@bgicli/bgicli 2.1.1 → 2.2.0

package/data/skills/tooluniverse-precision-oncology/SKILL.md

@@ -0,0 +1,1091 @@
+---
+name: tooluniverse-precision-oncology
+description: Provide actionable treatment recommendations for cancer patients based on molecular profile. Interprets tumor mutations, identifies FDA-approved therapies, finds resistance mechanisms, matches clinical trials. Use when oncologist asks about treatment options for specific mutations (EGFR, KRAS, BRAF, etc.), therapy resistance, or clinical trial eligibility.
+---
+
+# Precision Oncology Treatment Advisor
+
+Provide actionable treatment recommendations for cancer patients based on their molecular profile using CIViC, ClinVar, OpenTargets, ClinicalTrials.gov, and structure-based analysis.
+
+**KEY PRINCIPLES**:
+1. **Report-first** - Create report file FIRST, update progressively
+2. **Evidence-graded** - Every recommendation has evidence level
+3. **Actionable output** - Prioritized treatment options, not data dumps
+4. **Clinical focus** - Answer "what should we do?" not "what exists?"
+5. **English-first queries** - Always use English terms in tool calls (mutations, drug names, cancer types), even if the user writes in another language. Only try original-language terms as a fallback. Respond in the user's language
+
+---
+
+## When to Use
+
+Apply when user asks:
+- "Patient has [cancer] with [mutation] - what treatments?"
+- "What are options for EGFR-mutant lung cancer?"
+- "Patient failed [drug], what's next?"
+- "Clinical trials for KRAS G12C?"
+- "Why isn't [drug] working anymore?"
+
+---
+
+## Phase 0: Tool Verification
+
+**CRITICAL**: Verify tool parameters before first use.
+
+| Tool | WRONG | CORRECT |
+|------|-------|---------|
+| `civic_get_variant` | `variant_name` | `id` (numeric) |
+| `civic_get_evidence_item` | `variant_id` | `id` |
+| `OpenTargets_*` | `ensemblID` | `ensemblId` (camelCase) |
+| `search_clinical_trials` | `disease` | `condition` |
+
+---
+
+## Workflow Overview
+
+```
+Input: Cancer type + Molecular profile (mutations, fusions, amplifications)
+
+Phase 1: Profile Validation
+├── Validate variant nomenclature
+├── Resolve gene identifiers
+└── Confirm cancer type (EFO/ICD)
+
+Phase 2: Variant Interpretation
+├── CIViC → Evidence for each variant
+├── ClinVar → Pathogenicity
+├── COSMIC → Somatic mutation frequency
+├── GDC/TCGA → Real tumor data
+├── DepMap → Target essentiality
+├── OncoKB → FDA actionability levels (NEW)
+├── cBioPortal → Cross-study mutation data (NEW)
+├── Human Protein Atlas → Expression validation (NEW)
+├── OpenTargets → Target-disease evidence
+└── OUTPUT: Variant significance table + target validation + expression
+
+Phase 2.5: Tumor Expression Context (NEW)
+├── CELLxGENE → Cell-type specific expression in tumor
+├── ChIPAtlas → Regulatory context
+├── Cancer-specific expression patterns
+└── OUTPUT: Expression validation
+
+Phase 3: Treatment Options
+├── Approved therapies (FDA label)
+├── NCCN-recommended (literature)
+├── Off-label with evidence
+└── OUTPUT: Prioritized treatment list
+
+Phase 3.5: Pathway & Network Analysis (NEW)
+├── KEGG/Reactome → Pathway context
+├── IntAct → Protein interactions
+├── Drug combination rationale
+└── OUTPUT: Biological context for combinations
+
+Phase 4: Resistance Analysis (if prior therapy)
+├── Known resistance mechanisms
+├── Structure-based analysis (NvidiaNIM)
+├── Network-based bypass pathways (IntAct)
+└── OUTPUT: Resistance explanation + strategies
+
+Phase 5: Clinical Trial Matching
+├── Active trials for indication + biomarker
+├── Eligibility filtering
+└── OUTPUT: Matched trials
+
+Phase 5.5: Literature Evidence (NEW)
+├── PubMed → Published evidence
+├── BioRxiv/MedRxiv → Recent preprints
+├── OpenAlex → Citation analysis
+└── OUTPUT: Supporting literature
+
+Phase 6: Report Synthesis
+├── Executive summary
+├── Treatment recommendations (prioritized)
+└── Next steps
+```
+
+---
+
+## Phase 1: Profile Validation
+
+### 1.1 Resolve Gene Identifiers
+
+```python
+def resolve_gene(tu, gene_symbol):
+    """Resolve gene to all needed IDs."""
+    ids = {}
+    
+    # Ensembl ID (for OpenTargets)
+    gene_info = tu.tools.MyGene_query_genes(q=gene_symbol, species="human")
+    ids['ensembl'] = gene_info.get('ensembl', {}).get('gene')
+    
+    # UniProt (for structure)
+    uniprot = tu.tools.UniProt_search(query=gene_symbol, organism="human")
+    ids['uniprot'] = uniprot[0].get('primaryAccession') if uniprot else None
+    
+    # ChEMBL target
+    target = tu.tools.ChEMBL_search_targets(query=gene_symbol, organism="Homo sapiens")
+    ids['chembl_target'] = target[0].get('target_chembl_id') if target else None
+    
+    return ids
+```
+
+### 1.2 Validate Variant Nomenclature
+
+- **HGVS protein**: p.L858R, p.V600E
+- **cDNA**: c.2573T>G
+- **Common names**: T790M, G12C
+
+---
+
+## Phase 2: Variant Interpretation
+
+### 2.1 CIViC Evidence Query
+
+```python
+def get_civic_evidence(tu, gene_symbol, variant_name):
+    """Get CIViC evidence for variant."""
+    # Search for variant
+    variants = tu.tools.civic_search_variants(query=f"{gene_symbol} {variant_name}")
+    
+    evidence_items = []
+    for var in variants:
+        # Get evidence items for this variant
+        evi = tu.tools.civic_get_variant(id=var['id'])
+        evidence_items.extend(evi.get('evidence_items', []))
+    
+    # Categorize by evidence type
+    return {
+        'predictive': [e for e in evidence_items if e['evidence_type'] == 'Predictive'],
+        'prognostic': [e for e in evidence_items if e['evidence_type'] == 'Prognostic'],
+        'diagnostic': [e for e in evidence_items if e['evidence_type'] == 'Diagnostic']
+    }
+```
+
+### 2.2 COSMIC Somatic Mutation Analysis (NEW)
+
+```python
+def get_cosmic_mutations(tu, gene_symbol, variant_name=None):
+    """Get somatic mutation data from COSMIC database."""
+    
+    # Get all mutations for gene
+    gene_mutations = tu.tools.COSMIC_get_mutations_by_gene(
+        operation="get_by_gene",
+        gene=gene_symbol,
+        max_results=100,
+        genome_build=38
+    )
+    
+    # If specific variant, search for it
+    if variant_name:
+        specific = tu.tools.COSMIC_search_mutations(
+            operation="search",
+            terms=f"{gene_symbol} {variant_name}",
+            max_results=20
+        )
+        return {
+            'specific_variant': specific.get('results', []),
+            'all_gene_mutations': gene_mutations.get('results', [])
+        }
+    
+    return gene_mutations
+
+def get_cosmic_hotspots(tu, gene_symbol):
+    """Identify mutation hotspots in COSMIC."""
+    mutations = tu.tools.COSMIC_get_mutations_by_gene(
+        operation="get_by_gene",
+        gene=gene_symbol,
+        max_results=500
+    )
+    
+    # Count by position
+    position_counts = Counter(m['MutationAA'] for m in mutations.get('results', []))
+    hotspots = position_counts.most_common(10)
+    
+    return hotspots
+```
+
+**Why COSMIC matters**:
+- **Gold standard** for somatic cancer mutations
+- Provides cancer type distribution (which cancers have this mutation)
+- FATHMM pathogenicity prediction for novel variants
+- Identifies hotspots vs. rare mutations
+
+### 2.3 GDC/TCGA Pan-Cancer Analysis (NEW)
+
+Access real patient tumor data from The Cancer Genome Atlas:
+
+```python
+def get_tcga_mutation_data(tu, gene_symbol, cancer_type=None):
+    """
+    Get somatic mutations from TCGA via GDC.
+    
+    Answers: "How often is this mutation seen in real tumors?"
+    """
+    
+    # Get mutation frequency across all TCGA
+    frequency = tu.tools.GDC_get_mutation_frequency(
+        gene_symbol=gene_symbol
+    )
+    
+    # Get specific mutations
+    mutations = tu.tools.GDC_get_ssm_by_gene(
+        gene_symbol=gene_symbol,
+        project_id=f"TCGA-{cancer_type}" if cancer_type else None,
+        size=50
+    )
+    
+    return {
+        'frequency': frequency.get('data', {}),
+        'mutations': mutations.get('data', {}),
+        'note': 'Real patient tumor data from TCGA'
+    }
+
+def get_tcga_expression_profile(tu, gene_symbol, cancer_type):
+    """Get gene expression data from TCGA."""
+    
+    # Map cancer type to TCGA project
+    project_map = {
+        'lung': 'TCGA-LUAD',
+        'breast': 'TCGA-BRCA', 
+        'colorectal': 'TCGA-COAD',
+        'melanoma': 'TCGA-SKCM',
+        'glioblastoma': 'TCGA-GBM'
+    }
+    project_id = project_map.get(cancer_type.lower(), f'TCGA-{cancer_type.upper()}')
+    
+    expression = tu.tools.GDC_get_gene_expression(
+        project_id=project_id,
+        size=20
+    )
+    
+    return expression.get('data', {})
+
+def get_tcga_cnv_status(tu, gene_symbol, cancer_type):
+    """Get copy number status from TCGA."""
+    
+    project_map = {
+        'lung': 'TCGA-LUAD',
+        'breast': 'TCGA-BRCA'
+    }
+    project_id = project_map.get(cancer_type.lower(), f'TCGA-{cancer_type.upper()}')
+    
+    cnv = tu.tools.GDC_get_cnv_data(
+        project_id=project_id,
+        gene_symbol=gene_symbol,
+        size=20
+    )
+    
+    return cnv.get('data', {})
+```
+
+**GDC Tools Summary**:
+| Tool | Purpose | Key Parameters |
+|------|---------|----------------|
+| `GDC_get_mutation_frequency` | Pan-cancer mutation stats | `gene_symbol` |
+| `GDC_get_ssm_by_gene` | Specific mutations | `gene_symbol`, `project_id` |
+| `GDC_get_gene_expression` | RNA-seq data | `project_id` |
+| `GDC_get_cnv_data` | Copy number | `project_id`, `gene_symbol` |
+| `GDC_list_projects` | Find TCGA projects | `program="TCGA"` |
+
+**Why TCGA/GDC matters**:
+- **Real patient data** - Not cell line or curated, actual tumor sequencing
+- **Pan-cancer view** - Same gene across 33 cancer types
+- **Multi-omic** - Mutations, expression, CNV together
+- **Clinical correlation** - Survival data available
+
+### 2.4 DepMap Target Validation (NEW)
+
+Assess gene essentiality using CRISPR knockout data from cancer cell lines:
+
+```python
+def assess_target_essentiality(tu, gene_symbol, cancer_type=None):
+    """
+    Is this gene essential in cancer cell lines?
+    
+    Essential genes have negative dependency scores.
+    Answers: "If we target this gene, will cancer cells die?"
+    """
+    
+    # Get gene dependency data
+    dependencies = tu.tools.DepMap_get_gene_dependencies(
+        gene_symbol=gene_symbol
+    )
+    
+    # Get cell lines for specific cancer type
+    if cancer_type:
+        cell_lines = tu.tools.DepMap_get_cell_lines(
+            cancer_type=cancer_type,
+            page_size=20
+        )
+        return {
+            'gene': gene_symbol,
+            'dependencies': dependencies.get('data', {}),
+            'cell_lines': cell_lines.get('data', {}),
+            'interpretation': 'Negative scores = gene is essential for cell survival'
+        }
+    
+    return dependencies
+
+def get_depmap_drug_sensitivity(tu, drug_name, cancer_type=None):
+    """Get drug sensitivity data from DepMap."""
+    
+    drugs = tu.tools.DepMap_get_drug_response(
+        drug_name=drug_name
+    )
+    
+    return drugs.get('data', {})
+```
+
+**DepMap Tools Summary**:
+| Tool | Purpose | Key Parameters |
+|------|---------|----------------|
+| `DepMap_get_gene_dependencies` | CRISPR essentiality | `gene_symbol` |
+| `DepMap_get_cell_lines` | Cell line metadata | `cancer_type`, `tissue` |
+| `DepMap_search_cell_lines` | Search by name | `query` |
+| `DepMap_get_drug_response` | Drug sensitivity | `drug_name` |
+
+**Why DepMap matters for Precision Oncology**:
+- **Target validation** - Proves gene is essential for cancer survival
+- **Cancer selectivity** - Essential in cancer but not normal cells?
+- **Resistance prediction** - What other genes become essential when you knockout target?
+- **Combination rationale** - Identify synthetic lethal partners
+
+**Example Clinical Application**:
+```markdown
+### Target Essentiality Assessment (DepMap)
+
+**KRAS dependency in pancreatic cancer cell lines**:
+| Cell Line | KRAS Effect Score | Interpretation |
+|-----------|-------------------|----------------|
+| PANC-1 | -0.82 | Strongly essential |
+| MIA PaCa-2 | -0.75 | Essential |
+| BxPC-3 | -0.21 | Less dependent (KRAS WT) |
+
+*Interpretation: KRAS-mutant pancreatic cancer lines are highly dependent on KRAS - validates targeting strategy.*
+
+*Source: DepMap via `DepMap_get_gene_dependencies`*
+```
+
+### 2.5 OncoKB Actionability Assessment (NEW)
+
+OncoKB provides FDA-approved therapeutic actionability annotations:
+
+```python
+def get_oncokb_annotations(tu, gene_symbol, variant_name, tumor_type=None):
+    """
+    Get OncoKB actionability annotations.
+    
+    OncoKB Level of Evidence:
+    - Level 1: FDA-approved
+    - Level 2: Standard care
+    - Level 3A: Compelling clinical evidence
+    - Level 3B: Standard care in different tumor type
+    - Level 4: Biological evidence
+    - R1/R2: Resistance evidence
+    """
+    
+    # Annotate the specific variant
+    annotation = tu.tools.OncoKB_annotate_variant(
+        operation="annotate_variant",
+        gene=gene_symbol,
+        variant=variant_name,  # e.g., "V600E"
+        tumor_type=tumor_type  # OncoTree code e.g., "MEL", "LUAD"
+    )
+    
+    result = {
+        'oncogenic': annotation.get('data', {}).get('oncogenic'),
+        'mutation_effect': annotation.get('data', {}).get('mutationEffect'),
+        'highest_sensitive_level': annotation.get('data', {}).get('highestSensitiveLevel'),
+        'treatments': annotation.get('data', {}).get('treatments', [])
+    }
+    
+    # Get gene-level info
+    gene_info = tu.tools.OncoKB_get_gene_info(
+        operation="get_gene_info",
+        gene=gene_symbol
+    )
+    
+    result['is_oncogene'] = gene_info.get('data', {}).get('oncogene', False)
+    result['is_tumor_suppressor'] = gene_info.get('data', {}).get('tsg', False)
+    
+    return result
+
+def get_oncokb_cnv_annotation(tu, gene_symbol, alteration_type, tumor_type=None):
+    """Get OncoKB annotation for copy number alterations."""
+    
+    annotation = tu.tools.OncoKB_annotate_copy_number(
+        operation="annotate_copy_number",
+        gene=gene_symbol,
+        copy_number_type=alteration_type,  # "AMPLIFICATION" or "DELETION"
+        tumor_type=tumor_type
+    )
+    
+    return {
+        'oncogenic': annotation.get('data', {}).get('oncogenic'),
+        'treatments': annotation.get('data', {}).get('treatments', [])
+    }
+```
+
+**OncoKB Level Mapping**:
+| OncoKB Level | Our Tier | Description |
+|--------------|----------|-------------|
+| LEVEL_1 | ★★★ | FDA-recognized biomarker |
+| LEVEL_2 | ★★★ | Standard care |
+| LEVEL_3A | ★★☆ | Compelling clinical evidence |
+| LEVEL_3B | ★★☆ | Different tumor type |
+| LEVEL_4 | ★☆☆ | Biological evidence |
+| LEVEL_R1 | Resistance | FDA-approved resistance marker |
+| LEVEL_R2 | Resistance | Compelling resistance evidence |
+
+### 2.6 cBioPortal Cross-Study Analysis (NEW)
+
+Aggregate mutation data across multiple cancer studies:
+
+```python
+def get_cbioportal_mutations(tu, gene_symbols, study_id="brca_tcga"):
+    """
+    Get mutation data from cBioPortal across cancer studies.
+    
+    Provides: Mutation types, protein changes, co-mutations.
+    """
+    
+    # Get mutations for genes in study
+    mutations = tu.tools.cBioPortal_get_mutations(
+        study_id=study_id,
+        gene_list=",".join(gene_symbols)  # e.g., "EGFR,KRAS"
+    )
+    
+    # Parse results
+    results = []
+    for mut in mutations or []:
+        results.append({
+            'gene': mut.get('gene', {}).get('hugoGeneSymbol'),
+            'protein_change': mut.get('proteinChange'),
+            'mutation_type': mut.get('mutationType'),
+            'sample_id': mut.get('sampleId'),
+            'validation_status': mut.get('validationStatus')
+        })
+    
+    return results
+
+def get_cbioportal_cancer_studies(tu, cancer_type=None):
+    """Get available cancer studies from cBioPortal."""
+    
+    studies = tu.tools.cBioPortal_get_cancer_studies(limit=50)
+    
+    if cancer_type:
+        studies = [s for s in studies if cancer_type.lower() in s.get('cancerTypeId', '').lower()]
+    
+    return studies
+
+def analyze_co_mutations(tu, gene_symbol, study_id):
+    """Find frequently co-mutated genes."""
+    
+    # Get molecular profiles
+    profiles = tu.tools.cBioPortal_get_molecular_profiles(study_id=study_id)
+    
+    # Get mutation data
+    mutations = tu.tools.cBioPortal_get_mutations(
+        study_id=study_id,
+        gene_list=gene_symbol
+    )
+    
+    return {
+        'profiles': profiles,
+        'mutations': mutations,
+        'study_id': study_id
+    }
+```
+
+**cBioPortal Use Cases**:
+| Use Case | Tool | Parameters |
+|----------|------|------------|
+| Find mutation frequency | `cBioPortal_get_mutations` | `study_id`, `gene_list` |
+| List available studies | `cBioPortal_get_cancer_studies` | `limit` |
+| Get molecular profiles | `cBioPortal_get_molecular_profiles` | `study_id` |
+| Analyze co-mutations | Multiple tools | Combined analysis |
+
+### 2.7 Human Protein Atlas Expression (NEW)
+
+Validate target expression in tumor vs normal tissues:
+
+```python
+def get_hpa_expression(tu, gene_symbol):
+    """
+    Get protein expression data from Human Protein Atlas.
+    
+    Critical for validating:
+    - Target is expressed in tumor tissue
+    - Target has differential tumor vs normal expression
+    """
+    
+    # Search for gene
+    gene_info = tu.tools.HPA_search_genes_by_query(search_query=gene_symbol)
+    
+    if not gene_info:
+        return None
+    
+    # Get tissue expression data
+    ensembl_id = gene_info[0].get('Ensembl') if gene_info else None
+    
+    # Comparative expression in cancer cell lines
+    cell_line_data = tu.tools.HPA_get_comparative_expression_by_gene_and_cellline(
+        gene_name=gene_symbol,
+        cell_line="a549"  # Lung cancer cell line
+    )
+    
+    return {
+        'gene_info': gene_info,
+        'cell_line_expression': cell_line_data
+    }
+
+def check_tumor_specific_expression(tu, gene_symbol, cancer_type):
+    """Check if target has tumor-specific expression pattern."""
+    
+    # Map cancer type to cell line
+    cancer_to_cellline = {
+        'lung': 'a549',
+        'breast': 'mcf7',
+        'liver': 'hepg2',
+        'cervical': 'hela',
+        'prostate': 'pc3'
+    }
+    
+    cell_line = cancer_to_cellline.get(cancer_type.lower(), 'a549')
+    
+    expression = tu.tools.HPA_get_comparative_expression_by_gene_and_cellline(
+        gene_name=gene_symbol,
+        cell_line=cell_line
+    )
+    
+    return expression
+```
+
+**HPA Expression Validation Output**:
+```markdown
+### Expression Validation (Human Protein Atlas)
+
+| Gene | Tumor Cell Line | Expression | Normal Tissue | Differential |
+|------|-----------------|------------|---------------|--------------|
+| EGFR | A549 (lung) | High | Low-Medium | Tumor-elevated |
+| ALK | H3122 (lung) | High | Not detected | Tumor-specific |
+| HER2 | MCF7 (breast) | Medium | Low | Elevated |
+
+*Source: Human Protein Atlas via `HPA_get_comparative_expression_by_gene_and_cellline`*
+```
+
+### 2.8 Evidence Level Mapping
+
+| CIViC Level | Our Tier | Meaning |
+|-------------|----------|---------|
+| A | ★★★ | FDA-approved, guideline |
+| B | ★★☆ | Clinical evidence |
+| C | ★★☆ | Case study |
+| D | ★☆☆ | Preclinical |
+| E | ☆☆☆ | Inferential |
+
+### 2.4 Output Table
+
+```markdown
+## Variant Interpretation
+
+| Variant | Gene | Significance | Evidence Level | Clinical Implication |
+|---------|------|--------------|----------------|---------------------|
+| L858R | EGFR | Oncogenic driver | ★★★ (Level A) | Sensitive to EGFR TKIs |
+| T790M | EGFR | Resistance | ★★★ (Level A) | Resistant to 1st/2nd gen TKIs |
+
+### COSMIC Mutation Frequency
+
+| Gene | Mutation | COSMIC Count | Primary Cancer Types | FATHMM Prediction |
+|------|----------|--------------|---------------------|-------------------|
+| EGFR | L858R | 15,234 | Lung (85%), Colorectal (5%) | Pathogenic |
+| EGFR | T790M | 8,567 | Lung (95%) | Pathogenic |
+| BRAF | V600E | 45,678 | Melanoma (50%), Colorectal (15%) | Pathogenic |
+
+### TCGA/GDC Patient Tumor Data (NEW)
+
+| Gene | TCGA Project | SSM Cases | CNV Amp | CNV Del | % Samples |
+|------|-------------|-----------|---------|---------|-----------|
+| EGFR | TCGA-LUAD | 156 | 89 | 5 | 28% |
+| EGFR | TCGA-GBM | 45 | 312 | 2 | 57% |
+| KRAS | TCGA-PAAD | 134 | 8 | 1 | 92% |
+
+*Source: GDC via `GDC_get_mutation_frequency`, `GDC_get_cnv_data`*
+
+### DepMap Target Essentiality (NEW)
+
+| Gene | Mean Effect (All) | Mean Effect (Cancer Type) | Selectivity | Interpretation |
+|------|-------------------|---------------------------|-------------|----------------|
+| EGFR | -0.15 | -0.45 (lung) | Cancer-selective | Good target |
+| KRAS | -0.82 | -0.91 (pancreatic) | Essential | Hard to target |
+| MYC | -0.95 | -0.93 | Pan-essential | Challenging target |
+
+*Effect score <-0.5 = strongly essential for cell survival*
+*Source: DepMap via `DepMap_get_gene_dependencies`*
+
+*Combined Sources: CIViC, ClinVar, COSMIC, GDC/TCGA, DepMap*
+```
+
+---
+
+## Phase 2.5: Tumor Expression Context (NEW)
+
+### 2.5.1 Cell-Type Expression in Tumor (CELLxGENE)
+
+```python
+def get_tumor_expression_context(tu, gene_symbol, cancer_type):
+    """Get cell-type specific expression in tumor microenvironment."""
+    
+    # Get expression in tumor and normal cells
+    expression = tu.tools.CELLxGENE_get_expression_data(
+        gene=gene_symbol,
+        tissue=cancer_type  # e.g., "lung", "breast"
+    )
+    
+    # Cell metadata for context
+    cell_metadata = tu.tools.CELLxGENE_get_cell_metadata(
+        gene=gene_symbol
+    )
+    
+    # Identify tumor vs normal expression
+    tumor_expression = [c for c in expression if 'tumor' in c.get('cell_type', '').lower()]
+    normal_expression = [c for c in expression if 'normal' in c.get('cell_type', '').lower()]
+    
+    return {
+        'tumor_expression': tumor_expression,
+        'normal_expression': normal_expression,
+        'ratio': calculate_tumor_normal_ratio(tumor_expression, normal_expression)
+    }
+```
+
+**Why it matters**: 
+- Confirms target is expressed in tumor cells (not just stroma)
+- Identifies potential resistance from tumor heterogeneity
+- Supports drug selection based on expression patterns
+
+### 2.5.2 Output for Report
+
+```markdown
+## 2.5 Tumor Expression Context
+
+### Target Expression in Tumor Microenvironment (CELLxGENE)
+
+| Gene | Tumor Cells | Normal Cells | Tumor/Normal Ratio | Interpretation |
+|------|-------------|--------------|-------------------|----------------|
+| EGFR | High (TPM=85) | Medium (TPM=25) | 3.4x | Good target |
+| MET | Medium (TPM=35) | Low (TPM=8) | 4.4x | Potential bypass |
+| AXL | High (TPM=120) | Low (TPM=15) | 8.0x | Resistance marker |
+
+### Cell Type Distribution
+
+- **EGFR-high cells**: Tumor epithelial (85%), CAFs (10%), immune (5%)
+- **MET-high cells**: Tumor epithelial (70%), endothelial (20%), immune (10%)
+
+**Clinical Relevance**: EGFR highly expressed in tumor epithelial cells. AXL overexpression in tumor suggests potential resistance mechanism.
+
+*Source: CELLxGENE Census*
+```
+
+---
+
+## Phase 3: Treatment Options
+
+### 3.1 Approved Therapies
+
+Query order:
+1. `OpenTargets_get_associated_drugs_by_target_ensemblId` → Approved drugs
+2. `DailyMed_search_spls` → FDA label details
+3. `ChEMBL_get_drug_mechanisms_of_action_by_chemblId` → Mechanism
+
+### 3.2 Treatment Prioritization
+
+| Priority | Criteria |
+|----------|----------|
+| **1st Line** | FDA-approved for indication + biomarker (★★★) |
+| **2nd Line** | Clinical trial evidence, guideline-recommended (★★☆) |
+| **3rd Line** | Off-label with mechanistic rationale (★☆☆) |
+
+### 3.3 Output Format
+
+```markdown
+## Treatment Recommendations
+
+### First-Line Options
+**1. Osimertinib (Tagrisso)** ★★★
+- FDA-approved for EGFR T790M+ NSCLC
+- Evidence: AURA3 trial (ORR 71%, mPFS 10.1 mo)
+- Source: FDA label, PMID:27959700
+
+### Second-Line Options
+**2. Combination: Osimertinib + [Agent]** ★★☆
+- Evidence: Phase 2 data
+- Source: NCT04487080
+```
+
+---
+
+## Phase 3.5: Pathway & Network Analysis (NEW)
+
+### 3.5.1 Pathway Context (KEGG/Reactome)
+
+```python
+def get_pathway_context(tu, gene_symbols, cancer_type):
+    """Get pathway context for drug combinations and resistance."""
+    
+    pathway_map = {}
+    for gene in gene_symbols:
+        # KEGG pathways
+        kegg_gene = tu.tools.kegg_find_genes(query=f"hsa:{gene}")
+        if kegg_gene:
+            pathways = tu.tools.kegg_get_gene_info(gene_id=kegg_gene[0]['id'])
+            pathway_map[gene] = pathways.get('pathways', [])
+        
+        # Reactome disease score
+        reactome = tu.tools.reactome_disease_target_score(
+            disease=cancer_type,
+            target=gene
+        )
+        pathway_map[f"{gene}_reactome"] = reactome
+    
+    return pathway_map
+```
+
+### 3.5.2 Protein Interaction Network (IntAct)
+
+```python
+def get_resistance_network(tu, drug_target, bypass_candidates):
+    """Find protein interactions that may mediate resistance."""
+    
+    # Get interaction network for drug target
+    network = tu.tools.intact_get_interaction_network(
+        gene=drug_target,
+        depth=2  # Include 2nd degree connections
+    )
+    
+    # Find bypass pathway candidates in network
+    bypass_in_network = [
+        node for node in network['nodes']
+        if node['gene'] in bypass_candidates
+    ]
+    
+    return {
+        'network': network,
+        'bypass_connections': bypass_in_network,
+        'total_interactors': len(network['nodes'])
+    }
+```
+
+### 3.5.3 Output for Report
+
+```markdown
+## 3.5 Pathway & Network Analysis
+
+### Signaling Pathway Context (KEGG)
+
+| Pathway | Genes Involved | Relevance | Drug Targets |
+|---------|---------------|-----------|--------------|
+| EGFR signaling (hsa04012) | EGFR, MET, ERBB3 | Primary pathway | Osimertinib, Capmatinib |
+| PI3K-AKT (hsa04151) | PIK3CA, AKT1 | Downstream | Alpelisib |
+| RAS-MAPK (hsa04010) | KRAS, BRAF, MEK | Bypass potential | Sotorasib, Trametinib |
+
+### Drug Combination Rationale
+
+**Biological basis for combinations**:
+- EGFR inhibition → compensatory MET activation (60% of cases)
+- **Rationale for EGFR + MET inhibition**: Block primary and bypass pathways
+- Network shows direct EGFR-MET interaction (IntAct: MI-score 0.75)
+
+### Protein Interaction Network (IntAct)
+
+| Target | Direct Interactors | Key Partners | Relevance |
+|--------|-------------------|--------------|-----------|
+| EGFR | 156 | MET, ERBB2, ERBB3, GRB2 | Bypass pathways |
+| MET | 89 | EGFR, HGF, GAB1 | Resistance mediator |
+
+*Source: KEGG, Reactome, IntAct*
+```
+
+---
+
+## Phase 4: Resistance Analysis
+
+### 4.1 Known Mechanisms (Literature + CIViC)
+
+```python
+def analyze_resistance(tu, drug_name, gene_symbol):
+    """Find known resistance mechanisms."""
+    # CIViC resistance evidence
+    resistance = tu.tools.civic_search_evidence_items(
+        drug=drug_name,
+        evidence_type="Predictive",
+        clinical_significance="Resistance"
+    )
+    
+    # Literature search
+    papers = tu.tools.PubMed_search_articles(
+        query=f'"{drug_name}" AND "{gene_symbol}" AND resistance',
+        limit=20
+    )
+    
+    return {'civic': resistance, 'literature': papers}
+```
+
+### 4.2 Structure-Based Analysis (NvidiaNIM)
+
+When mutation affects drug binding:
+
+```python
+def model_resistance_mechanism(tu, gene_ids, mutation, drug_smiles):
+    """Model structural impact of resistance mutation."""
+    # Get/predict structure
+    structure = tu.tools.NvidiaNIM_alphafold2(sequence=wild_type_sequence)
+    
+    # Dock drug to wild-type
+    wt_docking = tu.tools.NvidiaNIM_diffdock(
+        protein=structure['structure'],
+        ligand=drug_smiles,
+        num_poses=5
+    )
+    
+    # Compare binding site changes
+    # Report: "T790M introduces bulky methionine, steric clash with erlotinib"
+```
+
+---
+
+## Phase 5: Clinical Trial Matching
+
+### 5.1 Search Strategy
+
+```python
+def find_trials(tu, condition, biomarker, location=None):
+    """Find matching clinical trials."""
+    # Search with biomarker
+    trials = tu.tools.search_clinical_trials(
+        condition=condition,
+        intervention=biomarker,  # e.g., "EGFR"
+        status="Recruiting",
+        pageSize=50
+    )
+    
+    # Get eligibility for top matches
+    nct_ids = [t['nct_id'] for t in trials[:20]]
+    eligibility = tu.tools.get_clinical_trial_eligibility_criteria(nct_ids=nct_ids)
+    
+    return trials, eligibility
+```
+
+### 5.2 Output Format
+
+```markdown
+## Clinical Trial Options
+
+| NCT ID | Phase | Agent | Biomarker Required | Status | Location |
+|--------|-------|-------|-------------------|--------|----------|
+| NCT04487080 | 2 | Amivantamab + lazertinib | EGFR T790M | Recruiting | US, EU |
+| NCT05388669 | 3 | Patritumab deruxtecan | Prior osimertinib | Recruiting | US |
+
+*Source: ClinicalTrials.gov*
+```
+
+---
+
+## Phase 5.5: Literature Evidence (NEW)
+
+### 5.5.1 Published Literature (PubMed)
+
+```python
+def search_treatment_literature(tu, cancer_type, biomarker, drug_name):
+    """Search for treatment evidence in literature."""
+    
+    # Drug + biomarker combination
+    drug_papers = tu.tools.PubMed_search_articles(
+        query=f'"{drug_name}" AND "{biomarker}" AND "{cancer_type}"',
+        limit=20
+    )
+    
+    # Resistance mechanisms
+    resistance_papers = tu.tools.PubMed_search_articles(
+        query=f'"{drug_name}" AND resistance AND mechanism',
+        limit=15
+    )
+    
+    return {
+        'treatment_evidence': drug_papers,
+        'resistance_literature': resistance_papers
+    }
+```
+
+### 5.5.2 Preprints (BioRxiv/MedRxiv)
+
+```python
+def search_preprints(tu, cancer_type, biomarker):
+    """Search preprints for cutting-edge findings."""
+    
+    # BioRxiv cancer research
+    biorxiv = tu.tools.BioRxiv_search_preprints(
+        query=f"{cancer_type} {biomarker} treatment",
+        limit=10
+    )
+    
+    # MedRxiv clinical studies
+    medrxiv = tu.tools.MedRxiv_search_preprints(
+        query=f"{cancer_type} {biomarker}",
+        limit=10
+    )
+    
+    return {
+        'biorxiv': biorxiv,
+        'medrxiv': medrxiv
+    }
+```
+
+### 5.5.3 Citation Analysis (OpenAlex)
+
+```python
+def analyze_key_papers(tu, key_papers):
+    """Get citation metrics for key evidence papers."""
+    
+    analyzed = []
+    for paper in key_papers[:10]:
+        work = tu.tools.openalex_search_works(
+            query=paper['title'],
+            limit=1
+        )
+        if work:
+            analyzed.append({
+                'title': paper['title'],
+                'citations': work[0].get('cited_by_count', 0),
+                'year': work[0].get('publication_year'),
+                'open_access': work[0].get('is_oa', False)
+            })
+    
+    return analyzed
+```
+
+### 5.5.4 Output for Report
+
+```markdown
+## 5.5 Literature Evidence
+
+### Key Clinical Studies
+
+| PMID | Title | Year | Citations | Evidence Type |
+|------|-------|------|-----------|---------------|
+| 27959700 | AURA3: Osimertinib vs chemotherapy... | 2017 | 2,450 | Phase 3 trial |
+| 30867819 | Mechanisms of osimertinib resistance... | 2019 | 680 | Review |
+| 34125020 | Amivantamab + lazertinib Phase 1... | 2021 | 320 | Phase 1 trial |
+
+### Recent Preprints (Not Peer-Reviewed)
+
+| Source | Title | Posted | Key Finding |
+|--------|-------|--------|-------------|
+| MedRxiv | Novel C797S resistance strategy... | 2024-01 | Fourth-gen TKI |
+| BioRxiv | scRNA-seq reveals resistance... | 2024-02 | Cell state switch |
+
+**⚠️ Note**: Preprints have NOT undergone peer review. Interpret with caution.
+
+### Evidence Summary
+
+| Category | Papers Found | High-Impact (>100 citations) |
+|----------|--------------|------------------------------|
+| Treatment efficacy | 25 | 8 |
+| Resistance mechanisms | 18 | 5 |
+| Combinations | 12 | 3 |
+
+*Source: PubMed, BioRxiv, MedRxiv, OpenAlex*
+```
+
+---
+
+## Report Template
+
+**File**: `[PATIENT_ID]_oncology_report.md`
+
+```markdown
+# Precision Oncology Report
+
+**Patient ID**: [ID] | **Date**: [Date]
+
+## Patient Profile
+- **Diagnosis**: [Cancer type, stage]
+- **Molecular Profile**: [Mutations, fusions]
+- **Prior Therapy**: [Previous treatments]
+
+---
+
+## Executive Summary
+[2-3 sentence summary of key findings and recommendation]
+
+---
+
+## 1. Variant Interpretation
+[Table with variants, significance, evidence levels]
+
+## 2. Treatment Recommendations
+### First-Line Options
+[Prioritized list with evidence]
+
+### Second-Line Options
+[Alternative approaches]
+
+## 3. Resistance Analysis (if applicable)
+[Mechanism explanation, strategies to overcome]
+
+## 4. Clinical Trial Options
+[Matched trials with eligibility]
+
+## 5. Next Steps
+1. [Specific actionable recommendation]
+2. [Follow-up testing if needed]
+3. [Referral if appropriate]
+
+---
+
+## Data Sources
+| Source | Query | Data Retrieved |
+|--------|-------|----------------|
+| CIViC | [gene] [variant] | Evidence items |
+| ClinicalTrials.gov | [condition] | Active trials |
+```
+
+---
+
+## Completeness Checklist
+
+Before finalizing report:
+
+- [ ] All variants interpreted with evidence levels
+- [ ] ≥1 first-line recommendation with ★★★ evidence (or explain why none)
+- [ ] Resistance mechanism addressed (if prior therapy failed)
+- [ ] ≥3 clinical trials listed (or "no matching trials")
+- [ ] Executive summary is actionable (says what to DO)
+- [ ] All recommendations have source citations
+
+---
+
+## Fallback Chains
+
+| Primary | Fallback | Use When |
+|---------|----------|----------|
+| CIViC variant | OncoKB (literature) | Variant not in CIViC |
+| OpenTargets drugs | ChEMBL activities | No approved drugs found |
+| ClinicalTrials.gov | WHO ICTRP | US trials insufficient |
+| NvidiaNIM_alphafold2 | AlphaFold DB | API unavailable |
+
+---
+
+## Evidence Grading
+
+| Tier | Symbol | Criteria | Example |
+|------|--------|----------|---------|
+| T1 | ★★★ | FDA-approved, Level A evidence | Osimertinib for T790M |
+| T2 | ★★☆ | Phase 2/3 data, Level B | Combination trials |
+| T3 | ★☆☆ | Preclinical, Level D | Novel mechanisms |
+| T4 | ☆☆☆ | Computational only | Docking predictions |
+
+---
+
+## Tool Reference
+
+See [TOOLS_REFERENCE.md](TOOLS_REFERENCE.md) for complete tool documentation.