The Immune Microenvironment Impacts Survival in Western Patients with Gastric Adenocarcinoma.

BACKGROUND: Expression of CD3+ T cells, CD8+ cytotoxic T cells, CD45RO+ memory T cells, and FOXP3+ regulatory T cells at the invasive margin (IM) and tumor center (TC) has correlated with survival in gastric adenocarcinoma (GA) patients from East Asia, independent of anatomic staging. The reason for improved survival in East Asians compared with Western patients is a subject of debate. This study examined the immune profiles of a cohort of Western patients with GA, and their association with overall survival (OS). METHODS: Immunohistochemistry (IHC) using antibodies to CD3, CD4, CD8, CD45RO, and FOXP3 was performed on a randomly selected resected GA specimens from 88 Western patients. Cutoffs for high or low expression of each marker were determined with maximally selected rank statistics, and multivariable Cox proportional-hazards models constructed to evaluate the relationship between OS and expression of each marker at the IM and TC. RESULTS: Immune cell density was independent of anatomic staging. High expression of CD3, CD4, CD8, and CD45RO at the IM along with CD4 and FOXP3 at the TC were associated with improved OS. A combined marker of CD3, CD8, CD45RO, and FOXP3 associated with OS in East Asian GA was also validated. DISCUSSION: This is the first report in US patients to demonstrate that high expression of multiple subsets of T lymphocytes in GA is associated with better OS independent of clinical factors and anatomic stage. Further evaluation of immune-modulating mechanisms may explain survival differences between Western and Eastern patients and provide opportunity for novel treatments.

Clinicopathological Features of Triple-Negative Breast Cancer Epigenetic Subtypes.

BACKGROUND/OBJECTIVE: Triple-negative breast cancer (TNBC) is a heterogeneous collection of breast tumors with numerous differences including morphological characteristics, genetic makeup, immune-cell infiltration, and response to systemic therapy. DNA methylation profiling is a robust tool to accurately identify disease-specific subtypes. We aimed to generate an epigenetic subclassification of TNBC tumors (epitypes) with utility for clinical decision-making. METHODS: Genome-wide DNA methylation profiles from TNBC patients generated in the Cancer Genome Atlas project were used to build machine learning-based epigenetic classifiers. Clinical and demographic variables, as well as gene expression and gene mutation data from the same cohort, were integrated to further refine the TNBC epitypes. RESULTS: This analysis indicated the existence of four TNBC epitypes, named as Epi-CL-A, Epi-CL-B, Epi-CL-C, and Epi-CL-D. Patients with Epi-CL-B tumors showed significantly shorter disease-free survival and overall survival [log rank; P = 0.01; hazard ratio (HR) 3.89, 95% confidence interval (CI) 1.3-11.63 and P = 0.003; HR 5.29, 95% CI 1.55-18.18, respectively]. Significant gene expression and mutation differences among the TNBC epitypes suggested alternative pathway activation that could be used as ancillary therapeutic targets. These epigenetic subtypes showed complementarity with the recently described TNBC transcriptomic subtypes. CONCLUSIONS: TNBC epigenetic subtypes exhibit significant clinical and molecular differences. The links between genetic make-up, gene expression programs, and epigenetic subtypes open new avenues in the development of laboratory tests to more efficiently stratify TNBC patients, helping optimize tailored treatment approaches.

Epigenetic profiling for the molecular classification of metastatic brain tumors.

Optimal treatment of brain metastases is often hindered by limitations in diagnostic capabilities. To meet this challenge, here we profile DNA methylomes of the three most frequent types of brain metastases: melanoma, breast, and lung cancers (n = 96). Using supervised machine learning and integration of DNA methylomes from normal, primary, and metastatic tumor specimens (n = 1860), we unravel epigenetic signatures specific to each type of metastatic brain tumor and constructed a three-step DNA methylation-based classifier (BrainMETH) that categorizes brain metastases according to the tissue of origin and therapeutically relevant subtypes. BrainMETH predictions are supported by routine histopathologic evaluation. We further characterize and validate the most predictive genomic regions in a large cohort of brain tumors (n = 165) using quantitative-methylation-specific PCR. Our study highlights the importance of brain tumor-defining epigenetic alterations, which can be utilized to further develop DNA methylation profiling as a critical tool in the histomolecular stratification of patients with brain metastases.

The Negative Impact of Body Mass Index on the Tumor Microenvironment in Colon Cancer: Results of a Prospective Trial.

BACKGROUND: The association between tumor mismatch repair status and obesity in colon cancer is not well understood. The authors of this study hypothesized that mismatch repair deficiency in colon cancer may be associated with a lower Body Mass Index (BMI) and improved patient outcome due to an enhanced tumor immune microenvironment. METHODS: For this study, 70 patients were randomly selected from a prospective trial evaluating nodal ultrastaging for colon cancer. The mismatch repair status of tumors and immunomarker expression were correlated with clinicopathologic characteristics and evaluated for disease-free survival. RESULTS: Patients with mismatch repair-deficient tumors (n = 11) had a lower mean BMI than those with mismatch repair-proficient tumors (n = 59) (22.16 vs. 26.30 kg/m2, respectively; p = 0.029).The findings showed that CD3+ T cells were inversely associated with mismatch repair proficiency (p = 0.048). Mismatch repair-proficient tumors in nonobese patients (BMI < 30 kg/m2) versus obese patients had a higher density of CD8+ (p = 0.008) and FOXP3+ (p = 0.005) T cells. Multivariable analysis linked CD4+ (hazard ratio [HR] 0.52; 95% confidence interval [CI] 0.35-0.76), CD8+ (HR 0.67; 95% CI 0.50-0.89), and number of tumor-positive lymph nodes (HR 1.19; 95% CI 1.03-1.36) to disease-free survival for patients with mismatch repair-proficient tumors. CONCLUSIONS: Tumor mismatch repair status and obesity are correlated in patients with colon cancer. Increased intratumoral T cells in nonobese patients suggests an unexplored link between tumor mismatch repair and immunoprofile.

Prognostic Utility of Immunoprofiling in Colon Cancer: Results from a Prospective, Multicenter Nodal Ultrastaging Trial.

BACKGROUND: Retrospective data indicate that immunoprofiling of T cell markers can be prognostic in colon cancer. Prospective T cell immunoprofiling of colon cancer has not been well defined for patients whose lymph nodes are ultrastaged. STUDY DESIGN: A prospective cohort was selected from patients enrolled in an ongoing phase II multicenter trial of nodal ultrastaging for colon cancer. Primary tumor specimens from 89 patients were analyzed by immunohistochemistry for the T cells CD3(+), CD4(+), CD8(+), and FOXP3(+). Lymphocyte populations were quantified with digital image analysis. Results were examined for their association with 5-year disease-free survival along with TNM stage and clinicopathologic variables. RESULTS: Longer disease-free survival was associated with higher CD3(+) counts at the invasive margin (IM) (p = 0.005), higher CD8(+) counts at the tumor center (TC) and IM (p = 0.002), a lower CD4(+)/CD8(+) ratio at the TC+IM (p = 0.027), and a higher CD8(+)/FOXP3(+) ratio at the TC+IM (p = 0.020). After multivariable analysis, CD8(+) at the TC+IM (p = 0.002), the CD8(+)/FOXP3(+) ratio at the TC+IM (p = 0.004), and the number of tumor-positive lymph nodes (p = 0.003) remained significant. CONCLUSIONS: This is the first prospective demonstration of the prognostic utility of immunoprofiling in colon cancer after nodal ultrastaging. Staging based on tumor immunoprofile can augment TNM staging and provide targets for specific immunotherapies.

Alectinib induced CNS radiation necrosis in an ALK+NSCLC patient with a remote (7 years) history of brain radiation.

Alectinib is a second generation ALK inhibitor that has significant clinical activity in central nervous system (CNS) metastases in anaplastic lymphoma kinase (ALK)-rearranged non-small cell lung cancer (NSCLC). Pseudoprogression (PsP) due to radiation necrosis during alecitnib treatment of central nervous system (CNS) metastases from ALK-rearranged NSCLC as been reported. Hence, distinguishing radiation-related PsP from alectinib-induced radiographic changes is important to avoid erroneous early trial discontinuation and abandonment of an effective treatment. However, it remains difficult to assess casuality of radiation necrosis is related to recent direct radiation or induced by alectinib treatment or both. It is also unknown how long from previous radiation can alectinib still induce radiation necrosis. Here we reported a crizotinib-refractory ALK-positive NSCLC patient who develop radiation necrosis in one of his metastatic CNS lesions after approximately 12 months of alectinib treatment who otherwise had on-going CNS response on alectinib. His most recent radiation to his CNS metastases was 7 years prior to the start of alectinib. This case illustrates that in the setting of pror CNS radiation, given the significant clinical activity of alectinib in CNS metastases in ALK-positive NSCLC patients the risk of CNS radiation necrosis remains long after previous radiation to the CNS metastases has been completed and can occur after durable response of treatment.

Cross-platform comparison of independent datasets identifies an immune signature associated with improved survival in metastatic melanoma.

Platform and study differences in prognostic signatures from metastatic melanoma (MM) gene expression reports often hinder consensus arrival. We performed survival/outcome-based pairwise comparisons of three independent MM gene expression profiles using the threshold-free algorithm rank-rank hypergeometric overlap analysis (RRHO). We found statistically significant overlap for genes overexpressed in favorable outcome (FO) groups, but no overlap for poor outcome (PO) groups. This "favorable outcome signature" (FOS) of 228 genes coinciding on all three overlapping gene lists showed immune function predominated in FO MM. Surprisingly, specific cell signature-enrichment analysis showed B cell-associated genes enriched in FO MM, along with T cell-associated genes. Higher levels of B and T cells (p<0.05) and their relative proximity (p<0.05) were detected in FO-to-PO tumor comparisons from an independent MM patients cohort. Finally, expression of FOS in two independent Stage III MM tumor datasets correctly predicted clinical outcome in 12/14 and 44/70 patients using a weighted gene voting classifier (area under the curve values 0.96 and 0.75, respectively). This RRHO-based, cross-study analysis emphasizes the RRHO approach power, confirms T cells relevance for prolonged MM survival, supports a favorable role for B cells in anti-melanoma immunity, and suggests B cells potential as means of intervention in melanoma treatment.

Immunoprofiling for prognostic assessment of colon cancer: a novel complement to ultrastaging.

BACKGROUND: Although AJCC/TNM staging remains the gold standard for prognostic assessment of colon cancer, stage-specific outcomes vary. We therefore prospectively evaluated the prognostic role of immunoprofiling. METHODS: Our cohort included 35 patients from an ongoing prospective trial of ultrastaging for colon cancer. Specimens were analyzed for T cell markers (CD3, CD4, CD8, and FoxP3). The number of tumor-infiltrating lymphocytes was analyzed at the tumor's margin and center and correlated with AJCC/TNM stage, clinicopathologic variables, and disease-free survival. RESULTS: There was a significant inverse association between number of CD3(+) cells in the tumor center and tumor stage (P = 0.05). The tumor center/margin ratio of CD3(+) cells also showed an inverse but non-significant relationship with nodal involvement (P = 0.07). Body mass index was inversely associated with numbers of CD3(+)(P = 0.04) and CD8(+)(P = 0.02) cells. Longer disease-free survival was correlated with higher CD8+ counts (P = 0.07), lower CD4(+)/CD8(+) ratios (P = 0.008), and higher CD8(+)/FoxP3(+) ratios (P = 0.02). CONCLUSIONS: This is the first prospective validation of immunoprofiling in patients whose colon cancer is staged with strict surgical and pathology quality measures. The apparent correlation between immunophenotypic response and clinical outcome warrants evaluation in a larger prospective trial.

Metastatic melanoma with striking adenocarcinomatous differentiation illustrating phenotypic plasticity in melanoma.

We report on the highly unusual case of a 75-year-old woman who developed a biphasic right axillary mass of apparent melanoma and adenocarcinoma 13 years after a diagnosis of primary melanoma on her right upper back. The differential diagnosis included a collision tumor and metastatic melanoma with adenocarcinomatous transdifferentiation. We utilized immunohistochemical staining, DNA sequencing, and comparative genomic hybridization (CGH) to characterize this unusual tumor. By immunohistochemistry, the melanomatous component was positive for S100 and Melan-A, and had patchy positivity for cytokeratin. The adenocarcinomatous component was negative for melanoma markers, but was strongly positive for cytokeratin. In addition, the glandular component was positive for CDX-2 and Ber-EP4, giving the distinct histologic and immunohistochemical impression of a gastrointestinal metastasis nested within a deposit of metastatic melanoma. Clinical and radiologic workup failed to reveal a primary gastrointestinal malignancy. Molecular genetic analysis, including DNA sequencing and CGH, revealed that both areas contained an identical NRAS Q61K mutation and had highly similar CGH profiles, including gains of chromosome 1q and losses of 1p, 4, 9, and 10, which are archetypical of melanoma. The NRAS mutation was also identified in a deposit of metastatic melanoma resected 12 years earlier, but was not seen in the patient's nontumorous tissue, indicating that it was somatically acquired. Genetic analyses demonstrate that 2 morphologically distinct tumors arose from a common ancestor melanoma cell that harbored an NRAS mutation and subsequently divergently evolved by the acquisition of additional genomic alterations. Our findings illustrate the ability of molecular analyses to resolve lineage in complex neoplasms and illustrate the phenotypic plasticity of cancer cells.

Comparative metabolism of the tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol by rat cytochrome P450 2A3 and human cytochrome P450 2A13.

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its carbonyl-reduction product, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), are potent lung carcinogens in rats and are presumed human lung carcinogens. NNK and NNAL are bioactivated to DNA-binding intermediates via hydroxylation of the carbon atoms adjacent to the nitroso moiety (i.e., alpha-hydroxylation) by cytochrome p450s (p450s). Therefore, it is important to delineate which p450s are efficient catalysts of this metabolic transformation. In this study, the kinetic parameters for NNK and NNAL metabolism were determined for two extrahepatic p450s that are expressed in the lung: rat p450 2A3 and human p450 2A13. p450s 2A3 and 2A13 exhibited Vmax values for NNK 4-hydroxylation of 10.8 +/- 0.4 and 13.8 +/- 0.8 pmol min-1 pmol P450-1, respectively; the corresponding Km values were 4.6 +/- 0.5 and 3.6 +/- 0.7 microM. The respective Vmax values for p450 2A3- and 2A13-mediated N-methyl hydroxylation of NNK were 8.2 +/- 0.3 and 4.6 +/- 0.2 pmol min-1 pmol p450-1. These data indicate that p450s 2A3 and 2A13 are both efficient catalysts of the metabolic activation of NNK and are, along with mouse p450 2A5, the best catalysts of this reaction currently known. Both enzymes also catalyzed the alpha-hydroxylation and N-oxidation of NNAL, and its oxidation to NNK. In general, Vmax/Km values for NNAL metabolism were 1 to 2 orders of magnitude lower than those for NNK metabolism, and p450 2A3 was a slightly better catalyst of NNAL metabolism than was p450 2A13. Given the exquisite sensitivity of the rat lung to NNK-induced carcinogenesis, the efficient bioactivation of NNK by rat p450 2A3, and the similar catalytic efficiency of p450s 2A3 and 2A13, p450 2A13 may be an important contributor to NNK bioactivation in the human lung.

Synthesis of stereospecifically deuterated 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) iastereomers and metabolism by A/J mouse lung microsomes and cytochrome p450 2A5.

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a lung carcinogen in mice and rats and is a putative human lung carcinogen. NNK undergoes cytochrome p450-mediated metabolic activation to DNA-binding intermediates but is also extensively reduced to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in vivo. Because NNAL is also tumorigenic, the carcinogenicity of NNK may actually be governed by the metabolic activation of NNAL, rather than direct activation of NNK. Metabolism of NNK and NNAL at the 4-position generates the same critical DNA lesion, O(6)-methylguanine, the levels of which are correlated to tumorigenicity in the A/J mouse model. In an effort to better understand the bioactivation of NNAL and the effect of carbinol-carbon stereochemistry on prochiral selectivity at the 4-position, (R)- and (S)-NNAL, along with the stereospecifically 4-deuterated diastereomers (1R,4R)-[4-(2)H(1)]NNAL, (1R,4S)-[4-(2)H(1)]NNAL, (1S,4R)-[4-(2)H(1)]NNAL, and (1S,4S)-[4-(2)H(1)]NNAL, were synthesized. The in vitro metabolism of these compounds was investigated using A/J mouse lung microsomes and Spodoptera frugiperda-expressed mouse cytochrome p450 2A5. Carbinol-carbon stereochemistry did not appreciably influence stereoselectivity at the 4-position in the metabolism of these compounds by mouse lung microsomes or p450 2A5 but did influence the regiochemistry of metabolism. The ratio of 4- to N-methyl hydroxylation was approximately 1:1 for the A/J mouse lung microsome-mediated metabolism of all substrates, but this ratio was higher for (1S) substrates than for their (1R) counterparts when p450 2A5 was used. Interestingly, p450 2A5 converted substrates with (1S) stereochemistry to the respective N-oxides, but this metabolite was not formed from substrates with (1R) stereochemistry. Furthermore, p450 2A5 catalyzed the formation of NNK from (1S) substrates at significantly greater maximal rates than from (1R) substrates. The implications of these differences in metabolism for the tumorigenic mechanism of NNAL are discussed.

Stereospecific deuterium substitution attenuates the tumorigenicity and metabolism of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK).

Stereochemical determinants of the tumorigenicity and metabolism of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were investigated using the stereospecifically deuterated isotopomers (4R)-[4-(2)H(1)]NNK and (4S)-[4-(2)H(1)]NNK. Upon ip administration to groups of 20 female A/J mice, NNK and (4S)-[4-(2)H(1)]NNK exhibited similar lung tumorigenicity at three different doses, whereas (4R)-[4-(2)H(1)]NNK was 2-fold less tumorigenic at all three doses. In a parallel experiment, levels of O(6)-methylguanine and 7-methylguanine were 2-fold lower in lung DNA of mice treated with (4R)-[4-(2)H(1)]NNK than in mice treated with NNK or (4S)-[4-(2)H(1)]NNK. To corroborate these in vivo data, the in vitro metabolism of these compounds was investigated using A/J mouse lung microsomes and Spodoptera frugiperda (Sf9)-expressed mouse cytochrome p450s 2A4 and 2A5. Kinetic isotope effects on the apparent V(max) ((D)V) for the product of NNK 4-hydroxylation, OPB, were 2.7 +/- 0.2 and 2.8 +/- 0.4 when (4R)- and (4S)-[4-(2)H(1)]NNK were incubated with mouse lung microsomes, respectively. The (D)V values for OPB formation were 3.2 +/- 0.2 and 2.2 +/- 0.2 when (4R)-[4-(2)H(1)]NNK was the substrate for p2A4 and 2A5, respectively, whereas they were 1.3 +/- 0.1 and 1.1 +/- 0.1 when (4S)-[4-(2)H(1)]NNK was the substrate for these respective enzymes. Analysis of an OPB derivative (10) for deuterium content by LC/MS confirmed the results from the kinetic assays and indicated that p450s 2A4 and 2A5 preferentially abstract the pro-R 4-hydrogen of NNK. The results obtained using Sf9-expressed p450s provide a rationale for the differences observed in the lung tumor and DNA adduct experiments, namely, that the attenuated tumorigenicity of (4R)-[4-(2)H(1)]NNK relative to (4S)-[4-(2)H(1)]NNK is due to prochiral selectivity during p450-catalyzed metabolic activation.

Reactions of formaldehyde plus acetaldehyde with deoxyguanosine and DNA: formation of cyclic deoxyguanosine adducts and formaldehyde cross-links.

We investigated the reactions of formaldehyde plus acetaldehyde with dGuo and DNA in order to determine whether certain 1,N(2)-propano-dGuo adducts could be formed. These adducts-3-(2'-deoxyribosyl)-5,6,7,8-tetrahydro-8-hydroxypyrimido[1,2-a]purine-(3H)-one (1) and 3-(2'-deoxyribosyl)-5,6,7,8-tetrahydro-6-hydroxypyrimido[1,2-a]purine-(3H)-one (3a,b)-have been previously characterized as products of the reaction of acrolein with dGuo and DNA. Adduct 1 predominates in certain model lipid peroxidation systems [Pan, J., and Chung, F. L. (2002) Chem. Res. Toxicol. 15, 367-372]. We hypothesized that this could be due to stepwise reactions of formaldehyde and acetaldehyde with dGuo, rather than by reaction of acrolein with dGuo. The results demonstrated that adducts 1 and 3a,b were relatively minor products of the reaction of formaldehyde and acetaldehyde with dGuo and that there was no selectivity in their formation. These findings did not support our hypothesis. However, substantial amounts of previously unknown cyclic dGuo adducts were identified in this reaction. The new adducts were characterized by their MS, UV, and NMR spectra as diastereomers of 3-(2'-deoxyribosyl)-6-methyl-1,3,5-diazinan[4,5-a]purin-10(3H)-one (10a,b). Adducts 10a,b were apparently formed by addition of formaldehyde to N1 of N(2)-ethylidene-dGuo, followed by cyclization. An analogous set of four diastereomers of 3-(2'-deoxyribosyl)-6,8-dimethyl-1,3,5-diazinan[4,5-a]purin-10(3H)-one (12a-d) were formed in the reactions of acetaldehyde with dGuo. These products are the first examples of exocyclic dGuo adducts of the pyrimido[1,2-a]purine type in which an oxygen atom is incorporated into the exocyclic ring. Formaldehyde-derived adducts were the other major products of the reactions of formaldehyde plus acetaldehyde with dGuo. Prominent among these were N(2)-hydroxymethyl-dGuo (9) and the cross-link di-(N(2)-deoxyguaonosyl)methane (13). We did not detect adducts 1, 3a,b, or 10a,b in enzymatic hydrolysates of DNA that had been allowed to react with formaldehyde plus acetaldehyde. However, we did detect substantial amounts of the formaldehyde cross-links di-(N(6)-deoxyadenosyl)methane (17), with lesser quantities of (N(6)-deoxyadenosyl-N(2)-deoxyguanosyl)methane (18), di-(N(2)-deoxyguanosyl)methane (13), and N(6)-hydroxymethyl-dAdo (19). Schiff base adducts of formaldehyde and acetaldehyde were also detected in these reactions. These results demonstrate that the reactions of formaldehyde plus acetaldehyde with dGuo are dominated by newly identified cyclic adducts and formaldehyde-derived products whereas the reactions with DNA result in the formation of formaldehyde cross-link adducts. The carcinogens formaldehdye and acetaldehyde occur in considerable quantities in the human body and in the environment. Therefore, further research is required to determine whether the adducts described here are formed in animals or humans exposed to these agents.