Fareeda Taher Nazer Hussain

High Detection Rates of Colorectal Neoplasia by Stool DNA Testing With a Novel Digital Melt Curve Assay

BACKGROUND & AIMS Current stool DNA tests identify about half of individuals with colorectal cancers and miss most individuals with advanced adenomas. We developed a digital melt curve (DMC) assay to quantify low-abundance mutations in stool samples for detection of colorectal neoplasms and compared this test with other approaches. METHODS We combined a melt curve assay with digital polymerase chain reaction and validated the quantitative range. We then evaluated its ability to detect neoplasms in 2 clinical studies. In study I, stool samples from patients with colorectal tumors with known mutations (KRAS, APC, BRAF, TP53) were assayed. In study II, archived stool samples from patients with advanced adenomas containing known KRAS mutations were assayed, along with controls. Results were compared with those from the stool DNA test PreGenPlus (Exact Sciences, Marlborough, MA), Hemoccult, and HemoccultSensa (both Beckman-Coulter, Fullerton, CA). RESULTS The DMC assay detected samples in which only 0.1% of target genes were mutated. In study I, the DMC assay detected known mutations in 28 (90%) of 31 tumor samples and 6 (75%) of 8 advanced adenoma samples. In study II, the DMC assay detected 16 (59%) of 27 advanced adenoma samples that contained KRAS mutations, compared with 7% with the Hemoccult, 15% with the HemoccultSensa, and 26% with the PreGenPlus assays (P < .05 for each, compared with the DMC assay); specificities did not differ significantly. CONCLUSIONS The DMC assay has a high level of sensitivity in detecting individuals with colon neoplasms and is better than current stool screening methods in detecting those with advanced adenomas. Further studies are indicated.

Detection of Colorectal Serrated Polyps by Stool DNA Testing: Comparison with Fecal Immunochemical Testing for Occult Blood (FIT)

Objectives Precursors to 1/3 of colorectal cancer (CRC), serrated polyps have been under-detected by screening due to their inconspicuous, non-hemorrhagic, and proximal nature. A new multi-target stool DNA test (multi-target sDNA) shows high sensitivity for both CRC and advanced adenomas. Screen detection of serrated polyps by this approach requires further validation. We sought to assess and compare noninvasive detection of sessile serrated polyps (SSP) ≥1 cm by sDNA and an occult blood fecal immunochemical test (FIT). Methods In a blinded prospective study, a single stool sample used for both tests was collected from 456 asymptomatic adults prior to screening or surveillance colonoscopy (criterion standard). All 29 patients with SSP≥1 cm were included as cases and all 232 with no neoplastic findings as controls. Buffered stool samples were processed and frozen on receipt; Exact Sciences performed sDNA in batches using optimized analytical methods. The sDNA multi-marker panel targets methylated BMP3 (mBMP3) and NDRG4, mutant KRAS, β-actin, and hemoglobin. FIT (Polymedco OC-FIT Check) was performed in separate lab ≤2 days post defecation and evaluated at cutoffs of 50 (FIT-50) and 100 ng/ml (FIT-100). Results Median ages: cases 61 (range 57–77), controls 62 (52–70), p = NS. Women comprised 59% and 51%, p = NS, respectively. SSP median size was 1.2 cm (1–3 cm), 93% were proximal, and 64% had synchronous diminutive polyps. Among multi-target sDNA markers, mBMP3 proved highly discriminant for detection of SSP≥1 cm (AUC = 0.87, p<0.00001); other DNA markers provided no incremental sensitivity. Hemoglobin alone showed no discrimination (AUC = 0.50, p = NS). At matched specificities, detection of SSP≥1 cm by stool mBMP3 was significantly greater than by FIT-50 (66% vs 10%, p = 0.0003) or FIT-100 (63% vs 0%, p<0.0001). Conclusions In a screening and surveillance setting, SSP≥1 cm can be detected noninvasively by stool assay of exfoliated DNA markers, especially mBMP3. FIT appears to have no value in SSP detection.

Students as Resurrectionists--A Multimodal Humanities Project in Anatomy Putting Ethics and Professionalism in Historical Context.

Because medical students have many different learning styles, the authors, medical students at Mayo Clinic, College of Medicine researched the history of anatomical specimen procurement, reviewing topic‐related film, academic literature, and novels, to write, direct, and perform a dramatization based on Robert Louis Stevensons The Body‐Snatcher. Into this performance, they incorporated dance, painting, instrumental and vocal performance, and creative writing. In preparation for the performance, each actor researched an aspect of the history of anatomy. These micro‐research projects were presented in a lecture before the play. Not intended to be a research study, this descriptive article discusses how student research and ethics discussions became a theatrical production. This addition to classroom and laboratory learning addresses the deep emotional response experienced by some students and provides an avenue to understand and express these feelings. This enhanced multimodal approach to“holistic learning” could be applied to any topic in the medical school curriculum, thoroughly adding to the didactics with history, humanities, and team dynamics. Anat Sci Educ 3:244–248, 2010.

Sole abnormalities of chromosome 7 in myeloid malignancies: Spectrum, histopathologic correlates, and prognostic implications

Among 6,565 consecutive abnormal cytogenetic reports at our institution, 3,192 (49%) constituted sole abnormalities, of which 230 (7%) involved chromosome 7: monosomy 7 (n = 98), 7q‐ (n = 51), der(1;7)(q10;p10) (n = 44), balanced translocations (n = 15), ring 7 (n = 13), and 7p‐ (n = 9). The most frequent histopathologic correlates were myelodysplastic syndromes (MDS; 28%), acute myeloid leukemia (AML; 17%), secondary or therapy‐related MDS/AML (13%), primary myelofibrosis (PMF; 7%), and chronic myelomonocytic leukemia (6%). Monosomy 7 was the most frequent in each one of these disease categories except PMF where 7q‐ was more frequent. In primary MDS, patients with der(1;7)(q10;p10) (n = 13), compared to those with monosomy 7 (n = 30) or 7q‐ (n = 15), were less likely (P = 0.04) to display excess blasts or multilineage dysplasia but overall and leukemia‐free survival adjusted for these variables revealed no significant difference between the three groups (P = 0.57 and 0.81, respectively). The current study does not prognostically distinguish monosomy 7 from 7q‐ or der(1;7), in MDS. Am. J. Hematol. 87:684–686, 2012.

T2036 Pan-Detection of Gastrointestinal Neoplasms By Stool DNA Testing: Establishment of Feasibility

Non-apoptotic human DNA (long DNA) in stool is a discriminant marker for colorectal cancer (Gastroenterology 2000; 119:1219). Fecal long DNA may originate from either the exfoliation of dysplastic cells or from the luminal hemorrhage or exudation of leukocytes. The contribution of these potential sources long DNA in stool has not been examined. If leukocytes are the major source, then fecal human DNA and occult blood levels should correlate. If exfoliation is the major source, then fecal human DNA and occult blood should be complementary markers for colorectal cancer. AIM: To quantify and correlate human DNA and hemoglobin in stools from patients with colorectal neoplasia, and to consider a simple stool assay for cancer screening. METHODS: Subjects comprised 74 patients with colorectal cancer, 27 with adenomas >1cm, and 100 with normal colonoscopy. One stool per subject was collected in stabilization buffer before or > 1 week after colonoscopy. Fecal long DNA and occult blood were quantified in blinded fashion by real-time Alu PCR (CEBP 2006 15:1115) and the HemoQuant assay (Mayo Medical Laboratories), respectively. HemoQuant assay results are not affected by fecal storage (Ann Intern Med 1984;101:297) and could be reliably applied to archival specimens for this study. RESULTS: Fecal long DNA and occult blood showed no correlation (R2=0). At 90% specificities, long DNA testing detected 70% of colorectal cancers and 46% of adenomas while occult blood testing detected 50% and 12%, respectively. Combining these tests detected 80% colorectal cancers and 46% of adenomas at 90% specificity. For detection of colorectal cancer, AUC values were 0.82, 0.78, and 0.90 for fecal long DNA, occult blood, and combination testing, respectively (p<0.05, long DNA or occult blood vs combination). The median (range) fecal long DNA level was 181 ng/g stool (0-7120 ng/g) with stage 1-2 cancers and 910 ng/g (0-19140 ng/ g) with stage 3-4 cancers, p<0.05. The median fecal hemoglobin level was 7.0 mg Hb/g (0.2-26.4 mg/g) with proximal cancers and 2.5 mg/g (0.1-36.1 mg/g) with distal ones, p<0.05. Median size was 4.0 cm (1.0-15.0) for neoplasms detected by the combined tests and 3.7 cm (1.0-7.0) for neoplasms missed, p=0.02. Neoplasm detection rates by combined tests were affected by neither tumor site nor stage. CONCLUSIONS: Long DNA and occult blood in stools are complementary stool markers for detection of colorectal cancer, and combined assay of these markers provides higher sensitivity for cancer than assay of either alone. The combined assay offers a simple and inexpensive screening approach that warrants further evaluation.

T2037 Quantitative Stool DNA Testing for Detection of Both Colorectal Cancer and Advanced Adenoma

Non-apoptotic human DNA (long DNA) in stool is a discriminant marker for colorectal cancer (Gastroenterology 2000; 119:1219). Fecal long DNA may originate from either the exfoliation of dysplastic cells or from the luminal hemorrhage or exudation of leukocytes. The contribution of these potential sources long DNA in stool has not been examined. If leukocytes are the major source, then fecal human DNA and occult blood levels should correlate. If exfoliation is the major source, then fecal human DNA and occult blood should be complementary markers for colorectal cancer. AIM: To quantify and correlate human DNA and hemoglobin in stools from patients with colorectal neoplasia, and to consider a simple stool assay for cancer screening. METHODS: Subjects comprised 74 patients with colorectal cancer, 27 with adenomas >1cm, and 100 with normal colonoscopy. One stool per subject was collected in stabilization buffer before or > 1 week after colonoscopy. Fecal long DNA and occult blood were quantified in blinded fashion by real-time Alu PCR (CEBP 2006 15:1115) and the HemoQuant assay (Mayo Medical Laboratories), respectively. HemoQuant assay results are not affected by fecal storage (Ann Intern Med 1984;101:297) and could be reliably applied to archival specimens for this study. RESULTS: Fecal long DNA and occult blood showed no correlation (R2=0). At 90% specificities, long DNA testing detected 70% of colorectal cancers and 46% of adenomas while occult blood testing detected 50% and 12%, respectively. Combining these tests detected 80% colorectal cancers and 46% of adenomas at 90% specificity. For detection of colorectal cancer, AUC values were 0.82, 0.78, and 0.90 for fecal long DNA, occult blood, and combination testing, respectively (p<0.05, long DNA or occult blood vs combination). The median (range) fecal long DNA level was 181 ng/g stool (0-7120 ng/g) with stage 1-2 cancers and 910 ng/g (0-19140 ng/ g) with stage 3-4 cancers, p<0.05. The median fecal hemoglobin level was 7.0 mg Hb/g (0.2-26.4 mg/g) with proximal cancers and 2.5 mg/g (0.1-36.1 mg/g) with distal ones, p<0.05. Median size was 4.0 cm (1.0-15.0) for neoplasms detected by the combined tests and 3.7 cm (1.0-7.0) for neoplasms missed, p=0.02. Neoplasm detection rates by combined tests were affected by neither tumor site nor stage. CONCLUSIONS: Long DNA and occult blood in stools are complementary stool markers for detection of colorectal cancer, and combined assay of these markers provides higher sensitivity for cancer than assay of either alone. The combined assay offers a simple and inexpensive screening approach that warrants further evaluation.

743 Noninvasive Detection of Serrated Colorectal Polyps by Stool Assay of Methylated Vimentin and Mutant BRAF Genes

Background: Serrated polyps are the critical precursors for 20-30% of colorectal cancers (CRCs) but have been largely excluded as screening outcomes to date. Serrated polyps, particularly the subset of sessile serrated adenomas (SSAs), are typically proximal, visually inconspicuous, rapid in growth, and characterized bymutant BRAF and aberrantlymethylated genes. Our preliminary data suggest that stool assay of mutant BRAF can detect SSAs (2010 Gastrointestinal Cancers Symposium, Orlando, FL). Aims: To assess detection of SSAs by stool assay of methylated vimentin (mVimentin) alone and in combination with mutant BRAF, and to compare detection accuracy with that of conventional fecal blood tests. Methods: In pre-study tissue analyses, mVimentin proved to discriminate SSAs from normal colonic mucosa and was selected for stool testing. Using our biospecimen archive from a multicenter screening study (Ann Intern Med 2008;149:441), unbuffered stools were identified from 14 cases with SSAs ≥1cm and blindly studied along with 19 matched controls without SSAs. Standard methods were used to extract stool DNA, and target genes were enriched by sequence capture. Following bisulfite treatment, mVimentin was quantified by methylation-specific PCR. Mutant BRAF was quantified by digital melt curve assay (Gastroenterology 2009;1136:459). Prospective Hemoccult and HemoccultSensa data (3 stools/patient) were available for comparison. Results: Median age was 68 (range 58-77) for SSA cases and 68 (55-74) for controls; respectively, 71% and 68% were female. Median SSA size was 1.5 cm (range 1-2) and 86% were proximal. Synchronous advanced adenomas were present in 36% of cases but in no controls. At 95% specificity cut-offs, stool assays of mVimentin and mutant BRAF respectively detected 57% and 50% of SSAs. In comparison, Hemoccult and HemoccultSensa each detected 7% (p<0.01 vs either stool DNA assay) with specificity for both of 95%. Combining stool mVimentin and mutant BRAF assays, SSA detection was 71% at a specificity of 89%. Combined stool DNA assays were positive in 5/5 (100%) of cases with synchronous advanced adenomas and in 5/9 (56%) without synchronous lesions; test positive SSAs were larger than negative ones (p=0.02). Conclusions: Based on this study using specimens from the screen setting, stool DNA assay targeting mVimentin or mutant BRAF markers appears to be feasible for detection of SSAs, and detection rates are substantially higher than by fecal blood testing. Combined stool assays of mVimentin and mutant BRAF increased SSA detection further but at a loss of specificity. These findings have important implications for CRC screening.

T2034 Stool DNA and Occult Blood for Detection of Colorectal Cancer: Complementary Markers

Non-apoptotic human DNA (long DNA) in stool is a discriminant marker for colorectal cancer (Gastroenterology 2000; 119:1219). Fecal long DNA may originate from either the exfoliation of dysplastic cells or from the luminal hemorrhage or exudation of leukocytes. The contribution of these potential sources long DNA in stool has not been examined. If leukocytes are the major source, then fecal human DNA and occult blood levels should correlate. If exfoliation is the major source, then fecal human DNA and occult blood should be complementary markers for colorectal cancer. AIM: To quantify and correlate human DNA and hemoglobin in stools from patients with colorectal neoplasia, and to consider a simple stool assay for cancer screening. METHODS: Subjects comprised 74 patients with colorectal cancer, 27 with adenomas >1cm, and 100 with normal colonoscopy. One stool per subject was collected in stabilization buffer before or > 1 week after colonoscopy. Fecal long DNA and occult blood were quantified in blinded fashion by real-time Alu PCR (CEBP 2006 15:1115) and the HemoQuant assay (Mayo Medical Laboratories), respectively. HemoQuant assay results are not affected by fecal storage (Ann Intern Med 1984;101:297) and could be reliably applied to archival specimens for this study. RESULTS: Fecal long DNA and occult blood showed no correlation (R2=0). At 90% specificities, long DNA testing detected 70% of colorectal cancers and 46% of adenomas while occult blood testing detected 50% and 12%, respectively. Combining these tests detected 80% colorectal cancers and 46% of adenomas at 90% specificity. For detection of colorectal cancer, AUC values were 0.82, 0.78, and 0.90 for fecal long DNA, occult blood, and combination testing, respectively (p<0.05, long DNA or occult blood vs combination). The median (range) fecal long DNA level was 181 ng/g stool (0-7120 ng/g) with stage 1-2 cancers and 910 ng/g (0-19140 ng/ g) with stage 3-4 cancers, p<0.05. The median fecal hemoglobin level was 7.0 mg Hb/g (0.2-26.4 mg/g) with proximal cancers and 2.5 mg/g (0.1-36.1 mg/g) with distal ones, p<0.05. Median size was 4.0 cm (1.0-15.0) for neoplasms detected by the combined tests and 3.7 cm (1.0-7.0) for neoplasms missed, p=0.02. Neoplasm detection rates by combined tests were affected by neither tumor site nor stage. CONCLUSIONS: Long DNA and occult blood in stools are complementary stool markers for detection of colorectal cancer, and combined assay of these markers provides higher sensitivity for cancer than assay of either alone. The combined assay offers a simple and inexpensive screening approach that warrants further evaluation.

Surgical Repair of Tetralogy of Fallot at Age 83

Tetralogy of Fallot (TOF) is a cyanotic congenital heart defect typically diagnosed in infancy and treated with early surgical correction. We report a patient with TOF diagnosed at age 78. Successful surgical repair was performed at age 83, the oldest reported age of surgical correction of this condition. Despite a complicated surgical and postoperative course, the patient is doing well almost four years later.

481 Noninvasive Detection of Colorectal Neoplasia (CRN) in Inflammatory Bowel Disease (IBD) by Stool DNA Testing: A Pilot Study

Noninvasive Detection of Colorectal Neoplasia (CRN) in Inflammatory Bowel Disease (IBD) by Stool DNA Testing: A Pilot Study John B. Kisiel, Tracy C. Yab, Fareeda Taher Nazer Hussain, William R. Taylor, Jonathan J. Harrington, Mary E. Devens, Julie A. Simonson, Megan Garrity-Park, William J. Sandborn, Edward V. Loftus, Bruce G. Wolff, David T. Rubin, Steven H. Itzkowitz, Hongzhi Zou, David A. Ahlquist