A machine learning-based gene signature of response to the novel alkylating agent LP-184 distinguishes its potential tumor indications.

BACKGROUND: Non-targeted cytotoxics with anticancer activity are often developed through preclinical stages using response criteria observed in cell lines and xenografts. A panel of the NCI-60 cell lines is frequently the first line to define tumor types that are optimally responsive. Open data on the gene expression of the NCI-60 cell lines, provides a unique opportunity to add another dimension to the preclinical development of such drugs by interrogating correlations with gene expression patterns. Machine learning can be used to reduce the complexity of whole genome gene expression patterns to derive manageable signatures of response. Application of machine learning in early phases of preclinical development is likely to allow a better positioning and ultimate clinical success of molecules. LP-184 is a highly potent novel alkylating agent where the preclinical development is being guided by a dedicated machine learning-derived response signature. We show the feasibility and the accuracy of such a signature of response by accurately predicting the response to LP-184 validated using wet lab derived IC50s on a panel of cell lines. RESULTS: We applied our proprietary RADR® platform to an NCI-60 discovery dataset encompassing LP-184 IC50s and publicly available gene expression data. We used multiple feature selection layers followed by the XGBoost regression model and reduced the complexity of 20,000 gene expression values to generate a 16-gene signature leading to the identification of a set of predictive candidate biomarkers which form an LP-184 response gene signature. We further validated this signature and predicted response to an additional panel of cell lines. Considering fold change differences and correlation between actual and predicted LP-184 IC50 values as validation performance measures, we obtained 86% accuracy at four-fold cut-off, and a strong (r = 0.70) and significant (p value 1.36e-06) correlation between actual and predicted LP-184 sensitivity. In agreement with the perceived mechanism of action of LP-184, PTGR1 emerged as the top weighted gene. CONCLUSION: Integration of a machine learning-derived signature of response with in vitro assessment of LP-184 efficacy facilitated the derivation of manageable yet robust biomarkers which can be used to predict drug sensitivity with high accuracy and clinical value.

The acylfulvene alkylating agent, LP-184, retains nanomolar potency in non-small cell lung cancer carrying otherwise therapy-refractory mutations.

More than 40% of non-small cell lung cancer (NSCLC) patients lack actionable targets and require non-targeted chemotherapeutics. Many become refractory to drugs due to underlying resistance-associated mutations. KEAP1 mutant NSCLCs further activate NRF2 and upregulate its client PTGR1. LP-184, a novel alkylating agent belonging to the acylfulvene class is a prodrug dependent upon PTGR1. We hypothesized that NSCLC with KEAP1 mutations would continue to remain sensitive to LP-184. LP-184 demonstrated highly potent anticancer activity both in primary NSCLC cell lines and in those originating from brain metastases of primary lung cancers. LP-184 activity correlated with PTGR1 transcript levels but was independent of mutations in key oncogenes (KRAS and KEAP1) and tumor suppressors (TP53 and STK11). LP-184 was orders of magnitude more potent in vitro than cisplatin and pemetrexed. Correlative analyses of sensitivity with cell line gene expression patterns indicated that alterations in NRF2, MET, EGFR and BRAF consistently modulated LP-184 sensitivity. These correlations were then extended to TCGA analysis of 517 lung adenocarcinoma patients, out of which 35% showed elevated PTGR1, and 40% of those further displayed statistically significant co-occurrence of KEAP1 mutations. The gene correlates of LP-184 sensitivity allow additional personalization of therapeutic options for future treatment of NSCLC.

Merkel cell carcinoma subgroups by Merkel cell polyomavirus DNA relative abundance and oncogene expression.

Merkel cell polyomavirus (MCPyV) was recently discovered in Merkel cell carcinoma (MCC), a clinically and pathologically heterogeneous malignancy of dermal neuroendocrine cells. To investigate this heterogeneity, we developed a tissue microarray (TMA) to characterize immunohistochemical staining of candidate tumor cell proteins and a quantitative PCR assay to detect MCPyV and measure viral loads. MCPyV was detected in 19 of 23 (74%) primary MCC tumors, but 8 of these had less than 1 viral copy per 300 cells. Viral abundance of 0.06-1.2 viral copies/cell was directly related to presence of retinoblastoma gene product (pRb) and terminal deoxyribonucleotidyl transferase (TdT) by immunohistochemical staining (p < or = 0.003). Higher viral abundance tumors tended to be associated with less p53 expression, younger age at diagnosis and longer survival (p < or = 0.08). These data suggest that MCC may arise through different oncogenic pathways, including ones independent of pRb and MCPyV.

Dopamine D2 receptor polymorphisms and adenoma recurrence in the Polyp Prevention Trial.

Epidemiological evidence suggests that obesity may be causally associated with colorectal cancer. Dopamine and the dopaminergic reward pathway have been implicated in drug and alcohol addiction as well as obesity. Polymorphisms within the D2 dopamine receptor gene (DRD2) have been shown to be associated with colorectal cancer risk. We investigated the association between DRD2 genotype at these loci and the risk of colorectal adenoma recurrence in the Polyp Prevention Trial. Odds ratios (OR) and 95% confidence intervals (CI) for risk of adenoma recurrence were calculated using unconditional logistic regression. Individuals with any, multiple (>or=2) or advanced adenoma recurrence after 4 years were compared to those without adenoma recurrence. Variation in intake of certain dietary components according to DRD2 genotype at 3 loci (rs1799732; rs6277; rs1800497) was also investigated. The DRD2 rs1799732 CT genotype was significantly associated with all adenoma recurrence (OR: 1.30; 95% CI: 1.01, 1.69). The rs1800497 TT genotype was also associated with a significantly increased risk of advanced adenoma recurrence (OR: 2.40; 95% CI: 1.11, 5.20). The rs1799732 CT and rs1800497 TT genotypes were significantly associated with adenoma recurrence in the Polyp Prevention Trial. Increased risk of adenoma recurrence as conferred by DRD2 genotypes may be related to difference in alcohol and fat intake across genotypes.

CYP3A43 Pro(340)Ala polymorphism and prostate cancer risk in African Americans and Caucasians.

The human cytochrome P450 3A subfamily of enzymes is involved in the metabolism of steroid hormones, carcinogens, and many drugs. A cytosine-to-guanine polymorphism in CYP3A43 results in a proline-to-alanine substitution at codon 340. Although the functional significance of this polymorphism is unknown, we postulate that the substitution of proline, an alpha-imino acid, with alanine, an amino acid, could be of biochemical significance. In a case-control study with 490 incident prostate cancer cases (124 African Americans and 358 Caucasians) and 494 controls (167 African Americans and 319 Caucasians), we examined the association between CYP3A43 Pro(340)Ala polymorphism and prostate cancer risk. When all subjects were considered, there was a 3-fold increase in risk of prostate cancer among individuals with the CYP3A43-Ala/Ala genotype (odds ratio, 3.0; 95% confidence interval, 1.2-7.2) compared with those with the CYP3A43-Pro/Pro genotype after adjusting for age, race, and smoking. The prevalence of the polymorphism was significantly higher in African Americans than Caucasians (45% versus 13%). In African Americans, there was a 2.6-fold increase in prostate cancer risk among individuals with the CYP3A43-Ala/Ala genotype (odds ratio, 2.6; 95% confidence interval, 1.0-7.0) compared with those with the CYP3A43-Pro/Pro genotype. Among Caucasians, the small number of homozygotes precluded computing risk estimates; there were only three individuals with the CYP3A43-Ala/Ala genotype. Our results suggest that the CYP3A43-Pro(340)Ala polymorphism contributes to prostate cancer risk.

Polymorphisms of XRCC1 and risk of esophageal and gastric cardia cancer.

BACKGROUND: Linxian, a rural county in North Central China, has among the highest rates of esophageal squamous cell carcinoma and gastric cardia adenocarcinoma in the world. In a nested case-cohort study that originated from two cancer prevention trials in Linxian, we examined the relationship between these cancers and two polymorphisms in the DNA repair gene XRCC1. METHODS: We conducted a case-cohort study among individuals in the cohort who were alive and cancer free in 1991, and had blood samples for DNA extraction. Real time Taqman analyses were conducted to genotype incident cancer cases (n = 221, 131 esophageal and 90 gastric cardia cancer cases) that developed through May 1996, and on an age- and sex-matched reference cohort (n = 454). We used Cox proportional hazard models to estimate relative risks (RR) and 95% confidence intervals (95% CI). RESULTS: We observed no association between the variant genotype in XRCC1 Arg194Trp (codon 194 arganine to tryptophan substitution) and esophageal or gastric cardia cancer. However, carrying at least one copy of the variant allele in XRCC1 Arg399Gln (codon 399 arganine to glutamine substitution) was associated with reduced risk of gastric cardia cancer (RR: 0.60, 95% CI: 0.37-0.97) and the combined category esophageal/gastric cancer (RR: 0.67, 95% CI: 0.48-0.95). In combined polymorphisms analyses, we observed a significant reduction in risk of combined esophageal/gastric cancer among individuals that had both the XRCC1 Arg194Trp and Arg399Gln variant genotyopes (RR: 0.47, 95% CI: 0.26-0.84). CONCLUSIONS: Our results suggest that the XRCC1 Arg399Gln variant genotype is associated with reduced risk of upper GI cancer and that individuals with both XRCC1 variant genotypes are also at significantly reduced risk of upper GI cancer in this high-risk Chinese population.

Effect modification by transferrin C2 polymorphism on lead exposure, hemoglobin levels, and IQ.

BACKGROUND: Iron deficiency and lead exposure remain significant public health issues in many parts of the world and are both independently associated with neurocognitive deficits. Polymorphisms in iron transport pathways have been shown to modify the absorption and toxicity of lead. OBJECTIVE: We hypothesized that the transferrin (TF) C2 polymorphism modifies the effects of lead and hemoglobin on intelligence. METHODS: Children aged 3-7 years (N=708) were enrolled from 12 primary schools in Chennai, India. The Binet-Kamath Scale of Intelligence were administered to ascertain intelligence quotient (IQ). Venous blood was analyzed for lead and hemoglobin levels. Genotyping for the TF C2 polymorphism (rs1049296) was carried out using a MassARRAY iPLEXTM platform. Stratified analyses and interaction models, using generalized estimating equations, were examined to explore interactions between lead, hemoglobin, and TF C2 categories. RESULTS: A one-unit increase in log blood lead and 1g/dl higher hemoglobin was associated with -77 (95% CI: -136, -18) and 17 (95% CI 14, 21) IQ points, respectively, among children carrying the C2 variant. In comparison, among children who had the homozygous wildtype allele, the same increment of lead and hemoglobin were associated with -21(95% CI: -65, 24) and 28 (95% CI: 15, 40) IQ points, respectively. There was a significant interaction between lead (p=0.04) and hemoglobin (p=0.07) with the C2 variant. CONCLUSION: Children who carry the TF C2 variant may be more susceptible to the neurotoxic effects of lead exposure and less protected by higher levels of hemoglobin.

Hemoglobin, lead exposure, and intelligence quotient: effect modification by the DRD2 Taq IA polymorphism.

BACKGROUND: Anemia and lead exposure remain significant public health issues in many parts of the world, often occurring together. Animal studies suggest that the dopamine D2 receptor (DRD2) mediates the effects of both lead and iron on cognition and behavior. OBJECTIVE: We tested the hypothesis that the DRD2 Taq IA polymorphism modifies the effects of lead and hemoglobin on intelligence quotient (IQ) among children. METHODS: Blood lead and hemoglobin were assessed in 717 children 3-7 years of age attending 12 schools in Chennai, India. IQ was determined using the Binet-Kamat scales of intelligence. Genotyping for the DRD2 polymorphism was carried out using a MassARRAY iPLEX platform. Stratified analyses and interaction models, using generalized estimating equations (GEEs), were used to explore interactions between lead and hemoglobin, and DRD2 Taq IA categories [homozygous variant (A1) vs. presence of wild-type allele (A2)]. RESULTS: After we controlled for potential confounders, a one-unit increase in log blood lead was associated with a decrease of 9 IQ points [95% confidence interval (CI), -18.08 to -0.16] in the homozygous-variant children (n = 73) compared with a decrease of 4 IQ points (95% CI, -7.21 to -0.69) among those with the wild-type allele (n = 644). Higher hemoglobin levels were associated with higher IQ in the children who carried the wild-type allele DRD2, but in children homozygous for the variant allele, an increase of 1 g/dL hemoglobin was associated with a decrease in 1.82 points of IQ (95% CI, -5.28 to 1.64; interaction term p-value = 0.02). CONCLUSION: The results of this study suggest that the DRD2 Taq IA polymorphism disrupts the protective effect of hemoglobin on cognition and may increase the susceptibility to the deficits in IQ due to lead exposure.

Merkel cell polyomavirus is not detected in mesotheliomas.

BACKGROUND: Merkel cell polyomavirus (MCPyV) is the first polyoma virus consistently linked to the etiology of a human cancer. Serological studies indicate that the virus is commonly acquired in childhood, with seroprevalence reaching 50% or higher among young adults. The modes of MCPyV transmission are still unclear, but it has been identified in respiratory tract samples. Given its respiratory tropism, we examined whether MCPyV could be detected in mesothelioma tissue, a malignancy induced in animal models by another polyomavirus, SV40. OBJECTIVE: To determine if MCPyV DNA can be detected in mesothelioma. STUDY DESIGN: DNA was extracted from 45 fresh-frozen mesothelioma samples. PCR was used to detect and quantify the abundance of MCPyV DNA, and a human control gene, in duplicates of the tissues. DNA from a sequence verified MCC tumor was used as a positive control. RESULTS: The human control gene was detected at high levels in all but three mesothelioma tissues. MCPyV DNA was detected in only one mesothelioma, and the level of viral DNA was very low. CONCLUSIONS: These results are inconsistent with the hypothesis that MCPyV is etiologically linked to mesothelioma.

Polynucleotide kinase 3' phosphatase variant, dietary variables and risk of adenoma recurrence in the Polyp Prevention Trial.

Polymorphisms in a number of genes encoding for DNA repair enzymes have been associated with altering the function of these enzymes and increasing risk of a number of cancers, including colon cancer. We have investigated the association between a common variant in polynucleotide kinase 3' phosphatase (PNKP), a putative DNA repair enzyme, and risk of adenoma recurrence in the Polyp Prevention Trial participants. We also investigated possible interaction or effect modification between carriage of the variant allele, dietary components and risk of adenoma recurrence. Unconditional logistic regression models were used to calculate the odds ratios and 95% confidence intervals for an association between the G/T polymorphism, PNKP T5644G and risk of adenoma recurrence. We observed no association between carriage of the variant allele and risk of adenoma recurrence. Furthermore, we found no effect modification between genotype, dietary components and risk of adenoma recurrence. The PNKP T5644G variant does not seem to be involved in adenoma recurrence in the Polyp Prevention Trial.

Folate and MTHFR: risk of adenoma recurrence in the Polyp Prevention Trial.

BACKGROUND: Low dietary folate intake has been associated with colorectal cancer risk and adenoma recurrence. A C/T transition at position 677 in the gene encoding methlylenetetrahydrofolate reductase (MTHFR C677T) has been reported to interact with folate intake to modulate colorectal adenoma recurrence or cancer risk. METHODS: We investigated the association between MTHFR, total folate, and the risk of colorectal adenoma recurrence in the Polyp Prevention Trial. We compared 625 individuals with any adenoma recurrence after 4 years (266 individuals with multiple (> or =2) recurrent adenomas and 101 individuals with advanced adenoma recurrence) to 978 individuals with no adenoma recurrence. Odds ratios (OR) and 95% confidence intervals (CI) for risk of adenoma recurrence were calculated using unconditional logistic regression. We also investigated effect modification of the MTHFR genotype associations by total folate intake. RESULTS: In general, no statistically significant associations were found between quartile of folate intake (dietary or total) and adenoma recurrence. The MTHFR CT genotype was associated with a significantly increased risk of multiple adenoma recurrence (OR: 1.34, 95% CI: 1.00, 1.81). No significant interaction was noted for total folate and MTHFR genotype, though an increased risk of recurrence noted for the MTHFR CT genotype was statistically significant only for those individuals with below median intake of total folate. CONCLUSION: We report that the MTHFR 677 CT genotype was associated with increased risk of adenoma recurrence (specifically multiple adenoma recurrence) 4 years after polypectomy.

FCGR3A and FCGR2A polymorphisms may not correlate with response to alemtuzumab in chronic lymphocytic leukemia.

The in vivo mechanism of action of alemtuzumab (anti-CD52; Campath-1H) remains unclear. With rituximab, FCGR3A and FCGR2A high-affinity polymorphisms have been associated with clinical response in lymphoma but not in CLL, suggesting potential divergent mechanisms of action between these 2 diseases. Herein, we examined FCGR3A (V/V, n = 4; V/F, n = 10; F/F, n = 19) and FCGR2A (A/A, n = 5; H/A, n = 22; H/H, n = 6) polymorphisms in 36 patients with relapsed CLL who were treated with thrice-weekly alemtuzumab for 12 weeks to assess the potential influence these high-affinity FcgammaR receptor polymorphisms had on response to alemtuzumab. Response to alemtuzumab was similar regardless of FCGR3A polymorphism (V/V, 25%; V/F, 40%; F/F, 32%) or FCGR2A polymorphism (A/A, 40%; H/A, 32%; H/H, 33%). These findings indicate that FCGR3A and FCGR2A polymorphisms may not predict response to alemtuzumab in CLL. Future studies examining larger cohorts of alemtuzumab-treated patients with CLL will be required to definitively determine the predictive value of specific FCGR polymorphisms to treatment response.

Fc gamma RIIIa and Fc gamma RIIa polymorphisms do not predict response to rituximab in B-cell chronic lymphocytic leukemia.

In follicular lymphoma (FL), genomic polymorphisms corresponding to the expression of valine (V) or phenylalanine (F) at amino acid 158 of Fc gamma RIIIa alter the binding affinity of immunoglobulin G1 (IgG1) to the receptor and have been associated with varied responses to rituximab. We examined Fc gamma RIIIa polymorphisms of 30 CLL patients with the phenotypes V/V (n = 6), V/F (n = 12), and F/F (n = 12) treated with thrice-weekly rituximab (375 mg/m(2)) for 4 weeks to correlate polymorphism type with infusion toxicity and response. Infusion toxicity (grade 3 or greater or hypoxia/hypotension requiring transient cessation of therapy) was observed equally among the groups (V/V, 50%; V/F, 33%; F/F, 41.6%; P =.78). The response to rituximab was also similar among the different polymorphism phenotypes (V/V, 33%; V/F, 41.6%; F/F, 50%). These data suggest that Fc gamma RIIIa polymorphisms are not predictive of response in CLL and that, unlike the case with FL, mechanisms of tumor clearance other than antibody-dependent cellular cytotoxicity may be more important.