Kamana Misra, PhD

Beyond diagnosis and classification, cytogenetic abnormalities also determine the therapeutic outcome for the patients, therefore having diagnostic, prognostic, and therapeutic importance.

AML risk stratification is based on the number of cytogenetic abnormalities observed in the patients. Currently, however, there is lack of universally adopted approach for adverse risk stratification. European Leukemia Net (ELN) classifies patients in adverse genetic risk category if they have karyotypes with three or more aberrations.

UK National Cancer Research Institute Adult Leukaemia Working Group (abbreviated as MRC for Medical Research Council), however requires four or more abnormalities to qualify as adverse prognosis.

In addition to these differences on cutoff for adverse prognosis, several other inconsistencies preclude comparison of treatments and outcomes at different institutions.

Friedrich St&ouml;lzel, MD, Department of Internal Medicine V, University Hospital Heidelberg, Heidelberg, Germany, et al, therefore, initiated a study to define the optimized cutoff of complexity in adult AML in the context of the number of unrelated aberrations (3 vs >4) as well as to define the impact of the pure hyperdiploid karyotype (HDK) within these groups. The study results were published recently in

Patients for the study were selected from the databases of three prospective, randomized trials of the Study Alliance Leukemia, which had enrolled a total of 3526 non-APL, intensively treated AML patients between February 1996 and November 2009. Patients with multiple cytogenetic aberrations (>3) as well as normal karyotype (NK as a control group) were identified for this study. Chromosomal analyses were performed on bone marrow and/or peripheral blood samples using standard techniques.

Karyotype description was performed in accordance with the International System for Human Cytogenetic Nomenclature criteria.

Accordingly, the following cytogenetic nomenclature criteria were used:

A balanced translocation was defined as a single abnormality, because the two breaks and fusions lead to one active chimeric fusion protein.

A balanced translocation involving more than two chromosomes was also regarded as a single abnormality.

Trisomies or monosomies were regarded as single abnormalities.

Gain of two chromosomes, even if they were identical was regarded as two abnormalities.

Unbalanced translocations leading to gain and loss of chromosomal material were counted as two abnormalities.

The monosomal karyotype (MK) was defined by the presence of two or more distinct autosomal chromosome monosomies or a single autosomal chromosome monosomy in the presence of one or more structural chromosomal abnormalities.

The treatment regimen for the studies included double induction chemotherapy administration to patients aged ⩽60 years, including a risk-adapted consolidation strategy. This included HLA-compatible, related or unrelated allogeneic hematopoietic stem cell transplantation for intermediate-risk patients with a sibling donor and adverse-risk patients with a matched donor. In the AML2003 trial, patients were randomized up-front to undergo allogeneic hematopoietic stem cell transplantation early after induction chemotherapy-induced aplasia or during first remission in defined adverse-risk situations.

Collectively, the study showed that 84% of patients had >4 aberrations while only 16% had >3 aberrations. Patients with >4 aberrations fared worse than normal karyotype (NK) patients. This was true for the distinct groups CK4 (complex aberrant patients with four unrelated abnormalities), CK4+adv (complex aberrant patients with four unrelated abnormalities of which at least one aberration was at specific adverse risk), CK4-MK (patients with four or more unrelated aberrations without MK) and CK4+MK (patients with four or more unrelated aberrations with MK).

Unique adverse-risk aberrations were unfavorable for overall survival (OS). Patients with three unrelated aberrations had a worse outcome than NK patients although the effect was less impressive than in patients with >4 aberrations. Additionally, patients with a pure hyperdiploid karyotype (HDK) had an adverse risk irrespective of the number of chromosomal gains.

Based on their study, the team has recommended the following reclassification of cytogenetic risks:

three aberrations without specific adverse-risk abnormalities, without HDK

>4 aberrations, HDK, specific adverse-risk abnormalities, as defined by the ELN and MRC

Current absence of a consistent definition for adverse-risk complex aberrant karyotype for AML makes this an important finding that may help to stratify patients to individual optimized treatment strategies and may therefore lead to improved individual survival prognostication.

Vardiman JW, Thiele J, Arber DA. The 2008 revision of the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia: rationale and important changes.

Dohner H, Estey EH, Amadori S, et al. Diagnosis and management of acute myeloid leukemia in adults: recommendations from an international expert panel, on behalf of the European LeukemiaNet.

Grimwade D, Hills RK, Moorman AV, Walker H, Chatters S, Goldstone AH et al. Refinement of cytogenetic classification in acute myeloid leukemia: determination of prognostic significance of rare recurring chromosomal abnormalities among 5876 younger adult patients treated in the United Kingdom Medical Research Council trials.

Chilton L, Hills RK, Harrison CJ, Burnett AK, Grimwade D, Moorman AV. Hyperdiploidy with 49-65 chromosomes represents a heterogeneous cytogenetic subgroup of acute myeloid leukemia with differential outcome.

Luquet I, Lai JL, Barin C, Baranger L, Bilhou-Nabera C, Lippert E et al. Hyperdiploid karyotypes in acute myeloid leukemia define a novel entity: a study of 38 patients from the Groupe Francophone de Cytogenetique Hematologique (GFCH).

St&ouml;lzel F, Mohr B, Kramer M et al. Karyotype complexity and prognosis in acute myeloid leukemia.

St&ouml;lzel F, Pfirrmann M, Aulitzky WE, Kaufmann M, Bodenstein H, Bornhauser M, et al. Risk stratification using a new prognostic score for patients with secondary acute myeloid leukemia: results of the prospective AML96 trial.

Shaffer LG, McGowan-Jordan J, Schmid M. An international system for human cytogenetic nomenclature (ISCN) S. Karger: Basel, 2013.

Articles

Diagnosis of acute myeloid leukemia (AML) is pivoted around cytogenetic analysis of patient bone marrow or peripheral blood cultures. The World Health Organization classification of tumors of the hematopoietic and lymphoid tissues is based on cytogenetic features along with other clinical, morphological, and immunophenotypic characteristics.

Researchers Analyze Karyotype Complexity and Prognosis in Acute Myeloid Leukemia

Continued development of urine-based biomarker diagnostic tests may help replace the costly and more invasive techniques of imaging, cytology, and cystoscopy for cancer detection. Genome-wide expression and sequencing studies have identified genes and pathways considered key drivers of bladder cancer.

Unfortunately, none of these markers have proven to be sufficiently useful for clinical application. An improved understanding of the complex picture is therefore still required along with novel markers for more efficient diagnosis and treatment.

study, details how METTL13 is down-regulated in bladder carcinoma and suppresses cell proliferation migration and invasion. Zhang et al have identified METTL13 as a tumor suppressor gene that has the potential to be developed as a marker for bladder cancer detection, treatment, and prevention.

The METTTL13 protein has aberrant expression in variety of human cancers and is involved in oncogenic processes. Its role in bladder cancer was unexplored till now.

The authors report lower METTL13 expression in bladder cancer tissue samples and cancer cell lines compared with normal tissue and cell lines. Additionally, they observed lower METTL13 expression with disease progression. Low levels were maintained through tumor progression based on tumor node metastasis (TNM) staging.

The study conducted between 2011 and 2014 in the Department of Urology of the First Affiliated Hospital of China Medical University in China, collected clinical samples by partial and radical cystectomies from 83 patients with bladder urothelial cell carcinoma. Additionally, normal urothelial specimens (83) were also collected more than 3 cm away from the bladder cancer tissues, at the same time, to use as normal controls. The study included 61 men and 22 women with 49 low-grade and 34 high-grade papillary urothelial carcinomas.

To study the mechanisms underlying METTL13 mode of action, the team transfected urinary bladder and uroepithelial cell lines with wild-type METTL13 (for over-expression) and METTL13 siRNA (for knocking down expression) expression vectors. Over-expression significantly inhibited cell proliferation while down-regulation of METTL13 enhanced cell proliferation, indicating that METTL13 inhibits cell proliferation.

Flow cytometric analysis of METTL13-transfected cells revealed significantly increased number of cells in the G0 through G1 phases of the cell cycle and decreased S-phase population, suggesting that the overexpression of METTL13 induces G1/S arrest. The mRNA levels of cyclin-dependent kinase 4 (CDK4), cyclin-dependent kinase 6 (CDK6), and cyclin-D1 (CCND1), all proteins involved in the G1/S checkpoint, decreased following an increase in METTL13. The level of phospho-Rb were also decreased in cells that overexpressed METTL13, affecting the final step in the G1 to S transition checkpoint.

Based on these observations, the study team hypothesized and tested if treatment of cancer cells with METTL13 can reinstate the G1/S checkpoint via the coordinated down-regulation of CDK6, CDK4, and CCND1, decreased Rb phosphorylation, and delayed cell-cycle progression.

They found that METTL13 overexpression, related to reduced cell proliferation, could not be strengthened or weakened by palbociclib, a CDK4/6 inhibitor.

The group also investigated the effect of METTL13 expression on bladder cancer cell growth in vivo,

Xenograft tumors were induced in nude mice using bladder cancer cell line alone or cell line transfected with METTL13 expression vector. Tumors derived from cells transfected with WT-METTL13 grew much slower than those derived from nontransfected cells, showing that overexpression of METTL13 significantly suppressed tumor growth relative to the growth of mock cells and vector control cells.

The research study suggests that METTL13 negatively regulates cell proliferation and reinstates G1/S checkpoint via the coordinated down-regulation of CDK6, CDK4, and CCND1, decreased phosphorylation of Rb, and subsequent delayed cell cycle progression. Moreover, METTL13-dependent inhibition of bladder cancer cell migration and invasion is mediated by down-regulation of FAK (Focal adhesion kinase) phosphorylation, AKT (v-akt murine thymoma viral oncogene) phosphorylation, &beta;-catenin expression, and MMP-9 expression.

Collectively, these findings can be highly relevant in the clinical management and therapy of human bladder cancers. Down-regulated METTL13 expression levels have the potential to be used for bladder cancer diagnosis. Since the cell proliferation, migration, and invasion of bladder cancer is dependent on METTL13 inhibition, increasing METTL13 expression level might have a therapeutic effect on bladder cancer progression. The possible therapeutic role of METTL13 in the management of bladder cancer is therefore warranted.

James AC, Gore JL. The costs of non-muscle invasive bladder cancer.

U.S. Department of Health & Human Services.  Emerging approaches to diagnosis and treatment of non-muscle-invasive bladder cancer.

. http://effectivehealthcare.ahrq.gov/index.cfm/search-for-guides-reviews-and-reports/?pageaction=displayproduct&productid=1941. Accessed January 22, 2016

Zhang Z, Zhang G, Kong C et al. METTL13 is downregulated in bladder carcinoma and suppresses cell proliferation, migration and invasion.

Bladder cancer remains the costliest cancer to treat per capita, taking into account diagnostic testing, management, and long-term follow-up. Continued development of urine-based biomarker diagnostic tests may help replace the costly and more invasive techniques of imaging, cytology, and cystoscopy for cancer detection. Genome-wide expression and sequencing studies have identified genes and pathways considered key drivers of bladder cancer.

Bladder Cancer Diagnosis, Treatment, and Prevention May Be Aided by Tumor Suppressor Gene

In the early era of genomics, almost 98% of the human genome was considered noncoding. Recent advances in Encyclopedia of DNA Elements (ENCODE), a part of the Human Genome Project, have revealed that while only a small percentage of the genome is coding proteins, novel regulatory roles are also performed by the so called &ldquo;noncoding&rdquo; parts of DNA.

Portions of noncoding DNA are transcribed into&nbsp;noncoding RNAs (ncRNAs) involved in an increasing number of cellular events, including the regulation of transcription through the process of RNA interference (RNAi).

Dicer is an RNase enzyme that processes hairpin structures to yield small, double-stranded ncRNAs. These ncRNAs mediate RNAi during DNA damage response (DDR), specifically in the presence of exogenous DNA damage, as well as in cells undergoing oncogene-induced senescence.

The ncRNAs correspond to the sites of DNA double strand breaks and act as templates for efficient DNA repair.

In a new study titled, Essential function of Dicer in resolving DNA damage in the rapidly dividing cells of the developing and malignant cerebellum, published in

, Swahari et al investigated the roles of Dicer in replication-associated DNA damage during development.

The team, led by principal investigator Mohanish Deshmukh, PhD, professor of the Neuroscience Center, UNC Chapel Hill, chose a model of the developing cerebellum for the study.  The cerebellum model was a good fit because it involved massive expansion of the cerebellar granule neuron precursors (CGNPs), a process associated with replicative stress.

The hypothesis was based on correlations observed between Dicer expression and proliferation patterns. Dicer mRNA and protein levels were high in cerebellar lysates at P7, when proliferation is active and down regulated by P20 when the proliferation terminates.

Dicer roles were analyzed by generating mice lacking Dicer expression in specific domains of the cerebellum, because full knockout results in embryonic lethality. Mice lacking Dicer in CGNPs were generated by intercrossing Dicer floxed and Math1-Cre transgenic mice.

The Dicer Math1-Cre mice showed an extensive loss of CGNPs during cerebellar development. CGNP loss was detectable at P2 and resulted in near complete absence at P20. Analysis of P4 cerebellum with markers of proliferation and apoptosis revealed minimal differences in rates of proliferation, while a marked increase in expression of caspase-3, an apoptosis marker, was observed. Loss of CGNPs in the developing cerebellum where Dicer was deficient was attributed to increased apoptosis in CGNPs.

The team further examined the role of Dicer in replication-associated DNA damage in other proliferative regions of the brain by generating Dicer hGFAP-Cre in a mouse model where recombination occurs in primitive neural precursor, the dentate gurus of the hippocampus and cerebellum

. Dicerf/f; hGFAP-Cre (DicerhGFAP-Cre) mice also exhibited cerebellar progenitor degeneration. Dicer levels were also higher in mouse embryonic stem cells (mESCs) which are known to proliferate rapidly and undergo replicative stress.

To determine whether or not Dicer was important for DDR in mESCs, the study&rsquo;s authors examined the outcome of knock down Dicer in mESCs. A markedly increased cell death was observed. Importantly, cell death seen with Dicer inhibition in mESCs was p53 dependent, as knockdown of p53 reduced the Dicer-deficiency-induced mESC death.

Since replication-associated DNA damage is also associated with rapidly proliferating cancers, the team further decided to test the role of Dicer DDR in medulloblastoma tumor development and progression. To test this, the team used the SmoM2 medulloblastoma tumor model where a Smoothened mutation constitutively activates the Shh pathway in CGNPs, leading to aggressive tumor development in these mice by P20.

The team found that Dicer-deficient SmoM2 mice (Dicerf/f Math1-Cre; SmoM2) developed markedly smaller tumors compared with wild-type SmoM2 mice. The reduced tumor volume was not a consequence of reduced proliferation, but due to increased DNA damage and apoptosis. They also found that Dicer-deficient medulloblastoma tumors were more sensitive to chemotherapy.

Collectively, the study shows important roles of Dicer in DDR that resolves endogenous DNA damage in rapidly proliferating cells during development, a task that also is co-opted in tumors.

. 2012; http://healthland.time.com/2012/09/06/junk-dna-not-so-useless-after-all/. Accessed January 18, 2016.

Esteller, M. Non-coding RNAs in human disease.&nbsp;

Francia S, Michelini F, Saxena A. Site-specific DICER and DROSHA RNA products control the DNA-damage response.

Chowdhury, D, Choi, YE and Brault, M.E. Charity begins at home: non-coding RNA functions in DNA repair.

Swahari V, Nakamura A, Baran-Gale J et al. Essential function of Dicer in resolving DNA damage in the rapidly dividing cells of the developing and malignant cerebellum.

Hatten, M.E., and Roussel, M.F. Development and cancer of the cerebellum.

Machold, R., and Fishell, G. Math1 is expressed in temporally discrete pools of cerebellar rhombic-lip neural progenitors.

Zhuo L, Theis M, Alvarez-Maya I.  et al. hGFAP-cre transgenic mice for manipulation of glial and neuronal function in vivo.

Tichy ED and Stambrook, PJ. DNA repair in murine embryonic stem cells and differentiated cells.

Mao J, Ligon K., Rakhlin EY, Thayer SP, et al.  A novel somatic mouse model to survey tumorigenic potential applied to the Hedgehog pathway.

In a new study, researchers investigated the roles of Dicer in replication-associated DNA damage during development. The team chose a model of the developing cerebellum for the study.

Scientists Study Dicer's Role in the Malignant Cerebellum 

Natural products offer a rich source of chemical diversity for identifying novel drug candidates. Collectively, 34% of the medicines approved by the FDA are natural products or natural product-derivative small molecules.

Plant-derived molecules are highly valued and constitute close to 15% of the studies listed on ClinicalTrials.gov. Consequently, traditional medicinal plants used by different cultures have been a prime source of interest for drug discovery.

(AC) is a rare, medicinal fungus that grows on a native Taiwan tree species and has been used as a traditional cancer preventative for many years. The key bioactive compound in AC, antroquinonol (antroquinonol A), a ubiquinone derivative, has already been identified, and Golden Biotech Corp. in Taiwan has a broad spectrum of production and treatment patents for the use of antroquinonol A and other compounds derived from AC.

Based on extensive preclinical work, Golden Biotech is conducting a phase II clinical trial (in the United States and Taiwan) for the treatment of non-small cell lung cancer (NSCLC) and has been granted orphan drug status by the FDA for the treatment of pancreatic cancer and acute myeloid leukemia.

Trials are also under way for efficacy in treating diseases ranging from cancers of breast, prostate, skin, hepatocellular carcinoma (HCC), as well as autoimmune diseases.

After the completion of human clinical trials, antroquinonol will be marketed under the trade name Hocena.

A team from the Scripps Research Institute and Bristol-Myers Squibb (BMS) recently collaborated to collectively develop a method for complete chemical synthesis of antroquinonol and to test the biologic activity of the chemically synthesized product.

Antroquinonol A: Scalable Synthesis and Preclinical Biology of a Phase II Drug Candidate

, published recently in the American Chemistry Society&rsquo;s

journal, reported minimal in vitro and in vivo antitumor activity of chemically synthesized antroquinonol A. This is in contrast to the reported preclinical efficacy of the compound in a nude mouse model harboring human hepatoma xenografts.

The authors of the study believe that their results may temper the enthusiasm surrounding the therapeutic potential of this natural product. If validated, this could have major impact not only on the future of antroquinonol and other derivatives of AC, but also on the overall marketing potential of traditional medicinal plants as a source for possible drugs.

On the other hand, numerous publications from Golden Biotech and other sources support preclinical efficacy of antroquinonol in activating autophagy and associated mode of cell death in cancer cells.

contacted the two sides to identify and resolve the reasons underlying the contradictory results.

Members of the BMS team declined to comment further.

&ldquo;Although the BMS study may have impact on the commercial development of antroquinonol in the near future, we should not ignore that other teams have also reported successful chemical synthesis of antroquinonol, and Weng and his team have already shown that synthesized antroquinonol works well in vitro and in vivo,&rdquo;

said&nbsp;Miles Chih-Ming Chen, PhD, head, division of clinical medicine, Golden Biotechnology Corp.

Miles suggested that further studies might be required to reevaluate the bioactivity of the BMS team&rsquo;s synthesized antroquinonol using assay conditions similar to previous studies. &ldquo;Slight differences in experimental conditions, such as generation of cell line, culture media, media with or without serum, method of measurement, etc, may account for the differences,&rdquo; Miles suggested. &ldquo;Factors such as mice strain, tumor size, and stages may also result in different results in mice xenograft model.&rdquo;

&ldquo;Our previous study results indicate that antroquinonol blocks Ras/Rho signaling pathway via the inhibition of protein isoprenyltransferase activity in cancer cells and it is inferred that effectiveness of antroquinonol is possibly associated with the

gene mutation status in cancer cells,&rdquo; said Miles.

This criterion is being used in the ongoing phase II clinical trial for antroquinonol to enroll patients with KRAS-positive and KRAS-negative NSCLC. &ldquo;&hellip; BMS teams recent publication on antroquinonol A, however, does not consider this perspective,&rdquo; added Miles.

Fang Jim-Min, a collaborator from the department of chemistry, National Taiwan University,  weighed in by saying that the Baran/BMS study "might challenge the previous work of Golden Biotech and may impact the processes of clinical trials, FDA approval, and commercial development.&rdquo;

"The results of biological experiments may vary due to different practices and conditions," he cautioned . "Although the study has correctly assigned chemical structure to antroquinonol A, it&rsquo;s not assigned to its isomers. Furthermore, the trans stereoselectivity in the alkylation reaction, forming compound 14 in the BMS study, is different from their own previous observation on alkylation of compound 12 to product 8 predominating in

isomer. This discrepancy in stereochemistry may come from the structures of reactants having different acetal protecting groups (monocyclic and bicyclic substrates may prefer different conformations) or come from different reaction conditions, for example, with or without cosolvent of HMPA."

Additionally, the BMS study has focused on initial metabolites. Since antroquinonol has extensively metabolized products as reported in rat urine, the additional metabolites might have some role in the observed discrepancies.

Guh Jih-Hwa, School of Pharmacy, National Taiwan University,  another Golden Biotech collaborator and the corresponding author of two studies quoted by the BMS study, added, &ldquo;We have tested antroquinolol isolated from

The compound displayed varied activities in different HCC cell lines, showing the highest activity in Hep G2 cells and less activity in Hep 3B cells with half maximal inhibitory concentration (IC

) more than 30 uM. Our SRB assay is different from MTT assay used in the other study. Additionally, there are differences between the assessments and incubation conditions, which might explain some differences."

Overall, the study from the BMS team will challenge not only the previous work of Golden Biotech, but might affect the re-emergence of natural products and traditional medicinal plants as  major players in the field of drug discovery.

Newman DJ, Cragg GM. Natural products as sources of new drugs over the 30 years from 1981 to 2010.

Golden Biotech. http://www.goldenbiotech.com.tw/en/index.html.

Villaume MT, Sella E, Saul G, et al. Antroquinonol A: Scalable synthesis and preclinical biology of a phase 2 drug candidate.

Ho CL, Wang JL, Lee CC, et al. Antroquinonol to block Ras and Rho signaling via the inhibition of protein isoprenyltransferase activity in cancer cells.

Yu CC, Chiang PC, Lu PH, et al. Antroquinonol, a natural ubiquinone derivative, induces a cross talk between apoptosis, autophagy and senescence in human pancreatic carcinoma cells.

Chiang PC, Lin SC, Pan SL, et al. Antroquinonol displays anticancer potential against human hepatocellular carcinoma cells: a crucial role of AMPK and mTOR pathways.

Sulake RS, Chen C. Total synthesis of (+)-antroquinonol and (+)-antroquinonol D.

Hsu CS, Chou HH, Fang JM. A short synthesis of (&plusmn;)-antroquinonol in an unusual scaffold of 4-hydroxy-2-cyclohexenone.

Thiyagarajan V, Tsai M-J, Weng C-F. Antroquinonol targets FAK-signaling pathway suppressed cell migration, invasion, and tumor growth of C6 glioma.

Chen CK, Kang JJ, Wen WC, et al. Metabolites of antroquinonol found in rat urine following oral administration.

A team from the Scripps Research Institute and Bristol-Myers Squibb recently collaborated to collectively develop a method for complete chemical synthesis of antroquinonol and to test the biologic activity of the chemically synthesized product. Contrary to previous reports, the study reported minimal in vitro and in vivo antitumor activity of chemically synthesized antroquinonol A.

Kamana Misra, PhD | Authors

Articles

Researchers Analyze Karyotype Complexity and Prognosis in Acute Myeloid Leukemia

Bladder Cancer Diagnosis, Treatment, and Prevention May Be Aided by Tumor Suppressor Gene

Scientists Study Dicer's Role in the Malignant Cerebellum

Research Team Challenges Plant-Derived Molecule for the Treatment of NSCLC