The aim of this prospective investigation was to assess the association of parameters derived from baseline 18F-FDG PET/CT with overall survival (OS) in men with castrate-resistant metastatic prostate cancer.
Eighty-seven men with castrate-resistant metastatic prostate cancer underwent 18F-FDG PET/CT and were followed prospectively for OS. Median follow-up in patients who were alive was 22.2 mo (range, 1.6–62.5 mo). OS was defined as the time between the PET/CT imaging or the start of chemotherapy, whichever was later, and death, with patients who were alive censored at the last follow-up date. PET parameters included maximum standardized uptake value (SUVmax) of the most active lesion, sum of SUVmax, and average SUVmax of all metabolically active lesions, after subtraction of patient-specific background-liver average SUV. Comparison of OS was based on univariate and multivariable Cox regression analyses of continuous PET parameters adjusted for standard clinical parameters (age, serum prostate-specific antigen level, alkaline phosphatase, use of pain medication, prior chemotherapy, and Gleason score at initial diagnosis). Survival curves based on Kaplan–Meier estimates are presented.
Among the 87 patients, 61 were dead at the time of last follow-up. Median OS was 16.5 mo (95% confidence interval [CI], 12.1–23.4 mo), and the OS probability at 24 mo was 39% ± 6%. For the univariate analysis, the hazard ratios associated with each unit increase were 1.01 (95% CI, 1.006–1.02) for sum of SUVmax (P = 0.002), 1.11 (95% CI, 1.03–1.18) for maximum SUVmax (P = 0.010), and 1.13 (95% CI, 0.99–1.30) for average SUVmax (P = 0.095). For the multivariable analysis adjusting for relevant clinical parameters, the continuous parameter sum of SUVmax remained significant (P = 0.053), with a hazard ratio of 1.01 (95% CI, 1.001–1.02). When sum of SUVmax was grouped into quartile ranges, there was poorer survival probability for the patients in the fourth-quartile range than for those in the first-quartile range, with a univariate hazard ratio of 3.8 (95% CI, 1.8–7.9).
Sum of SUVmax derived from 18F-FDG PET/CT contributes independent prognostic information on OS in men with castrate-resistant metastatic prostate cancer, and this information may be useful in assessing the comparative effectiveness of various conventional and emerging treatment strategies.
18F-FDG; prostate; cancer; castrate-resistant; survival
Eribulin mesylate (E7389) is an analog of halichondrin B with a unique mechanism of microtubule binding. The activity and toxicity of eribulin were assessed in patients with advanced non-small cell lung cancer (NSCLC) previously treated with a taxane.
An open-label phase II study included patients with NSCLC previously treated with platinum and taxane-based therapy, with up to two prior cytotoxic regimens, given for metastatic disease or as adjuvant therapy. Patients were stratified by taxane-sensitivity: taxane-sensitive (TS, progression > 90 days after taxane) or taxane-resistant (TR, progression ≤ 90 days after taxane). Patients received an intravenous infusion of eribulin at 1.4 mg/m2 on days 1 and 8 every 21 days. The primary endpoint was objective response rate (ORR) and secondary endpoints included progression-free survival (PFS) and overall survival (OS).
Sixty-six patients were accrued. The ORR was 5% with a median duration of response of 7.8 months. In the TS arm, 3 out of 45 patients (7%) achieved a partial response (PR) and another 11 out of 45 (24%) achieved stable disease (SD) for at least 3 months, whereas in the TR arm, no patients achieved a PR and 4 out of 21 (19%) achieved SD for at least 3 months. Median PFS was 2.9 months in the TS subgroup and 1.2 months in the TR subgroup. The median OS was 12.6 months in the TS subgroup and 8.9 months in the TR subgroup. Toxicities were primarily hematologic; only two patients developed grade 3 neuropathy.
Eribulin mesylate is well tolerated and demonstrates activity in pre-treated, taxane-sensitive NSCLC.
Halichondrin B; Eribulin Mesylate; Non-Small Cell Lung Cancer; Taxane-Refractory; Taxane-Sensitive
Retrospective studies suggest that p53 alteration is prognostic for recurrence in patients with urothelial bladder cancer and predictive for benefit from combination methotrexate, vinblastine, doxorubicin, and cisplatin (MVAC) adjuvant chemotherapy.
Patients and Methods
Patients with pT1/T2N0M0 disease whose tumors demonstrated ≥ 10% nuclear reactivity on centrally performed immunohistochemistry for p53 were offered random assignment to three cycles of adjuvant MVAC versus observation; p53-negative patients were observed. By using a log-rank test with one-sided α = .05 and β = .10, 190 p53-positive patients were planned to be randomly assigned to detect an absolute improvement in probability of recurring by 3 years from 0.50 to 0.30.
A total of 521 patients were registered, 499 underwent p53 assessment, 272 (55%) were positive, and 114 (42%) were randomly assigned. Accrual was halted on the basis of the data and safety monitoring board review of a futility analysis. Overall 5-year probability of recurring was 0.20 (95% CI, 0.16 to 0.24) with no difference on the basis of p53 status. Only 67% of patients randomly assigned to MVAC received all three cycles with 12 patients receiving no treatment. There was no difference in recurrence in the randomly assigned patients (hazard ratio, 0.78; 95% CI, 0.29 to 2.08; P = .62).
Neither the prognostic value of p53 nor the benefit of MVAC chemotherapy in patients with p53-positive tumors was confirmed, but the high patient refusal rate, lower than expected event rate, and failures to receive assigned therapy severely compromised study power.
Zoledronic acid, a bisphosphonate, delays progression of bone metastases in adult malignancies. Bone is a common metastatic site of advanced neuroblastoma. We previously reported efficacy of zoledronic acid in a murine model of neuroblastoma bone invasion prompting this Phase I trial of zoledronic acid with cyclophosphamide in children with neuroblastoma and bone metastases. The primary objective was to determine recommended dosing of zoledronic acid for future trials.
Escalating doses of intravenous zoledronic acid were given every 28 days with oral metronomic cyclophosphamide (25 mg/m2/day). Toxicity, response, zoledronic acid pharmacokinetics, bone turnover markers, serum IL-6, and sIL-6R were evaluated.
Twenty-one patients, median age 7.5 (range 0.8 - 25.6) years were treated with 2 mg/m2 (n=4), 3 mg/m2 (n=3), or 4 mg/m2 (n=14) zoledronic acid. Fourteen patients were evaluable for dose escalation. A median of one (range 1-18) courses was given. Two dose limiting toxicities (Grade 3 hypophosphatemia) occurred at 4 mg/m2 zoledronic acid. Other Grade 3-4 toxicities included hypocalcemia (n=2), elevated transaminases (n=1), neutropenia (n=2), anemia (n=1), lymphopenia (n=1), and hypokalemia (n=1). Osteosclerosis contributed to fractures in one patient after 18 courses. Responses in evaluable patients included 1 partial response, 9 stable disease (median 4.5 courses, range 3-18), and 10 progressions. Zoledronic acid pharmacokinetics were similar to adults. Markers of osteoclast activity and serum IL-6 levels decreased with therapy.
Zoledronic acid with metronomic cyclophosphamide is well tolerated with clinical and biologic responses in recurrent/refractory neuroblastoma. The recommended dose of zoledronic acid is 4 mg/m2 every 28 days.
Phase I; neuroblastoma; bisphosphonate
This study aimed to perform a prospective evaluation of 18F-NaF and 18F-FDG PET/CT in the detection of occult metastatic disease in men with prostate cancer and biochemical relapse.
Thirty-seven men with prostate-specific antigen (PSA) relapse (median, 3.2 ng/mL; range, 0.5–40.2 ng/mL) after definitive therapy for localized prostate cancer [26 radical prostatectomy (RP), 11 external beam radiation therapy] and negative conventional imaging underwent 18F-FDG and 18F-NaF PET/CT on 2 separate days within the same week. Studies were interpreted by 2 experienced radiologists in consensus for abnormal uptake suspicious for metastatic disease. The reference standard was a combination of imaging and clinical follow-up. Rank of PSA values for positive and negative PET/CT was compared using analysis of variance adjusting for primary therapy. Association between PSA and scan positivity in patients with RP was evaluated using Wilcoxon rank sum test.
Result of the 18F-FDG PET/CT scan was positive for nodal disease in 2 patients. True-positive detection rate for occult osseous metastases by 18F-NaF PET/CT was 16.2%. Median PSA levels for positive versus negative PET/CT scans were 4.4 and 2.9 ng/mL, respectively, with the difference marginally significant in prostatectomized men (P = 0.072). Percentages of patients with either 18F-NaF– or 18F-FDG–positive PET/CT in RP and external beam radiation therapy were 10% (n = 10) and undefined (n = 0) for a PSA of 2 ng/mL or less, 29% (n = 7) and 50% (n = 2) for PSA greater than 2 ng/mL but 4 ng/mL or less,60% (n = 5) and 40%(n = 5) for PSA greater than 4 ng/mL but 10 ng/mL or less, and 25% (n = 4) and 25% (n = 4) for PSA greater than 10 ng/mL, respectively.
In biochemical relapse of prostate cancer, 18F-NaF PET/CT is useful in the detection of occult osseous metastases, whereas the yield of 18F-FDG PET/CT is relatively limited. 18F-NaF PET/CT positivity tends to associate with increasing PSA level in prostatectomized men and may occur in lower PSA ranges than conventionally recognized.
18F-NaF; 18F-FDG; prostate; cancer; PSA
Regions in the 8q24 gene desert contribute significantly to the risk of prostate cancer and other adult cancers. This region contains several DNA regions with enhancer activity in cultured cells. One such segment, histone acetylation peak 10 (AcP10), contains a risk single nucleotide polymorphism (SNP) that is significantly associated with the pathogenesis of colorectal, prostate and other cancers. The mechanism by which AcP10 influences cancer risk remains unknown. Here we show that AcP10 contains a sequence that is highly conserved across terrestrial vertebrates and is capable in transgenic mice of directing reporter gene expression to a subset of prostate lumenal epithelial cells. These cells include a small population of Nkx3.1-positive cells that persist even after androgen ablation. Castration-resistant Nkx3.1-positive (CARN) cells were shown by others to function both as stem cells and cells of origin of prostate cancer. Our results thus provide a mechanism by which AcP10 could influence prostate cancer risk.
GRP78/BiP is a multifunctional protein which plays a major role in endoplasmic reticulum (ER) protein processing, protein quality control, maintaining ER homeostasis and controlling cell signaling and viability. Previously, using a transgene-induced mammary tumor model, we demonstrated that Grp78 heterozygosity not only impeded cancer growth through suppression of tumor cell proliferation and promotion of apoptosis, the Grp78+/− mice exhibited dramatic reduction (70%) in the microvessel density (MVD) of the endogenous mammary tumors while having no effect on the MVD of normal organs. This observation suggests that GRP78 may critically regulate the function of the host vasculature within the tumor microenvironment. In this report, we interrogated the role of GRP78 in the tumor microenvironment. In mouse tumor models where wild-type, syngeneic mammary tumor cells were injected into the host, we showed that Grp78+/− mice suppressed tumor growth and angiogenesis during the early but not late phase of tumor growth. Growth of metastatic lesions of wild-type, syngeneic melanoma cells in the Grp78+/− mice was potently suppressed. We created conditional heterozygous knockout of GRP78 in the host endothelial cells and demonstrated severe reduction of tumor angiogenesis and metastatic growth with minimal effect on normal tissue MVD. Furthermore, knockdown of GRP78 expression in immortalized human endothelial cells demonstrated that GRP78 is a critical mediator of angiogenesis by regulating cell proliferation, survival, and migration. Our findings suggest that concomitant use of current chemotherapeutic agents and novel therapies against GRP78 may offer a powerful dual approach to arrest cancer initiation, progression and metastasis.
GRP78; tumor angiogenesis; conditional knockout; mouse model; metastatic growth; endothelial cells
Few predictive markers exist for response to adjuvant chemotherapy in breast cancer. The 78-kD glucose-regulated protein (GRP78) is a potent anti-apoptotic factor, conferring drug resistance. Recently, we reported that high GRP78 expression in breast cancer specimens predict a shorter recurrence-free survival in patients who received doxorubicin-based adjuvant chemotherapy. Interestingly, the opposite effect was observed in 25 patients who additionally received a taxane. To confirm this potentially paradigm shifting finding, we investigated whether GRP78 is associated with recurrence-free survival in an independent cohort of taxane-treated breast cancer patients. Immunohistochemical staining of GRP78 was performed on archival paraffin-embedded formalin-fixed tumor specimens obtained from 48 female breast cancer patients before chemotherapy treatment. These patients received doxorubicin and cyclophosphamide, followed by paclitaxel or docetaxel on a clinical trial. GRP78 expression level was evaluated by a pathologist, masked to all clinical and outcome data. Association between GRP78 expression and recurrence-free survival was evaluated. GRP78 positivity predicts a better recurrence-free survival, independent of other prognostic factors [hazard ratio (HR) for moderate positivity: 0.40 (95% confidence interval (CI): 0.087–1.83); HR for strong positivity: 0.16 (95% CI: 0.018–1.50); Ptrend=0.053]. In a pooled analysis with the previous 25 patients, almost identical HRs were obtained with Ptrend=0.024. This provides further evidence that GRP78 is a potential independent predictor for response to taxane-based adjuvant chemotherapy in breast cancer.
131I-Metaiodobenzylguanidine (131I-MIBG) provides targeted radiotherapy for children with neuroblastoma, a malignancy of the sympathetic nervous system. Dissociated radioactive iodide may concentrate in the thyroid, and MIBG is concentrated in the liver after MIBG therapy. The aim of our study was to analyze the effects of 131I-MIBG therapy on thyroid and liver function.
Pre and post therapy thyroid and liver functions were reviewed in a total of 194 neuroblastoma patients treated with 131I-MIBG therapy. The cumulative incidence over time was estimated for both thyroid and liver toxicities. The relationship to cumulative dose/kg, number of treatments, time from treatment to follow-up, sex, and patient age was examined.
In patients who presented with Grade 0 or Grade 1 thyroid toxicity at baseline, 12±4% experienced onset or worsening to Grade 2 hypothyroidism and one patient developed Grade 2 hyperthyroidism by two years after 131I-MIBG therapy. At two years post 131I-MIBG therapy, 76±4% patients experienced onset or worsening of hepatic toxicity to any grade, and 23±5% experienced onset of or worsening to Grade 3 or 4 liver toxicity. Liver toxicity usually was transient asymptomatic transaminase elevation, frequently confounded by disease progression and other therapies.
The prophylactic regimen of potassium iodide and potassium perchlorate with 131I-MIBG therapy resulted in a low rate of significant hypothyroidism. Liver abnormalities following 131I-MIBG therapy were primarily reversible and did not result in late toxicity. 131I-MIBG therapy is a promising treatment for children with relapsed neuroblastoma with a relatively low rate of symptomatic thyroid or hepatic dysfunction.
Neuroblastoma; 131I-MIBG; Hypothyroidism
The identification of sensitive biomarkers for the detection of ovarian cancer is of high clinical relevance for early detection and/or monitoring of disease recurrence. We developed a systematic multi-step biomarker discovery and verification strategy to identify candidate DNA methylation markers for the blood-based detection of ovarian cancer.
We used the Illumina Infinium platform to analyze the DNA methylation status of 27,578 CpG sites in 41 ovarian tumors. We employed a marker selection strategy that emphasized sensitivity by requiring consistency of methylation across tumors, while achieving specificity by excluding markers with methylation in control leukocyte or serum DNA. Our verification strategy involved testing the ability of identified markers to monitor disease burden in serially collected serum samples from ovarian cancer patients who had undergone surgical tumor resection compared to CA-125 levels.
We identified one marker, IFFO1 promoter methylation (IFFO1-M), that is frequently methylated in ovarian tumors and that is rarely detected in the blood of normal controls. When tested in 127 serially collected sera from ovarian cancer patients, IFFO1-M showed post-resection kinetics significantly correlated with serum CA-125 measurements in six out of 16 patients.
We implemented an effective marker screening and verification strategy, leading to the identification of IFFO1-M as a blood-based candidate marker for sensitive detection of ovarian cancer. Serum levels of IFFO1-M displayed post-resection kinetics consistent with a reflection of disease burden. We anticipate that IFFO1-M and other candidate markers emerging from this marker development pipeline may provide disease detection capabilities that complement existing biomarkers.
The aim of this study is to investigate the relationships between hypertension, hypertension medication and bladder cancer risk in a population-based case–control study conducted in Los Angeles. Non-Asians between the ages of 25 and 64 years with histologically confirmed bladder cancers diagnosed between 1987 and 1996 were identified through the Los Angeles County Cancer Surveillance Program. A total of 1585 cases and their age-, gender- and race-matched neighborhood controls were included in the analyses. Conditional logistic regression models were used to examine the relationship between history of hypertension, medication use and bladder cancer risk. A history of hypertension was not related to bladder cancer; however, among hypertensive individuals, there was a significant difference in bladder cancer risk related to the use of diuretics or antihypertensive drugs (P for heterogeneity = 0.004). Compared with individuals without hypertension, hypertensive individuals who regularly used diuretics/antihypertensives had a similar risk [odds ratio (OR) 1.06; 95% confidence interval (CI) 0.86–1.30], whereas untreated hypertensive subjects had a 35% reduction in risk (OR: 0.65; 95% CI: 0.48–0.88). A greater reduction in bladder cancer risk was observed among current-smokers (OR: 0.43; 95% CI: 0.27–0.71) and carriers of GSTM1-null (homozygous absence) genotypes (OR: 0.43; 95% CI: 0.22–0.85). Similarly, among smokers with GSTM1-null genotype, levels of 4-aminobiphenyl-hemoglobin adducts were significantly lower among untreated hypertensive individuals (45.7 pg/g Hb) compared with individuals without hypertension (79.8 pg/g Hb) (P = 0.009). In conclusion, untreated hypertension was associated with a reduced risk of bladder cancer.
Sensitive detection and characterization of circulating tumor cell (CTC) could revolutionize the approach to patients with early stage and metastatic cancer. The current methodologies have significant limitations including limited capture efficiency and ability to characterize captured cells. Here, we report the development of a novel parylene membrane filter-based portable microdevice for size-based isolation with high recovery rate and direct on-chip characterization of captured CTC from human peripheral blood.
We evaluated the sensitivity and efficiency of CTC capture in a model system using blood samples from healthy donors spiked with tumor cell lines. 59 model system samples were tested for determining the recovery rate of the microdevice. Moreover, 10 model system samples and 57 blood samples from cancer patients were subjected to both membrane microfilter device and CellSearch® platform enumeration for direct comparison.
Using the model system, the microdevice achieved >90% recovery with probability of 95% recovering at least one cell when 5 are seeded in 7.5 ml of blood. CTCs were identified in 51 out of 57 patients using the microdevice, compared to only 26 patients with the CellSearch® method. When CTC were detected by both methods, greater numbers were recovered by the microfilter device in all but 5 patients.
This filter-based microdevice is both a capture and analysis platform, capable of multiplexed imaging and genetic analysis. The microdevice presented here has the potential to enable routine CTC analysis in clinical setting for effective management of cancer patients.
Despite the success of modern chemotherapy in the treatment of large bowel cancers, patients with metastatic gastric cancer continue to have a dismal outcome. Identifying predictive and prognostic markers is an important step to improving current treatment approaches and extending survival.
Extracting data from the US NCI's Surveillance, Epidemiology, and End Results (SEER) registries, we compared overall survival for patients with metastatic gastric cancer by gender, age, and ethnicity using Cox proportional hazards models. 13,840 patients (≥ 18 years) were identified from 1988-2004. Males and females were categorized by age grouping and ethnicity.
19% of Hispanic patients were diagnosed < 45 years of age as compared to 5.5% of Caucasians. Caucasian patients and men were more likely to be diagnosed with tumors in the gastric cardia (P<0.001). In our survival analysis, we found that women had a lower risk of dying as compared to men (P<0.001). Overall survival diminished with age (P<0.001). The median overall survival was 6 months in patients of ≤ 44 years old as compared to 3 months in patients 75 years and older. Gender differences in overall survival significantly varied by race and tumor grade/differentiation (P for interaction = 0.003 and 0.005, respectively).
This is the largest study of metastatic gastric cancer patients from the SEER registry to show that age, gender, and tumor location are significant independent prognostic factors for overall survival in patients with metastatic gastric cancer.
gastric cancer; gender; age; ethnicity; survival
The optimal design of phase II studies continues to be the subject of vigorous debate, especially with regards to studies of newer molecularly targeted agents. The observations that many new therapeutics ‘fail’ in definitive phase III studies, coupled with the numbers of new agents to be tested as well as the increasing costs and complexity of clinical trials further emphasizes the critical importance of robust and efficient phase II design.
The Clinical Trial Design Task Force(CTD-TF)of the NCI Investigational Drug Steering Committee (IDSC) has published a series of discussion papers on Phase II trial design in Clinical Cancer Research. The IDSC has developed formal recommendations regarding aspects of phase II trial design which are the subject of frequent debate such as endpoints(response vs. progression free survival), randomization(single arm designs vs. randomization), inclusion of biomarkers, biomarker based patient enrichment strategies, and statistical design(e.g. two stage designs vs. multiple-group adaptive designs).
While these recommendations in general encourage the use of progression-free survival as the primary endpoint, the use of randomization, the inclusion of biomarkers and the incorporation of newer designs, we acknowledge that objective response as an endpoint, and single arm designs, remain relevant in certain situations. The design of any clinical trial should always be carefully evaluated and justified based on the characteristic specific to the situation.
Currently, tumor grade is the best predictor of outcome at first presentation of noninvasive papillary (Ta) bladder cancer. However, reliable predictors of Ta tumor recurrence and progression for individual patients, which could optimize treatment and follow-up schedules based on specific tumor biology, are yet to be identified.
To identify genes predictive for recurrence and progression in Ta bladder cancer at first presentation using a quantitative, pathway-specific approach.
Design, setting, and participants
Retrospective study of patients with Ta G2/3 bladder tumors at initial presentation with three distinct clinical outcomes: absence of recurrence (n = 16), recurrence without progression (n = 16), and progression to carcinoma in situ or invasive disease (n = 16).
Expressions of 24 genes that feature in relevant pathways that are deregulated in bladder cancer were quantified by real-time polymerase chain reaction on tumor biopsies from the patients at initial presentation.
Results and limitations
CCND3 (p = 0.003) and HRAS (p = 0.01) were predictive for recurrence by univariate analysis. In a multivariable model based on CCND3 expression, sensitivity and specificity for recurrence were 97% and 63%, respectively. HRAS (p < 0.001), E2F1 (p = 0.017), BIRC5/Survivin (p = 0.038), and VEGFR2 (p = 0.047) were predictive for progression by univariate analysis. Multivariable analysis based on HRAS, VEGFR2, and VEGF identified progression with 81% sensitivity and 94% specificity. Since this is a small retrospective study using medium-throughput profiling, larger confirmatory studies are needed.
Gene expression profiling across relevant cancer pathways appears to be a promising approach for Ta bladder tumor outcome prediction at initial diagnosis. These results could help differentiate between patients who need aggressive versus expectant management.
Noninvasive urothelial carcinoma; First presentation; Recurrence; Progression; CCND3; HRAS; E2F1; Survivin; VEGFR2; VEGF
Children with relapsed neuroblastoma have poor survival. It is crucial to have a reliable method for evaluating functional response to new therapies. In this study, we compared two functional imaging modalities for neuroblastoma: metaiodobenzylguanidine (MIBG) scan for uptake by the norepinephrine transporter and [18F]fluorodeoxyglucose positron emission tomography (FDG-PET) uptake for glucose metabolic activity.
Patients and Methods
Patients enrolled onto a phase I study of sequential infusion of iodine-131 (131I) MIBG (NANT-2000-01) were eligible for inclusion if they had concomitant FDG-PET and MIBG scans. 131I-MIBG therapy was administered on days 0 and 14. For each patient, we compared all lesions identified on concomitant FDG-PET and MIBG scans and gave scans a semiquantitative score.
The overall concordance of positive lesions on concomitant MIBG and FDG-PET scans was 39.6% when examining the 139 unique anatomic lesions. MIBG imaging was significantly more sensitive than FDG-PET overall and for the detection of bone lesions (P < .001). There was a trend for increased sensitivity of FDG-PET for detection of soft tissue lesions. Both modalities showed similar improvement in number of lesions identified from day 0 to day 56 scan and in semiquantitative scores that correlated with overall response. FDG-PET scans became completely negative more often than MIBG scans after treatment.
MIBG scan is significantly more sensitive for individual lesion detection in relapsed neuroblastoma than FDG-PET, though FDG-PET can sometimes play a complementary role, particularly in soft tissue lesions. Complete response by FDG-PET metabolic evaluation did not always correlate with complete response by MIBG uptake.
This study sought to determine if alterations in molecular pathways could supplement TNM staging to more accurately predict clinical outcome in patients with urothelial carcinoma (UC).
Patients and Methods
Expressions of 69 genes involved in known cancer pathways were quantified on bladder specimens from 58 patients with UC (stages Ta-T4) and five normal urothelium controls. All tumor transcript values beyond two standard deviations from the normal mean expression were designated as over- or underexpressed. Univariate and multivariable analyses were conducted to obtain a predictive expression signature. A published external data set was used to confirm the potential of the prognostic gene panels.
In univariate analysis, six genes were significantly associated with time to recurrence, and 10 with overall survival. Recursive partitioning identified three genes as significant determinants for recurrence, and three for overall survival. Of all genes identified by either univariate or partitioning analysis, four were found to significantly predict both recurrence and survival (JUN, MAP2K6, STAT3, and ICAM1); overexpression was associated with worse outcome. Comparing the favorable (low or normal) expression of ≥ three of four versus ≤ two of four of these oncogenes showed 5-year recurrence probability of 41% versus 88%, respectively (P < .001), and 5-year overall survival probability of 61% versus 5%, respectively (P < .001). The prognostic potential of this four-gene panel was confirmed in a large independent external cohort (disease-specific survival, P = .039).
We have documented the generation of a concise, biologically relevant four-gene panel that significantly predicts recurrence and survival and may also identify potential therapeutic targets for UC.
Multiple clinical trials are investigating the use of the DNA methylation inhibitors azacitidine and decitabine for the treatment of solid tumors. Clinical trials in hematological malignancies have shown that optimal activity does not occur at their maximum tolerated doses but selection of an optimal biological dose and schedule for use in solid tumor patients is hampered by the difficulty of obtaining tumor tissue to measure their activity. Here we investigate the feasibility of using plasma DNA to measure the demethylating activity of the DNA methylation inhibitors in patients with solid tumors. We compared four methods to measure LINE-1 and MAGE-A1 promoter methylation in T24 and HCT116 cancer cells treated with decitabine treatment and selected Pyrosequencing for its greater reproducibility and higher signal to noise ratio. We then obtained DNA from plasma, peripheral blood mononuclear cells, buccal mucosa cells and saliva from ten patients with metastatic solid tumors at two different time points, without any intervening treatment. DNA methylation measurements were not significantly different between time point 1 and time point 2 in patient samples. We conclude that measurement of LINE-1 methylation in DNA extracted from the plasma of patients with advanced solid tumors, using Pyrosequencing, is feasible and has low within patient variability. Ongoing studies will determine whether changes in LINE-1 methylation in plasma DNA occur as a result of treatment with DNA methylation inhibitors and parallel changes in tumor tissue DNA.
DNA methylation; plasma DNA; biomarker; cancer; repetitive elements; DNA methylation inhibitors; solid tumors; repetitive DNA elements; LINE; MAGE-A1
Irinotecan and temozolomide have single-agent activity and schedule-dependent synergy against neuroblastoma. Because protracted administration of intravenous irinotecan is costly and inconvenient, we sought to determine the maximum-tolerated dose (MTD) of oral irinotecan combined with temozolomide in children with recurrent/resistant high-risk neuroblastoma.
Patients and Methods
Patients received oral temozolomide on days 1 through 5 combined with oral irinotecan on days 1 through 5 and 8 through 12 in 3-week courses. Daily oral cefixime was used to reduce irinotecan-associated diarrhea.
Fourteen assessable patients received 75 courses. Because neutropenia and thrombocytopenia were initially dose-limiting, temozolomide was reduced from 100 to 75 mg/m2/d for subsequent patients. Irinotecan was then escalated from 30 to 60 mg/m2/d. First-course grade 3 diarrhea was dose-limiting in one of six patients treated at the irinotecan MTD of 60 mg/m2/d. Other toxicities were mild and reversible. The median SN-38 lactone area under the plasma concentration versus time curve at this dose was 72 ng · hr/mL. One patient with bulky soft tissue disease had a complete response through six courses. Six additional patients received a median of seven courses (range, three to 22 courses) before progression.
This all-oral regimen was feasible and well tolerated in heavily pretreated children with resistant neuroblastoma, and seven (50%) of 14 assessable patients had response or disease stabilization for three or more courses in this phase I trial. SN-38 lactone exposures were similar to those reported with protracted intravenous irinotecan. The dosages recommended for further study in this patient population are temozolomide 75 mg/m2/d plus irinotecan 60 mg/m2/d when given with cefixime.
Iodine-131—metaiodobenzylguanidine (131I-MIBG) provides targeted radiotherapy with more than 30% response rate in refractory neuroblastoma, but activity infused is limited by radiation safety and hematologic toxicity. The goal was to determine the maximum-tolerated dose of 131I-MIBG in two consecutive infusions at a 2-week interval, supported by autologous stem-cell rescue (ASCR) 2 weeks after the second dose.
Patients and Methods
The 131I-MIBG dose was escalated using a 3 + 3 phase I trial design, with levels calculated by cumulative red marrow radiation index (RMI) from both infusions. Using dosimetry, the second infusion was adjusted to achieve the target RMI, except at level 4, where the second infusion was capped at 21 mCi/kg.
Twenty-one patients were enrolled onto the study at levels 1 to 4, with 18 patients assessable for toxicity and 20 patients assessable for response. Cumulative 131I-MIBG given to achieve the target RMI ranged from 22 to 50 mCi/kg, with cumulative RMI of 3.2 to 8.92 Gy. No patient had a dose-limiting toxicity. Reversible grade 3 nonhematologic toxicity occurred in six patients at level 4, establishing the recommended cumulative dose as 36 mCi/kg. The median time to absolute neutrophil count more than 500/μL after ASCR was 13 days (4 to 27 days) and to platelet independence was 17 days (6 to 47 days). Responses included two partial responses, eight mixed responses, three stable disease, and seven progressive disease. Responses by semiquantitative MIBG score occurred in eight patients, soft tissue responses occurred in five of 11 patients, but bone marrow responses occurred in only two of 13 patients.
The lack of toxicity with this approach allowed dramatic dose intensification of 131I-MIBG, with minimal toxicity and promising activity.
Despite the significant progress made in colon cancer chemotherapy, advanced disease remains largely incurable and novel efficacious chemotherapies are urgently needed. Histone deacetylase inhibitors (HDACi) represent a novel class of agents which have demonstrated promising preclinical activity and are undergoing clinical evaluation in colon cancer. The goal of this study was to identify genes in colon cancer cells that are differentially regulated by two clinically advanced hydroxamic acid HDACi, vorinostat and LBH589 to provide rationale for novel drug combination partners and identify a core set of HDACi-regulated genes.
HCT116 and HT29 colon cancer cells were treated with LBH589 or vorinostat and growth inhibition, acetylation status and apoptosis were analyzed in response to treatment using MTS, Western blotting and flow cytometric analyses. In addition, gene expression was analyzed using the Illumina Human-6 V2 BeadChip array and Ingenuity® Pathway Analysis.
Treatment with either vorinostat or LBH589 rapidly induced histone acetylation, cell cycle arrest and inhibited the growth of both HCT116 and HT29 cells. Bioinformatic analysis of the microarray profiling revealed significant similarity in the genes altered in expression following treatment with the two HDACi tested within each cell line. However, analysis of genes that were altered in expression in the HCT116 and HT29 cells revealed cell-line-specific responses to HDACi treatment. In addition a core cassette of 11 genes modulated by both vorinostat and LBH589 were identified in both colon cancer cell lines analyzed.
This study identified HDACi-induced alterations in critical genes involved in nucleotide metabolism, angiogenesis, mitosis and cell survival which may represent potential intervention points for novel therapeutic combinations in colon cancer. This information will assist in the identification of novel pathways and targets that are modulated by HDACi, providing much-needed information on HDACi mechanism of action and providing rationale for novel drug combination partners. We identified a core signature of 11 genes which were modulated by both vorinostat and LBH589 in a similar manner in both cell lines. These core genes will assist in the development and validation of a common gene set which may represent a molecular signature of HDAC inhibition in colon cancer.
Neuroblastoma, the second most common solid tumor in children, frequently metastasizes to the bone marrow and the bone. Neuroblastoma cells present in the bone marrow stimulate the expression of interleukin-6 (IL-6) by bone marrow stromal cells (BMSC) to activate osteoclasts. Here we have examined whether stromal-derived IL-6 also has a paracrine effect on neuroblastoma cells. An analysis of the expression of IL-6 and its receptor IL-6R in 11 neuroblastoma cell lines indicated the expression of IL-6 in 7 cell lines and of IL-6R in 9 cell lines. Treatment of IL-6R positive cells with rhIL-6 resulted in STAT-3 and Erk 1/2 activation. Culturing IL-6R positive neuroblastoma cells in the presence of BMSC or rhIL-6 increased proliferation and protected tumor cells from etoposide-induced apoptosis, whereas it had no effect on IL-6R negative tumor cells. In vivo, neuroblastoma tumors grew faster in the presence of a paracrine source of IL-6. IL-6 induced the expression of cyclooxygenase-2 in neuroblastoma cells with concomitant release of prostaglandin-E2, that increased the expression of IL-6 by BMSC. Supporting a role for stromal-derived IL-6 in patients with neuroblastoma bone metastasis, we observed elevated levels of IL-6 in the serum and bone marrow of 16 patients with neuroblastoma bone metastasis, and in BMSC derived from these patients. Altogether the data indicate that stromal-derived IL-6 contributes to the formation of a bone marrow microenvironment favorable to the progression of metastatic neuroblastoma.
neuroblastoma; interleukin-6; STAT-3; cell proliferation; apoptosis; tumor microenvironment
Patients with malignant mesothelioma (MM), an aggressive cancer associated with asbestos exposure, usually present clinically with advanced disease and this greatly reduces the likelihood of curative treatment. MM is difficult to diagnose without invasive techniques; the development of non-invasively detectable molecular markers would therefore be highly beneficial. DNA methylation changes in cancer cells provide powerful markers that are potentially detectable non-invasively in DNA shed into bodily fluids. Here we examined the methylation status of 28 loci in 52 MM tumors to investigate their potential as molecular markers for MM. To exclude candidate MM markers that might be positive in biopsies/pleural fluid due to contaminating surrounding non-tumor lung tissue/DNA, we also examined the methylation of these markers in lung samples (age- or environmentally-induced hypermethylation is frequently observed in non-cancerous lung). Statistically significantly increased methylation in MM vs. non-tumor lung samples was found for estrogen receptor 1 (ESR1; p=0.0002), solute carrier family 6 member 20 (SLC6A20; p=0.0022) and spleen tyrosine kinase (SYK; p=0.0003). Examination of associations between methylation levels of the 28 loci and clinical parameters suggest associations of the methylation status of metallothionein genes with gender, histology, asbestos exposure, and lymph node involvement, and the methylation status of leucine zipper tumor suppressor 1 (LZTS1) and SLC6A20 with survival.
APC; CpG islands; DNA methylation; ESR1; lung; mesothelioma; metallothionein; SLC6A20; SYK
Exposure to tobacco smoke is associated with increased DNA methylation at certain genes in both lung and bladder tumors. We sought to identify interactions in bladder cancer between DNA methylation and a history of smoking, along with any possible effect of aging. We measured DNA methylation in 342 transitional cell carcinoma (TCC) tumors at BCL2, PTGS2 (COX2), DAPK, CDH1 (ECAD), EDNRB, RASSF1A, RUNX3, TERT, and TIMP3. The prevalence of methylation at RUNX3, a polycomb target gene, increased as a function of age at diagnosis (p=0.031) and a history of smoking (p=0.015). RUNX3 methylation also preceded methylation at the other 8 genes (p<0.001). It has been proposed that DNA methylation patterns constitute a “molecular clock” and can be used to determine the “age” of normal tissues, i.e., the number of times the cells have divided. Since RUNX3 methylation increases with age, is not present in normal urothelium, and occurs early in tumorigenesis, it can be used for the first time as a molecular clock in order to determine the age of a bladder tumor. Doing so reveals that tumors from smokers are “older” than tumors from nonsmokers (p=0.009) either due to tumors in smokers initiating earlier or undergoing more rapid cell divisions. Since RUNX3 methylation is acquired early on in tumorigenesis then its detection in biopsy or urine specimens could provide a marker to screen cigarette smokers long before any symptoms of bladder cancer are present.
RUNX3; methylation; bladder cancer; tobacco smoking; age
In an initial epigenetic characterization of diffuse large B-cell lymphoma (DLBCL), we evaluated the DNA methylation levels of over 500 CpG islands. Twelve CpG islands (AR, CDKN1C, DLC1, DRD2, GATA4, GDNF, GRIN2B, MTHFR, MYOD1, NEUROD1, ONECUT2, and TFAP2A) showed significant methylation in over 85% of tumors. Interestingly, the methylation levels of a CpG island proximal to FLJ21062 differed between the activated B-cell-like (ABC-DLBCL) and germinal center B-cell-like (GCB-DLBCL) subtypes. In addition, we compared the methylation and expression status of sixty-seven genes proximal (within 500-bp) to the methylation assays. We frequently observed that hypermethylated CpG islands are proximal to genes that are expressed at low or undetectable levels in tumors. However, many of these same genes were also poorly expressed in DLBCL tumors where their cognate CpG islands were hypomethylated. Nevertheless, the proportional reductions in BNIP3, MGMT, RBP1, GATA4, IGSF4, CRABP1, and FLJ21062 expression with increasing methylation suggests that epigenetic processes strongly influence these genes. Lastly, the moderate expression of several genes proximal to hypermethylated CpG tracts suggests that DNA methylation assays are not always accurate predictors of gene silencing. Overall, further investigation of the highlighted CpG islands as potential clinical biomarkers is warranted.
epigenetics; genomics; CpG island; microarray; gene expression