Aakash Gajjar

Methods for detecting circulating cancer stem cells (CCSCs) as a novel approach for diagnosis of colon cancer relapse/metastasis.

Cancer stem cells (CSCs) are believed to be resistant to currently available therapies and may be responsible for relapse of cancer in patients. Measuring circulating tumor cells (CTCs) in the blood of patients has emerged as a non-invasive diagnostic procedure for screening patients who may be at high risk for developing metastatic cancers or relapse of the cancer disease. However, accurate detection of CTCs has remained a problem, as epithelial-cell markers used to date are not always reliable for detecting CTCs, especially during epithelial–mesenchymal transition. As CSCs are required to initiate metastatic tumors, our goal was to optimize and standardize a method for identifying circulating CSCs (CCSCs) in patients, using established CSC markers. Here, we report for the first time the detection of CCSCs in the blood of athymic nude mice, bearing metastatic tumors, and in the blood of patients positive for colonic adenocarcinomas. Using a simple and non-expensive method, we isolated a relatively pure population of CSCs (CD45−/CK19+), free of red blood cells and largely free of contaminating CD45+ white blood cells. Enriched CCSCs from patients with colon adenocarcinomas had a malignant phenotype and co-expressed CSC markers (DCLK1/LGR5) with CD44/Annexin A2. CSCs were not found in the blood of non-cancer patients, free of colonic growths. Enriched CCSCs from colon cancer patients grew primary spheroids, suggesting the presence of tumor-initiating cells in the blood of these patients. In conclusion, we have developed a novel diagnostic assay for detecting CSCs in circulation, which may more accurately predict the risk of relapse or metastatic disease in patients. As CSCs can potentially initiate metastatic growths, patients positive for CCSCs can be treated with inhibitory agents that selectively target CSCs, besides conventional treatments, to reduce the risk of relapse/metastatic disease for improving clinical outcomes.

An innovative approach for sternal closure.

PURPOSE Midline sternotomy remains the preferred technique for access in cardiac surgery. Application of steel wires has been the preferred method of closure. Because of associated complications, such as superficial and deep infections, as well as bony nonunion complications, an alternative technique is being proposed. The purpose of this study is to evaluate results of a new device for sternal closure. DESCRIPTION The Sternal Talon (KLS Martin Group, Jacksonville, FL), a lightweight titanium closure device is designed to encircle the sternum, thus yielding a stable closure by effectively distributing the strength of closure over the entire length of the sternotomy. After multiple strength tests demonstrated its superiority over wires, and cadaver tests confirmed its ease of placement, the Food and Drug Administration recently approved the device for its unrestricted use. Eight institutions were chosen to perform initial placements. Patient selection was limited to patients at high risk for sternotomy complications. EVALUATION In 42 patients who underwent placement of the Sternal Talon (KLS Martin Group) after sternotomy, no wound infections or dehiscence, nonunions, or returns to the operating room were observed. Three postoperative deaths were reported, none of which were device related. The device is magnetic resonance imaging compatible and there are no reported problems with computed tomographic scatter or chest roentgenogram visualization. CONCLUSIONS These initial cases prove the safety and efficacy of the Sternal Talon device for sternum closure in high-risk patients and may be regarded as an alternative to conventional wire closure. Future prospective studies are warranted to prove the superiority of the device in terms of long-term stability and sternum union rates, as well as decreased infection rates specifically in the high-risk patient population undergoing sternotomy.

Epigenetic changes and alternate promoter usage by human colon cancers for expressing DCLK1-isoforms: Clinical Implications

DCLK1 specifically marks colon/pancreatic cancers in mice, and is expressed by human colon adenocarcinomas (hCRCs). Down-regulation of DCLK1 results in loss of cancer-stem-cells (CSCs), and inhibits spheroidal/xenograft growths from hCRC-cells. The 5′-promoter of DCLK1-gene is reportedly hypermethylated in hCRCs, resulting in loss of expression of DCLK1-transcripts, originating from 5′(α)-promoter (termed DCLK1-L, in here). However, in mouse colon-tumors, 5′-promoter of DCLK1-gene remains unchanged, and DCLK1-L, originating from 5′(α)-promoter, is expressed. We hypothesized that elevated levels of DCLK1-protein in hCRC-cells, may be transcribed/translated from an alternate-promoter. Several in silico and molecular biology approaches were used to test our hypothesis. We report for the first time that majority of hCRCs express short-transcripts of DCLK1 (termed DCLK1-S, in here) from an alternate β-promoter in IntronV of the gene, while normal-colons mainly express DCLK1-L from 5′(α)-promoter. We additionally report an important role of β-catenin and TCF4/LEF binding-sites for activating (α)-promoter, while activated NF-κBp65 (bound to NF-κB-cis-element), activates (β)-promoter in cancer-cells. DCLK1-S expression was examined in a cohort of 92 CRC patients; high-expressors had significantly worse overall-survival compared to low-expressors. Our novel findings’ regarding usage of alternate (β)-promoter by hCRCs, suggests that DCLK1-S may represent an important target for preventing/inhibiting colon-cancers, and for eliminating colon-CSCs.

Abstract 1857: Dysregulation of transsulfuration enzymes contribute to malignant transformation in a murine model of colitis-associated carcinogenesis

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA Introduction: Ulcerative colitis (UC) is a highly morbid, chronic inflammatory disease characterized by mucosal ulceration of the colonic mucosa and is associated with the higher risk of colitis-associated cancer (CAC). The exact mechanism(s) causing UC progression to CAC is currently unknown. The balanced activity of transsulfuration pathway enzymes, cystathionine-gamma-lyase (CSE) and cystathionine-beta-synthase (CBS), and their production of endogenous hydrogen sulfide gas, are critical to maintenance of the colonic homeostasis. CSE activity is suggested to be important for wound healing and mucosal protection. Consistent with this, we have previously showed a decrease CSE expression in human colonic mucosa obtained from patients with chronic UC, compared to normal colonic mucosa by immunocytochemistry. By contrast, increased CBS expression is implicated in the progression of sporadic colorectal carcinoma. However, the role of these enzymes in CAC is unknown. We hypothesize that the dysregulation in CSE/CBS expression and activity is important to the progression from UC to CAC. Methods: CSE null mice and wild type Sv129/B6 (control) mice were used in azoxymethane-dextran sodium sulfate (AOM-DSS) colon cancer model which mimics human CAC. The disease development was followed up to day 80. Confocal microscopy and Western blot was used to assess the gene expression during cancer development. Size, number, and time interval to tumor formation, as well inflammation were assessed. Results: Abrogation of CSE expression using CSE null animals in AOM-DSS model of CAC accelerated the time to tumor development and resulted in an increase in both tumor size (p<0.001) and number (p<0.001) compared to wild-type controls. CBS protein expression was increased within the colonic tumor when compared to the normal margin in AOM-DSS treated animals by Western blot analysis and tissue immunostaining. Interestingly, CAC liver metastases, an exceedingly rare finding in this mouse model, were identified. Conclusion: Taken together, our human and murine data suggest that dysregulation in the transsulfuration pathway enzymes CSE and CBS expression/activity may be critical contributor to the CAC development in UC and may serve as a potential biomarker for disease progression in the future. Citation Format: Paul Johnson, Ches’Que M. Phillips, Carl Grim, John R. Zatarain, Aakash H. Gajjar, Suimin Qiu, Rui Wang, Celia Chao, Iryna V. Pinchuk, Mark R. Hellmich. Dysregulation of transsulfuration enzymes contribute to malignant transformation in a murine model of colitis-associated carcinogenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1857.

Inside the USCAP Journals

Amplification of the 12q13–15 chromosomal region involving the murine double minute-2 (MDM2) locus is characteristic of both well-differentiated and dedifferentiated liposarcoma. Dedifferentiated liposarcoma, the more aggressive form, can be graded using the Fédération Nationale des Centres de Lutte Contre le Cancer (FNCLCC, or “French”) system. The system has three grades—low (1), intermediate (2), and high (3)—based on combined scores for tumor differentiation, mitotic rate, and level of tumor necrosis. The fold amplification of MDM2 is high but variable between cases. Jour and colleagues used fluorescence in situ hybridization to characterize the amplification level of MDM2. The technique is very helpful diagnostically in cases when the biopsy lacks the characteristic lower-grade adipocytic component. Although the grade 3 dedifferentiated liposarcomas behaved more aggressively in terms of local recurrence than lower-grade liposarcomas on multivariate analysis, higher MDM2 amplification levels did not correlate with local recurrence independently. Grading consequences of PTEN loss in prostate biopsies See page 128

Abstract 543: A short isoform of DCLK1, transcribed from an alternate promoter in human colon cancers, represents a novel biomarker and target for diagnostic and treatment purposes

DCLK1 is a specific marker of colon and pancreatic cancers in mice, and is expressed by human colon adenocarcinomas (hCRCs). A sub-set of DCLK1 +ve colon cancer-stem-cells, are resistant to chemopreventive/chemotherapeutic reagents and undergo autophagic survival, resulting in the relapse of colon-cancer disease. However, down-regulation of DCLK1, along with chemopreventive agents, inhibits spheroidal/xenograft growths from hCRC cells, and eliminates both colon-cancer-stem cells and relapse of the disease. We now know that the 5′-promoter of DCLK1 gene is hypermethylated in hCRCs, but not in mouse colon-tumors, resulting in the loss of expression of DCLK1 long-transcripts (DCLK1-L), from the 5′promoter of DCLK1-gene in humans, but not in mice. We hypothesized that elevated levels of DCLK1 protein, detected in hCRCs, are likely transcribed from an alternate-promoter in humans. We used several in silico and molecular biology approaches to test our hypothesis, and report for the first time that hCRCs express short-transcripts of DCLK1 (DCLK1-S) from an alternate promoter in IntronV of the gene, while normal human colons express the long transcript (DCLK1-L) from 5′-promoter. We additionally report an important role of β-catenin and TCF4/LEF binding-sites for activating 5′-promoter, while NF-κBp65 binding to NF-κB cis element, activates the TATA box containing IntronV-promoter in cancer cells. DCLK1-S expression was examined in a cohort of 92 CRC patients, in relation to overall survival and clinicopathological parameters. High expressors had significantly worse overall-survival compared to low expressors, and DCLK1-S expression was found to be an independent prognostic factor. Conclusions. Our novel findings regarding alternate promoter usage by normal colons vs hCRCs suggest that we can develop strategies for specifically targeting DCLK1-S to eliminate colon cancer-stem-cells, while sparing DCLK1-L functions in neurons and normal cells. Our findings further suggest prognostic/diagnostic value of measuring DCLK1-S in CRC patients. Loss of DCLK1-L expression can also be used as a diagnostic marker for indicating the on-set of epigenetic changes associated with colon carcinogenesis in humans, as an early marker. Citation Format: Malaney R. O9Connell, Shubhashish Sarkar, Gurinder Luthra, Yoshinaga Okugawa, Yuji Toiyama, Ajay Goel, Aakash Gajjar, Suimin Qiu, Lawrence Sowers, Pomila Singh. A short isoform of DCLK1, transcribed from an alternate promoter in human colon cancers, represents a novel biomarker and target for diagnostic and treatment purposes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 543. doi:10.1158/1538-7445.AM2015-543