A Phase II Trial of Guadecitabine plus Atezolizumab in Metastatic Urothelial Carcinoma Progressing after Initial Immune Checkpoint Inhibitor Therapy.

PURPOSE: On the basis of preclinical evidence of epigenetic contribution to sensitivity and resistance to immune checkpoint inhibitors (ICI), we hypothesized that guadecitabine (hypomethylating agent) and atezolizumab [anti-programmed cell death ligand 1 (PD-L1)] together would potentiate a clinical response in patients with metastatic urothelial carcinoma (UC) unresponsive to initial immune checkpoint blockade therapy. PATIENTS AND METHODS: We designed a single arm phase II study (NCT03179943) with a safety run-in to identify the recommended phase II dose of the combination therapy of guadecitabine and atezolizumab. Patients with recurrent/advanced UC who had previously progressed on ICI therapy with programmed cell death protein 1 or PD-L1 targeting agents were eligible. Preplanned correlative analysis was performed to characterize peripheral immune dynamics and global DNA methylation, transcriptome, and immune infiltration dynamics of patient tumors. RESULTS: Safety run-in enrolled 6 patients and phase II enrolled 15 patients before the trial was closed for futility. No dose-limiting toxicity was observed. Four patients, with best response of stable disease (SD), exhibited extended tumor control (8-11 months) and survival (>14 months). Correlative analysis revealed lack of DNA demethylation in tumors after 2 cycles of treatment. Increased peripheral immune activation and immune infiltration in tumors after treatment correlated with progression-free survival and SD. Furthermore, high IL6 and IL8 levels in the patients' plasma was associated with short survival. CONCLUSIONS: No RECIST responses were observed after combination therapy in this trial. Although we could not detect the anticipated tumor-intrinsic effects of guadecitabine, the addition of hypomethylating agent to ICI therapy induced immune activation in a few patients, which associated with longer patient survival.

Identifying aggressive prostate cancer foci using a DNA methylation classifier.

BACKGROUND: Slow-growing prostate cancer (PC) can be aggressive in a subset of cases. Therefore, prognostic tools to guide clinical decision-making and avoid overtreatment of indolent PC and undertreatment of aggressive disease are urgently needed. PC has a propensity to be multifocal with several different cancerous foci per gland. RESULTS: Here, we have taken advantage of the multifocal propensity of PC and categorized aggressiveness of individual PC foci based on DNA methylation patterns in primary PC foci and matched lymph node metastases. In a set of 14 patients, we demonstrate that over half of the cases have multiple epigenetically distinct subclones and determine the primary subclone from which the metastatic lesion(s) originated. Furthermore, we develop an aggressiveness classifier consisting of 25 DNA methylation probes to determine aggressive and non-aggressive subclones. Upon validation of the classifier in an independent cohort, the predicted aggressive tumors are significantly associated with the presence of lymph node metastases and invasive tumor stages. CONCLUSIONS: Overall, this study provides molecular-based support for determining PC aggressiveness with the potential to impact clinical decision-making, such as targeted biopsy approaches for early diagnosis and active surveillance, in addition to focal therapy.

Improving needle biopsy accuracy in small renal mass using tumor-specific DNA methylation markers.

PURPOSE: The clinical management of small renal masses (SRMs) is challenging since the current methods for distinguishing between benign masses and malignant renal cell carcinomas (RCCs) are frequently inaccurate or inconclusive. In addition, renal cancer subtypes also have different treatments and outcomes. High false negative rates increase the risk of cancer progression and indeterminate diagnoses result in unnecessary and potentially morbid surgical procedures. EXPERIMENTAL DESIGN: We built a predictive classification model for kidney tumors using 697 DNA methylation profiles from six different subgroups: clear cell, papillary and chromophobe RCC, benign angiomylolipomas, oncocytomas, and normal kidney tissues. Furthermore, the DNA methylation-dependent classifier has been validated in 272 ex vivo needle biopsy samples from 100 renal masses (71% SRMs). RESULTS: In general, the results were highly reproducible (89%, n=70) in predicting identical malignant subtypes from biopsies. Overall, 98% of adjacent-normals (n=102) were correctly classified as normal, while 92% of tumors (n=71) were correctly classified malignant and 86% of benign (n=29) were correctly classified benign by this classification model. CONCLUSIONS: Overall, this study provides molecular-based support for using routine needle biopsies to determine tumor classification of SRMs and support the clinical decision-making.

Identification of DNA Methylation-Independent Epigenetic Events Underlying Clear Cell Renal Cell Carcinoma.

Alterations in chromatin accessibility independent of DNA methylation can affect cancer-related gene expression, but are often overlooked in conventional epigenomic profiling approaches. In this study, we describe a cost-effective and computationally simple assay called AcceSssIble to simultaneously interrogate DNA methylation and chromatin accessibility alterations in primary human clear cell renal cell carcinomas (ccRCC). Our study revealed significant perturbations to the ccRCC epigenome and identified gene expression changes that were specifically attributed to the chromatin accessibility status whether or not DNA methylation was involved. Compared with commonly mutated genes in ccRCC, such as the von Hippel-Lindau (VHL) tumor suppressor, the genes identified by AcceSssIble comprised distinct pathways and more frequently underwent epigenetic changes, suggesting that genetic and epigenetic alterations could be independent events in ccRCC. Specifically, we found unique DNA methylation-independent promoter accessibility alterations in pathways mimicking VHL deficiency. Overall, this study provides a novel approach for identifying new epigenetic-based therapeutic targets, previously undetectable by DNA methylation studies alone, that may complement current genetic-based treatment strategies. Cancer Res; 76(7); 1954-64. ©2016 AACR.

The pollution removal and stormwater reduction performance of street-side bioretention basins after ten years in operation.

This study evaluated the pollution removal and hydrologic performance of five, 10-year old street-side bioretention systems. The bioretention basins were subjected to a series of simulated rainfall events using synthetic stormwater. Four different pollution concentrations were tested on three of the bioretention basins. The four concentrations tested were: A) no pollution; B) typical Australian urban pollutant loads; C) double the typical pollution loads, and; D) five times the typical pollution loads. Tests were also undertaken to determine the levels of contaminant and heavy metals build-up that occurred in the filter media over the 10 year operational life of the bioretention systems. Although highly variable, the overall hydrological performance of the basins was found to be positive, with all basins attenuating flows, reducing both peak flow rates and total outflow volumes. Total suspended solids removal performance was variable for all tests and no correlation was found between performance and dosage. Total nitrogen (TN) removal was positive for Tests B, C and D. However, the TN removal results for Test A were found to be negative. Total phosphorus (TP) was the only pollutant to be effectively removed from all basins for all four synthetic stormwater tests. The study bioretention basins were found to export pollutants during tests where no pollutants were added to the simulated inflow water (Test A). Heavy metal and hydrocarbon testing undertaken on the bioretention systems found that the pollution levels of the filter media were still within acceptable limits after 10 years in operation. This field study has shown bioretention basin pollution removal performance to be highly variable and dependant on a range of factors including inflow pollution concentrations, filter media, construction methods and environmental factors. Further research is required in order to fully understand the potential stormwater management benefits of these systems.

Do sediment type and test durations affect results of laboratory-based, accelerated testing studies of permeable pavement clogging?

Previous studies have attempted to quantify the clogging processes of Permeable Interlocking Concrete Pavers (PICPs) using accelerated testing methods. However, the results have been variable. This study investigated the effects that three different sediment types (natural and silica), and different simulated rainfall intensities, and testing durations had on the observed clogging processes (and measured surface infiltration rates) of laboratory-based, accelerated PICP testing studies. Results showed that accelerated simulated laboratory testing results are highly dependent on the type, and size of sediment used in the experiments. For example, when using real stormwater sediment up to 1.18 mm in size, the results showed that neither testing duration, nor stormwater application rate had any significant effect on PICP clogging. However, the study clearly showed that shorter testing durations generally increased clogging and reduced the surface infiltration rates of the models when artificial silica sediment was used. Longer testing durations also generally increased clogging of the models when using fine sediment (<300 µm). Results from this study will help researchers and designers better anticipate when and why PICPs are susceptible to clogging, reduce maintenance and extend the useful life of these increasingly common stormwater best management practices.

DNA methylation directly silences genes with non-CpG island promoters and establishes a nucleosome occupied promoter.

Despite the fact that 45% of all human gene promoters do not contain CpG islands, the role of DNA methylation in control of non-CpG island promoters is controversial and its relevance in normal and pathological processes is poorly understood. Among the few studies which investigate the correlation between DNA methylation and expression of genes with non-CpG island promoters, the majority do not support the view that DNA methylation directly leads to transcription silencing of these genes. Our reporter assays and gene reactivation by 5-aza-2'-deoxycytidine, a DNA demethylating agent, show that DNA methylation occurring at CpG poor LAMB3 promoter and RUNX3 promoter 1(RUNX3 P1) can directly lead to transcriptional silencing in cells competent to express these genes in vitro. Using Nucleosome Occupancy Methylome- Sequencing, NOMe-Seq, a single-molecule, high-resolution nucleosome positioning assay, we demonstrate that active, but not inactive, non-CpG island promoters display a nucleosome-depleted region (NDR) immediately upstream of the transcription start site (TSS). Furthermore, using NOMe-Seq and clonal analysis, we show that in RUNX3 expressing 623 melanoma cells, RUNX3 P1 has two distinct chromatin configurations: one is unmethylated with an NDR upstream of the TSS; another is methylated and nucleosome occupied, indicating that RUNX3 P1 is monoallelically methylated. Together, these results demonstrate that the epigenetic signatures comprising DNA methylation, histone marks and nucleosome occupancy of non-CpG island promoters are almost identical to CpG island promoters, suggesting that aberrant methylation patterns of non-CpG island promoters may also contribute to tumorigenesis and should therefore be included in analyses of cancer epigenetics.

Lymph node dissection technique is more important than lymph node count in identifying nodal metastases in radical cystectomy patients: a comparative mapping study.

BACKGROUND: The value of lymph node dissection (LND) in the treatment of bladder urothelial carcinoma is well established. However, standards for the quality of LND remain controversial. OBJECTIVE: We compared the distribution of lymph node (LN) metastases in a two-institution cohort of patients undergoing radical cystectomy (RC) using a uniformly applied extended LND template. DESIGN, SETTING, AND PARTICIPANTS: Patients undergoing RC at the University of Southern California (USC) Institute of Urology and at Oregon Health Sciences University (OHSU) were included if they met the following criteria: (1) no prior pelvic radiotherapy or LND; (2) lymphatic tissue submitted from all nine predesignated regions, including the paracaval and para-aortic LNs; (3) bladder primary; and (4) category M0 disease. The number and location of LN metastases were prospectively entered into corresponding databases. MEASUREMENTS: LN maps were constructed and correlated with preoperative and pathologic characteristics. Kaplan-Meier curves were constructed to estimate overall survival (OS) and recurrence free survival (RFS) among LN-positive (LN+) patients. RESULTS AND LIMITATIONS: Inclusion criteria were met by 646 patients (439 USC, 207 OHSU), and 23% had LN metastases at time of cystectomy. Although there was a difference in the median per-patient LN count between institutions, there were no significant interinstitutional differences in the incidence or distribution of positive LNs, which were found in 11% of patients with ≤pT2b and in 44% of patients with ≥pT3a tumors. Among LN+ patients, 41% had positive LNs above the common iliac bifurcation. Estimated 5-yr RFS and OS rates for LN+ patients were 45% and 33%, respectively, and did not differ significantly between institutions. CONCLUSIONS: LN metastases in regions outside the boundaries of standard LND are common. Adherence to meticulous dissection technique within an extended template is likely more important than total LN count for achieving optimal oncologic outcomes.

Hypomethylation of a LINE-1 promoter activates an alternate transcript of the MET oncogene in bladders with cancer.

It was recently shown that a large portion of the human transcriptome can originate from within repetitive elements, leading to ectopic expression of protein-coding genes. However the mechanism of transcriptional activation of repetitive elements has not been definitively elucidated. For the first time, we directly demonstrate that hypomethylation of retrotransposons can cause altered gene expression in humans. We also reveal that active LINE-1s switch from a tetranucleosome to dinucleosome structure, acquiring H2A.Z- and nucleosome-free regions upstream of TSSs, previously shown only at active single-copy genes. Hypomethylation of a specific LINE-1 promoter was also found to induce an alternate transcript of the MET oncogene in bladder tumors and across the entire urothelium of tumor-bearing bladders. These data show that, in addition to contributing to chromosomal instability, hypomethylation of LINE-1s can alter the functional transcriptome and plays a role not only in human disease but also in disease predisposition.

Urethral recurrence in a woman after continent orthotopic urinary diversion for bladder cancer.

We describe a 55-year-old woman with a urethral recurrence of transitional cell carcinoma of her bladder 4 years after she underwent radical cystectomy with negative margins and no evidence of invasion of the bladder neck, anterior vaginal wall, or proximal urethra. This was the first urethral recurrence in an appropriately selected female patient at our institution. This report emphasizes the need for long-term follow-up in patients after radical cystectomy and the need for guidelines for appropriate selection of female patients for continent orthotopic urinary diversion.

Radical cystectomy for primary neuroendocrine tumors of the bladder: the university of southern california experience.

PURPOSE: Primary neuroendocrine tumors of the bladder are rare and they include small and large cell variants. We reviewed our experience with treating these tumors with radical cystectomy to evaluate their histopathological characteristics and clinical outcomes. MATERIALS AND METHODS: From August 1971 to June 2004, 2,005 patients underwent radical cystectomy for primary bladder cancer at our institution, of whom 25 (1.2%) had neuroendocrine tumors of the bladder, including small cell carcinoma in 20 and large cell carcinoma in 5. Pure neuroendocrine-type histology was identified in 16 cases, including 1 with small and large cell features, while the remaining 9 had mixed histology, that is transitional cell carcinoma in 8 and adenocarcinoma in 1. Multi-agent chemotherapy was administered to 14 patients. RESULTS: Median patient age was 68 years (range 40 to 82) and 19 patients were male (76%). A total of 19 patients (76%) had lymph node involvement, of whom 2 had small liver metastases found intraoperatively, while only 4 (16%) had organ confined tumors and 2 (8%) had extravesical, node negative disease. These tumors tended to have a flat, ulcerative gross appearance with lymphovascular invasion, carcinoma in situ and necrosis present microscopically. Median followup was 11.8 years (range 18 days to 15.1 years). Five-year overall and recurrence-free survival was 10% and 13%, respectively. There was no significant survival difference between small and large cell carcinoma. Mixed histologies tended to do better than pure neuroendocrine tumors, although this did not attain statistical significance (p = 0.064). Patients receiving multimodality therapy had significantly better overall (p = 0.051) and recurrence-free (p = 0.003) survival than those treated with cystectomy alone. CONCLUSIONS: Neuroendocrine tumors of the bladder usually present with advanced pathological stage and portend a poor prognosis. Adjuvant chemotherapy protocols may provide improved survival compared with cystectomy alone.

Prognostic significance of lymphovascular invasion of bladder cancer treated with radical cystectomy.

PURPOSE: We determined the prognostic significance of lymphovascular invasion (LVI) in patients treated for invasive transitional cell carcinoma of the bladder with radical cystectomy. MATERIALS AND METHODS: From August 1971 to June 2004, 2,005 patients underwent radical cystectomy for primary bladder cancer with intent to cure. All patients with nontransitional cell carcinoma histology, palliative procedures, unknown lymphovascular status, less than pT1 pathological stage, or any neoadjuvant or adjuvant chemotherapy/radiation therapy were excluded, leaving 702 comprising the study cohort. Of the 702 patients 249 (36%) had LVI. RESULTS: Median followup was 11.0 years (range 8 days to 23.2 years). Overall 5 and 10-year survival was 51% and 34%, while 5 and 10-year recurrence-free survival was 66% and 64%, respectively. Ten-year recurrence-free survival in patients without LVI was 74% compared with 42% in those with LVI (p <0.0001). Similarly 10-year overall survival was 43% in patients without LVI compared with 18% in those with LVI (p <0.0001). In the organ confined/lymph node negative and lymph node positive pathological subgroups survival outcomes were significantly worse if LVI was present. Although a trend was observed, LVI status was not statistically significant in patients with extravesical node negative disease. Stepwise Cox regression analysis revealed that pathological subgroup (organ confined, extravesical and lymph node positive) (p <0.0001) and LVI status (p = 0.0004) were independent prognostic variables for recurrence-free and overall survival. CONCLUSIONS: Lymphovascular invasion appears to be an important and independent prognostic variable in patients with invasive bladder cancer treated with radical cystectomy. LVI status should be determined in cystectomy specimens, which may provide further risk stratification in patients following radical cystectomy.

Ethnic differences in neuroendocrine cell expression in normal human prostatic tissue.

OBJECTIVES: To determine whether any differences exist in neuroendocrine (NE) cell differentiation in normal prostates among various ethnic groups because the incidence and mortality from prostate cancer vary across racial groups. METHODS: Archived paraffin-embedded prostate samples not containing any malignancy were obtained from cystoprostatectomy specimens. Prostatic tissue was obtained from 15 African Americans, 13 Hispanics, 15 Asians, and 16 whites. NE cells were identified based on immunoreactivity for chromogranin A. The mean number of NE cells per high power field (HPF) for each patient was determined using a visual quantitative method by two observers who were unaware of the race of the patients. RESULTS: The geometric mean number of NE cells was 6.1/HPF for Asians, 5.6/HPF for whites, 4.0/HPF for Hispanics, and 0.7/HPF for African Americans. A highly significant difference was observed in the distribution of NE cells between African Americans and each of the other races (P < or =0.003). A trend toward greater NE expression was noted in Asians and whites compared with Hispanics; however, this difference did not reach statistical significance. CONCLUSIONS: We found a fivefold to eightfold difference in the distribution of NE cells in the normal prostates of African-American men compared with that of other races. The comparatively low NE cell expression in African-American men may play a role in the greater rate of prostate carcinogenesis seen in this ethnic group.

Prescription drug use and risk of acute myeloid leukemia by French-American-British subtype: results from a Los Angeles County case-control study.

Chemotherapy is a well-established risk factor for acute myeloid leukemia (AML) but little is known about other prescription drugs and AML risk. We report data from a population-based Los Angeles County study in which 299 matched case-control pairs had complete data on prescription drug use and 88% of cases were subtyped according to the French-American-British (FAB) criteria. Cases were diagnosed between 1987 and 1994. Prescription nonsteroidal anti-inflammatory drug (NSAID) use for at least 4 weeks in the 2 to 10 years before diagnosis was associated with decreased risk (odds ratio = 0.5, 95% confidence interval=0.3, 1.0; p=0.04) with dose-response most evident for FAB subtype M2 (OR = 0.6, CI: 0.1, 2.9 for duration < or =6 months; OR = 0.2, CI: 0.0, 1.6 for >6 months). For subtype M4, ORs increased with increasing duration of benzodiazepine use in the 2 to 10 years before diagnosis (OR = 1.5, CI: 0.3, 9.0 for < or =6 months vs. OR = 5.0, CI: 0.6, 42.8 for >6 months). These results suggest that prescription drugs other than chemotherapy may have FAB subtype-specific effects on AML risk.

Detection of methylated apoptosis-associated genes in urine sediments of bladder cancer patients.

PURPOSE: There is increasing evidence for a fundamental role for epigenetic silencing of apoptotic pathways in cancer. Changes in DNA methylation can be detected with a high degree of sensitivity, so we used the MethyLight assay to determine how methylation patterns of apoptosis-associated genes change during bladder carcinogenesis and whether DNA methylation could be detected in urine sediments. EXPERIMENTAL DESIGN: We analyzed the methylation status of the 5' regions of 12 apoptosis-associated genes (ARF, FADD, TNFRSF21, BAX, LITAF, DAPK, TMS-1, BCL2, RASSF1A, TERT, TNFRSF25, and EDNRB) in 18 bladder cancer cell lines, 127 bladder cancer samples, and 37 samples of adjacent normal bladder mucosa using the quantitative MethyLight assay. We also analyzed the methylation status in urine sediments of 20 cancer-free volunteers and 37 bladder cancer patients. RESULTS: The 5' regions of DAPK, BCL2, TERT, RASSFIA, and TNFRSF25 showed significant increases in methylation levels when compared with nonmalignant adjacent tissue (P < or = 0.01). Methylation levels of BCL2 were significantly associated with tumor staging and grading (P < or = 0.01), whereas methylation levels of RASSF1A and ARF were only associated with tumor stage (P < or = 0.04), and TERT methylation and EDNRB methylation were predictors of tumor grade (P < or = 0.02). To investigate clinical usefulness for noninvasive bladder cancer detection, we further analyzed the methylation status of the markers in urine samples of patients with bladder cancer. Methylation of DAPK, BCL2, and TERT in urine sediment DNA from bladder cancer patients was detected in the majority of samples (78%), whereas they were unmethylated in the urine sediment DNA from age-matched cancer-free individuals. CONCLUSIONS: Our results indicate that methylation of the 5' region of apoptosis-associated genes is a common finding in patients with bladder carcinoma. The ability to detect methylation not only in bladder tissue, but also in urine sediments, suggests that methylation markers are promising tools for noninvasive detection of bladder cancers. Our results also indicate that some methylation markers, such as those in regions of RASSF1A and TNFRSF25, might be of limited use for detection because they are also methylated in normal bladder tissues.

Alcohol consumption and risk of adult-onset acute myeloid leukemia: results from a Los Angeles County case-control study.

Few studies have examined the role of alcohol consumption in risk of adult acute myeloid leukemia (AML). Two previous case-control studies resulted in inconsistent findings. We report data from a Los Angeles County population-based case-control study in which 164 matched case-control pairs were asked about lifetime history of alcohol consumption and 136 cases were subtyped according to the French-American-British (FAB) criteria. Estimated categorical odds ratios (OR) adjusted for smoking and education were suggestive of a possible protective effect but trend tests were non-significant. Analyses by FAB subtype did not reveal subtype-specific associations but generally suffered from lack of power. Larger studies are needed to more thoroughly investigate the relationship between alcohol consumption and AML risk.

Prognosis of seminal vesicle involvement by transitional cell carcinoma of the bladder.

PURPOSE: Transitional cell carcinoma (TCC) of the bladder that extends directly into contiguous organs (pT4) portends a poor prognosis. The 2002 American Joint Committee on Cancer staging system does not include seminal vesicle involvement by primary TCC of the bladder. In this analysis we evaluated the clinical outcomes and prognostic significance of seminal vesicle involvement with TCC of the bladder after radical cystectomy. MATERIALS AND METHODS: From 1971 to 2001, 1,682 patients underwent radical cystectomy and pelvic lymphadenectomy for bladder cancer. Only those tumors that involved adjacent organs through the bladder wall (pT4) were included. Overall 132 male patients with a median age of 68 years (range 36 to 98) qualified for analysis. Patients were stratified into 4 subgroups of 1) direct extravesical prostatic stromal involvement only in 37 patients (28%), 2) prostatic stroma and seminal vesicle involvement in 37 patients (28%), 3) seminal vesicle involvement only in 10 patients (8%) and 4) other contiguous pelvic organ involvement (stage pT4b) in 48 patients (36%). Overall 88 patients (67%) received some form of adjuvant therapy. At a median followup of 12.5 years (range 0 to 15.2) clinical outcomes were analyzed including overall and recurrence-free survival using Kaplan-Meier plots. RESULTS: There was no significant difference in clinical outcomes or prognosis for groups 2 and 3, thus they were combined for analysis. Five-year overall survival for any seminal vesicle involvement (10%) was significantly worse than prostatic stromal involvement only (38%) but was similar to pT4b tumors (7%, p <0.0001). The 5-year recurrence-free survival for seminal vesicle involvement (14%) was also significantly worse than prostatic stromal involvement alone (68%) but similar to that pT4b disease (25%, p = 0.01). Results were controlled for lymph node status. CONCLUSIONS: Patients with extravesical tumor extension into seminal vesicles and contiguous pelvic organs are at high risk for recurrence and progression. Involvement of the seminal vesicles by direct extension of bladder TCC portends a prognosis similar to that of pT4b disease and should, therefore, be classified as such.

Smoking and risk of acute myeloid leukemia: results from a Los Angeles County case-control study.

Acute myelogenous leukemia (AML) is a heterogeneous disease with distinct histologic subtypes likely to have distinct risk factors. The authors examined smoking and the risk of adult AML by French-American-British (FAB) subtype in a Los Angeles County, California, population-based case-control study of 412 cases diagnosed between 1987 and 1994 and 412 matched controls. Consistent with previous studies, smoking was not a substantial risk factor for AML overall (odds ratio (OR) = 1.2, 95% confidence interval (CI): 0.9, 1.6). However, increased risk was observed for FAB subtype M2 (OR = 2.3, 95% CI: 1.1, 4.4), particularly for subjects aged 60-75 years (OR = 3.3, 95% CI: 1.1, 10.0). For M2, significant dose-response was associated with total years smoked (p = 0.02), cigarettes per day (p = 0.007), and product filter status (filtered vs. nonfiltered; p = 0.03). The authors estimate that 42% (standard error = 13%) of M2 cases are attributable to smoking. There were no or weak associations between smoking and increased AML risk for other FAB subtypes. The finding by this study of an association between smoking and FAB subtype M2 confirms a previously published report and suggests that earlier findings of no or weak smoking-AML associations may have been due to lack of subtype-specific analysis.