma characteristic of pfp deficiency.

Parathyroid Hormone–Related Protein Protects against Mammary Tumor Emergence and Is Associated with Monocyte Infiltration in Ductal Carcinoma In situ

Parathyroid hormone-related protein (PTHrP) is required for mammary gland development and promotes the growth of breast cancer metastases within bone. However, there are conflicting reports of the prognostic significance of its expression in primary breast cancers. To study the role of PTHrP in early breast cancer, the effect of conditional deletion of PTHrP was examined in the context of neu-induced mammary tumorigenesis. Loss of PTHrP resulted in a higher tumor incidence. Transcriptional profiling of the tumors revealed that PTHrP influenced genes relevant to heterotypic cell signaling, including regulators of monocyte recruitment. Immunohistochemical analysis of human breast cancers revealed that PTHrP expression was associated with both HER-2/neu expression and macrophage infiltration in preinvasive ductal carcinoma in situ. The gene expression signature associated with loss of PTHrP expression in vivo correlated with poorer outcome in human breast cancer. Together, these data indicate that loss of PTHrP accelerates mammary tumorigenesis possibly by a non-cell-autonomous tumor suppressor pathway.

Nipple aspiration and ductal lavage in women with a germline BRCA1 or BRCA2 mutation

IntroductionThe aim of this study was to collect serial samples of nipple aspirate (NA) and ductal lavage (DL) fluid from women with germline BRCA1/2 mutations in order to create a biorepository for use in identifying biomarkers of breast cancer risk.MethodsBetween March 2003 and February 2005, 52 women with germline BRCA1 or BRCA2 mutations (median age 43 years, range 27 to 65 years) were scheduled for six-monthly NA, DL and venesection. DL was attempted for all NA fluid-yielding (FY) and any non-FY ducts that could be located at each visit.ResultsTwenty-seven (52%) women were postmenopausal, predominantly (19/27) from risk reducing bilateral salpingo-oophorectomy (BSO). FY ducts were identified in 60% of all women, 76% of premenopausal women versus 44% of postmenopausal (P = 0.026). Eighty-five percent of women had successful DL. Success was most likely in women with FY ducts (FY 94% versus non-FY 71% (P = 0.049). DL samples were more likely to be cellular if collected from FY ducts (FY 68% versus non-FY 43%; P = 0.037). Total cell counts were associated with FY status (FY median cell count 30,996, range 0 to >1,000,000 versus non-FY median cell count 0, range 0 to 173,577; P = 0.002). Four women (8%) had ducts with severe atypia with or without additional ducts with mild epithelial atypia; seven others had ducts with mild atypia alone (11/52 (21%) in total). Median total cell count was greater from ducts with atypia (105,870, range 1920 to >1,000,000) than those with no atypia (174, 0 to >1,000,000; P ≤ 0.001).ConclusionIt is feasible to collect serial NA and DL samples from women at high genetic risk of breast cancer, and we are creating a unique, prospective collection of ductal samples that have the potential to be used for discovery of biomarkers of breast cancer risk and evaluate the ongoing effects of risk reducing BSO. DL cellular atypia was not predictive of a current breast cancer and longer follow up is needed to determine whether atypia is an additional marker of future breast cancer risk in this population already at high genetic risk of breast cancer.

Absence of retroviral vector-mediated transformation of gene-modified T cells after long-term engraftment in mice

There is considerable concern regarding the transforming potential of retroviral vectors currently used for gene therapy, with evidence that retroviral integration can lead to leukemia in recipients of gene-modified stem cells. However, it is not clear whether retroviral-mediated transduction of T cells can lead to malignancy. We transduced mouse T cells with a Moloney murine retroviral gene construct and transferred them into congenic mice, which were preconditioned to enhance the engraftment of transferred T cells. Recipients were then observed long-term for evidence of cancer. Transferred T cells persisted in mice throughout life at levels up to 17% with gene copy numbers up to 5.89 × 105 per million splenocytes. Mice receiving gene-modified T cells developed tumors at a similar rate as control mice that did not receive T cells, and tumors in both groups of mice were of a similar range of histologies. Hematological malignancies comprised approximately 60% of cancers, and the remaining cancers consisted largely of carcinomas. Importantly, the incidence of lymphomas was similar in both groups of mice, and no lymphomas were found to be of donor T-cell origin. This study indicates that the use of retroviral vectors to transduce T cells does not lead to malignant transformation.