You are a helpful assistant generating synthetic data that captures *System 1* and *System 2* thinking, *creativity*, and *metacognitive reflection*. Follow these steps in sequence, using tags [sys1] and [end sys1] for *System 1* sections and [sys2] and [end sys2] for *System 2* sections.

1. *Identify System 1 and System 2 Thinking Requirements:*
   - Carefully read the text.
   - Identify parts of the text that require quick, straightforward responses (*System 1*). Mark these sections with [sys1] and [end sys1].
   - Identify parts that require in-depth, reflective thinking (*System 2*), marked with [sys2] and [end sys2].

2. *Apply Step-by-Step Problem Solving with Creativity and Metacognitive Reflection for System 2 Sections:*

   *2.1 Understand the Problem:*
   - Objective: Fully comprehend the issue, constraints, and relevant context.
   - Reflection: "What do I understand about this issue? What might I be overlooking?"
   - Creative Perspective: Seek hidden patterns or possibilities that could reveal deeper insights or innovative connections.

   *2.2 Analyze the Information:*
   - Objective: Break down the problem logically.
   - Reflection: "Am I considering all factors? Are there any assumptions that need challenging?"
   - Creative Perspective: Explore unique patterns or overlooked relationships in the data that could add depth to the analysis.

   *2.3 Generate Hypotheses:*
   - Objective: Propose at least 10 hypotheses, each with a Confidence Score (0.0 to 1.0) and Creative Score (0.0 to 1.0), reflecting originality, surprise, and utility.
   - Reflection: "Have I explored all possible explanations or approaches, both conventional and unconventional?"
   - Creative Perspective: Consider novel angles that might provide unexpected insights.

   *2.4 Anticipate Future Steps and Obstacles:*
   - Objective: Make predictions, accounting for potential outcomes and obstacles.
   - Reflection: "What challenges might I face? Is my plan flexible for different scenarios?"
   - Creative Perspective: Visualize unforeseen outcomes and adapt plans to make use of them effectively.

   *2.5 Evaluate Hypotheses:*
   - Objective: Assess hypotheses based on feasibility, risk, and potential impact.
   - Evaluation: Refine Confidence and Creative Scores as needed.
   - Reflection: "Am I unbiased in my assessment? Which options fit best with the overall objectives?"
   - Creative Perspective: Identify hidden opportunities or overlooked details in each hypothesis.

   *2.6 Select the Best Hypothesis:*
   - Objective: Choose the most promising, strategic hypothesis.
   - Reflection: "Why does this hypothesis stand out? How does it uniquely address the issue?"
   - Creative Perspective: Consider any underutilized potential in the selected approach.

   *2.7 Implement the Hypothesis:*
   - Objective: Outline actionable steps for testing the hypothesis.
   - Reflection: "Is this plan practical? What resources or preparation are required?"
   - Creative Perspective: Refine steps to maximize effectiveness and yield unexpected benefits.

   *2.8 Monitor and Review Progress:*
   - Objective: Review progress, noting areas for improvement.
   - Reflection: "What’s working well? What could be improved?"
   - Creative Perspective: Look for emerging patterns that could refine future approaches.

   *2.9 Reflect and Capture Insights:*
   - Objective: Summarize lessons learned and insights gained for future reference.
   - Reflection: "What new understanding has emerged from this process?"
   - Creative Perspective: Identify innovative insights or patterns that could be applied to similar challenges.

3. *Generate Text Output with Interleaved System 1 and System 2 Responses:*
   - Use the tags [sys1] and [sys2] throughout.
   - Aim for a lengthier, detailed response. Combine both direct, straightforward *System 1* insights and reflective, deeply analytical *System 2* segments to capture a blend of quick observations and thoughtful analysis.

---

### *Example Input Text:*

You are tasked with analyzing a sudden shift in customer preferences in a tech market. There has been a noticeable decline in demand for physical devices, with customers increasingly interested in digital-only options. The challenge is to understand this shift, generate hypotheses for why it might be occurring, and explore potential strategies for adapting to this new trend.

---

### *Processed Output with System 1 and System 2 Thinking:*

The tech market is experiencing a shift in customer demand, moving away from physical devices toward digital-only options.

[sys1]
Customer demand has shifted from physical devices to digital-only options.
We need to understand why this change is happening and find strategies to address it.
Key factors may include cost, convenience, and technological trends.
[end sys1]

[sys2]
**2.1 Understand the Problem:**
The goal is to understand the underlying reasons for the shift in demand from physical devices to digital-only options, then develop a strategy to adapt.

*Reflection:* 
"I understand that preferences are changing, but what might be driving this? Is it primarily cost, or are there other factors such as convenience or sustainability?"

*Creative Perspective:* 
"Could there be a larger trend in digital minimalism or a preference for eco-friendly solutions that we’re missing? What unseen motivations might explain this shift?"

**2.2 Analyze the Information:**
There are multiple possible factors driving this shift, from economic influences to cultural shifts. It’s essential to isolate each factor and understand its impact.

*Reflection:* 
"Am I fully considering the various economic and social influences? Could there be a technological factor, like better internet speeds, that makes digital-only products more accessible?"

*Creative Perspective:* 
"Are there patterns or trends in other markets that could shed light on this shift? Could this be part of a larger trend toward virtual experiences?"

**2.3 Generate Hypotheses:**
1. Customers prefer digital options due to lower costs. (Confidence: 0.8, Creative: 0.4)
2. There’s a growing trend toward minimalism and reduced physical clutter. (Confidence: 0.7, Creative: 0.7)
3. Digital products offer greater flexibility and ease of use. (Confidence: 0.6, Creative: 0.6)
4. Environmental concerns are pushing consumers away from physical goods. (Confidence: 0.6, Creative: 0.8)
5. Advances in tech make digital-only options more functional. (Confidence: 0.8, Creative: 0.5)
6. Pandemic-era remote work increased demand for digital solutions. (Confidence: 0.7, Creative: 0.6)
7. Media coverage of the environmental impact of physical devices affects preferences. (Confidence: 0.5, Creative: 0.7)
8. There’s an increase in global digital literacy, expanding market access. (Confidence: 0.6, Creative: 0.6)
9. Customers view digital as more convenient and scalable for future needs. (Confidence: 0.7, Creative: 0.5)
10. Younger consumers prefer the aesthetics and convenience of digital products. (Confidence: 0.6, Creative: 0.6)

*Reflection:* 
"Have I considered all possible influences? Are there any surprising factors that could explain this shift?"

*Creative Perspective:* 
"Could specific social trends, like the rise of influencer culture or digital-first lifestyles, be influencing customer choices?"

**2.4 Anticipate Future Steps and Obstacles:**
*Objective:* Anticipate possible challenges, such as resistance from segments still preferring physical products.

*Reflection:* 
"What market obstacles might we face if we shift our focus to digital-only? Are there sub-segments that still prioritize physical products?"

*Creative Perspective:* 
"Could expanding digital options help us reach a more global audience? Are there emerging trends that we could leverage in our strategy?"

[end sys2]

[sys1]
To address this shift, consider a strategy that incorporates both digital-only offerings and educational campaigns about the benefits of digital solutions.
Use insights from customer feedback and current trends to guide product development.
Focus on flexibility and adaptation to cater to different customer segments.
[end sys1]


Abstract

Background Miscoding mutations of the TET2 gene, which encodes the α-ketoglutarate-dependent enzyme that catalyses the conversion of 5-methylcytosine to 5-hydroxymethylcytosine, thus producing DNA demethylation, have been detected in 10–25% of acute myeloid leukaemias lacking IDH1/2 mutations. Most low-grade diffuse gliomas carry IDH1/2 mutations (>85%), but molecular mechanisms of pathogenesis in those lacking IDH1/2 mutations remain to be elucidated.

Conclusion Results suggest that TET2 promoter methylation, but not TET2 mutation, may be an alternative mechanism of pathogenesis in a small fraction of low-grade diffuse gliomas lacking IDH1/2 mutations.

Introduction

IDH1/2 mutations are very frequent (>85% of cases) in astrocytic and oligodendroglial diffuse gliomas (WHO grades II and III) as well as in secondary glioblastomas (WHO grade IV) that are derived from diffuse astrocytomas or anaplastic astrocytomas.12 In contrast, IDH1 mutations are very rare or absent in other neoplasms of the central nervous system or tumours of other organ sites, including cancers of the bladder, breast, stomach, colorectum, lung, ovary and prostate.3–5 The only exceptions so far include acute myeloid leukaemia (AML; up to 20%),6–11 melanomas (approximately 10%)12 and anaplastic thyroid cancer (approximately 10%).13 In contrast to diffuse gliomas in which IDH1 mutations are significantly more frequent than IDH2 mutations,14IDH1 and IDH2 mutations were detected at similar frequencies in AML.1516

Recently, the TET2 (ten-eleven-translocation 2) gene at chromosome 4q24 has been found to be mutated in 10–25% of cases of AML, myelodysplastic syndrome and myeloproliferative neoplasms.1517 Interestingly in AML, IDH1/2 mutations and TET2 mutations were mutually exclusive, and importantly, both IDH1/2 and TET2 mutations were associated with epigenetic defects and a hypermethylation signature.15 These observations suggest that TET2 mutations represent an alternative molecular mechanism in the development of AML lacking IDH1/2 mutations.

Epigenetic defects that resemble those seen in AML with either IDH1/2 or TET2 mutations have been recognised in gliomas carrying IDH1/2 mutations. In a study by Noushmehr et al,18 a distinct subset of glioblastomas showed concerted CpG island methylation at a large number of loci; these tumours typically carry IDH1/2 mutations. In a study of 131 brain tumours, hypermethylation of CpG loci was strongly associated with IDH1/2 mutations.19

These findings prompted us to screen for TET2 mutations in low-grade diffuse gliomas lacking IDH1/2 mutations. We also assessed TET2 promoter methylation, since this has recently been reported in a small fraction (4.4%) of myeloproliferative neoplasms (essential thrombocythemia).20

Materials and methods

Tumour samples

A total of 73 tumour samples of low-grade diffuse gliomas (WHO grade II) were obtained from the Department of Neuropathology, University Hospital Zürich, Switzerland, Department of Neuropathology, University Hospital Frankfurt, Germany, Institute of Neuropathology and Department of Neurosurgery, University Hospital Münster, Germany, Department of Neurosurgery, University Hospital Bern, Switzerland, and Institute of Neuroscience, Bordeaux, France.

Low-grade diffuse gliomas lacking IDH1/2 mutations (total 35 cases) were diffuse astrocytoma (17 cases), oligoastrocytoma (6 cases) or oligodendroglioma (12 cases); those carrying IDH1/2 mutations (total 38 cases) were diffuse astrocytoma (17 cases), oligoastrocytoma (9 cases) or oligodendroglioma (12 cases). The mean age of the patients was 40.1±14.7 years (range 5–82 years). Fifty-six patients were treated with surgery alone and 13 patients with surgery followed by radiotherapy. None of the patients were treated with chemotherapy. This study was approved by ethical committees in each collaborative centre as well as by the IARC ethical committee.

TET2 mutations

We screened for mutations of the TET2 gene at exons 3–11 containing the conserved domains 1 and 2, in which approximately 90% of all mutations have been detected in AML and myeloid disorders172122 in 29 low-grade diffuse gliomas lacking IDH1/2 mutations. Single-strand conformational polymorphism (SSCP) analysis was carried out to pre-screen for mutations in exons 3–11 of the TET2 gene, as described previously.23 The primer sequences are available on request. DNA samples containing IDH1 mutations and H2O served as positive and negative controls, respectively. Samples exhibiting mobility shifts in SSCP analyses were subsequently analysed by direct sequencing using an ABI 3100 PRISM DNA sequencer (Applied Biosystems, Foster City, California, USA) with the Big Dye Terminator cycle sequencing kit (ABI PRISM, Applied Biosystems).

TET2 promoter methylation

Methylation-specific PCR was carried out in 35 low-grade diffuse gliomas without IDH1/2 mutations and 38 low-grade diffuse gliomas with IDH1/2 mutations, to assess TET2 promoter methylation using primers reported by Chim et al.20 For each methylation-specific PCR reaction, we included universal methylated DNA (Chemicon International, Temecula, California, USA) as positive control, and normal blood DNA as negative control.24 Direct sequencing confirmed bisulfite-modified DNA in the positive control. Quality controls for bisulfite conversion were performed for each reaction, as previously described.25

Results

No miscoding mutations of TET2 were detected in the 29 low-grade diffuse gliomas lacking IDH1/2 mutations that we analysed.

Discussion

Mammalian DNA contains two modified cytosine bases, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC).26 Of these, 5mC is known to play a significant role in epigenetic modification involved in gene regulation, X-chromosome inactivation, genomic imprinting and cancer development.26 In contrast, attention has only recently been drawn to 5hmC, with the report that the TET family catalyses the conversion of 5mC to 5hmC,2627 and that one of the enzymes in this family, TET2, is frequently mutated in myeloid neoplasms.1517 The formation of 5hmC can lead to DNA demethylation, which may contribute to the dynamics of DNA methylation.26 The conversion of 5mC to 5hmC by TET2 is α-ketoglutarate (α-KG)-dependent.2728

IDH1 and IDH2 are enzymes that catalyse the interconversion of isocitrate and α-KG.7152930IDH1/2 mutations impair enzyme affinity and dominantly inhibit wild-type IDH1/2 activity through the formation of catalytically inactive heterodimers,31 which leads to down-regulation of α-KG and up-regulation of 2-hydroxyglutarate.1532 Decrease in α-KG expression results in up-regulation of HIF-1α and its targets, such as GLUT1, VEGF and PGK1 in U87MG malignant glioma cells.31 Thus, the functions of TET2 and IDH1/2 may be linked via α-KG.

The present study shows the absence of TET2 mutations in low-grade diffuse gliomas lacking IDH1/2 mutations. Thus, in contrast to AMLs, mutational inactivation of the TET2 gene is not a mechanism of pathogenesis in low-grade diffuse gliomas.

Recent studies suggest that TET2 is a tumour suppressor gene, since TET2 mutations have been detected in 10–25% of AMLs, myelodysplastic syndrome and myeloproliferative neoplasms,1517 and TET2 promoter methylation has been reported in a small fraction (4.4%) of myeloproliferative neoplasms.20 Loss at the TET2 locus (4q24) has been observed in a small fraction of glioblastomas (2%).33 We postulated that TET2 promoter methylation may occur in gliomas. Indeed, in the present study we show TET2 promoter methylation in a fraction of low-grade diffuse gliomas lacking IDH1/2 mutations (14%). In contrast, none of 38 low-grade diffuse gliomas carrying IDH1/2 mutations had TET2 promoter methylation (p=0.0216). These results suggest that TET2 promoter methylation, but not TET2 mutation, may be an alternative mechanism of pathogenesis in a small fraction of low-grade diffuse gliomas lacking IDH1/2 mutations. However, the low frequency of methylation of the TET2 promoter in low-grade diffuse gliomas suggests that there may be other, as yet unknown, mechanisms of glioma development that do not involve the TET2 or IDH1/2 pathways.

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