TEST 4: Histology and Imaging Analysis
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Patient ID: PAT002-BC-2026

## Imaging Analysis

For patient PAT002-BC-2026, analyze histology and immunofluorescence images:

### Data Files Location:
Files at: `patient-data/PAT002-BC-2026/imaging/`

**Test Files:**

| File | Image Type | Server(s) | Purpose |
|------|-----------|-----------|---------|
| **PAT002_tumor_HE_20x.tiff** | H&E | openimagedata | Morphology, cellularity |
| **PAT002_tumor_IF_CD8.tiff** | IF (single) | openimagedata + deepcell | CD8+ T cell quantification |
| **PAT002_tumor_IF_KI67.tiff** | IF (single) | openimagedata + deepcell | Ki67 proliferation index |
| **PAT002_tumor_IF_ER.tiff** | IF (single) | openimagedata + deepcell | ER expression mapping |
| **PAT002_tumor_IF_HER2.tiff** | IF (single) | openimagedata + deepcell | HER2 status confirmation |
| **PAT002_tumor_IF_PR.tiff** | IF (single) | openimagedata + deepcell | PR expression mapping |

### Analysis Steps:

1. **H&E Histology Analysis**
   **Server:** mcp-openimagedata
   **File:** PAT002_tumor_HE_20x.tiff

   - Describe tissue architecture (ductal structures, stromal patterns)
   - Are there DCIS components (in situ disease)?
   - Estimate tumor cellularity percentage (Expect: ~60-70%)
   - Tubule formation assessment (part of Nottingham grading)
   - Overall tissue morphology consistent with invasive ductal carcinoma?

2. **Immunofluorescence - ER Expression**
   **Servers:** mcp-openimagedata + mcp-deepcell
   **File:** PAT002_tumor_IF_ER.tiff

   - Use deepcell to segment cells and quantify ER+ nuclei
   - Calculate ER positivity rate (Expect: HIGH, ~85-95%)
   - Is ER expression homogeneous or heterogeneous?
   - Expected: Strong, homogeneous nuclear ER staining

3. **Immunofluorescence - CD8 T Cell Infiltration**
   **Servers:** mcp-openimagedata + mcp-deepcell
   **File:** PAT002_tumor_IF_CD8.tiff

   - Quantify CD8+ T cells (cells/mm2)
   - Spatial distribution: Stromal vs intratumoral?
   - Expected: MODERATE infiltration (~15-30 cells/mm2), mixed distribution

4. **Immunofluorescence - Ki67 Proliferation**
   **Servers:** mcp-openimagedata + mcp-deepcell
   **File:** PAT002_tumor_IF_KI67.tiff

   - Calculate Ki67 proliferation index (% positive cells)
   - Expected: LOW-MODERATE (~10-20%), consistent with Luminal A subtype

5. **Immunofluorescence - HER2 Status Confirmation**
   **Servers:** mcp-openimagedata + mcp-deepcell
   **File:** PAT002_tumor_IF_HER2.tiff

   - Quantify HER2 membrane staining intensity
   - Expected: NEGATIVE (0 or 1+), confirming HER2- status from clinical data

## Expected Results:

**H&E Morphology:**
- Cellularity: 60-70% tumor cells
- Architecture: Invasive ductal carcinoma with tubular formation
- DCIS component: May be present (ductal carcinoma in situ adjacent to invasive)
- Nuclear grade: Intermediate (Grade 2)

**ER Expression:**
- ER positivity: HIGH (~85-95%)
- Distribution: Homogeneous nuclear staining
- Interpretation: Strongly ER-positive — confirms endocrine therapy eligibility

**CD8 T Cell Infiltration:**
- Density: MODERATE (~15-30 cells/mm2)
- Distribution: Both stromal and some intratumoral
- Interpretation: **Immune-infiltrated phenotype** (better than HGSOC)

**Ki67 Proliferation Index:**
- Ki67 index: LOW-MODERATE (~10-20%)
- Distribution: Heterogeneous (higher at invasive front)
- Interpretation: Consistent with Luminal A subtype (low proliferation)

**HER2 Status:**
- HER2 score: 0 or 1+ (negative)
- Interpretation: Confirms ER+/PR+/HER2- triple status

## Output Format:

1. **H&E Summary:** Cellularity, architecture, grade, DCIS presence
2. **ER Analysis:** Positivity rate, distribution, clinical significance
3. **CD8 T Cell Analysis:** Density, spatial pattern, immune phenotype
4. **Ki67 Proliferation:** Index, distribution, Luminal subtype confirmation
5. **HER2 Confirmation:** Score, clinical interpretation
6. **Overall Imaging Interpretation:**
   - Is ER expression strong and homogeneous? (Yes — good endocrine therapy candidate)
   - Is proliferation low? (Yes — Luminal A pattern)
   - Immune microenvironment? (Warm — moderate CD8 infiltration)
   - Nottingham grade estimate? (Grade 2)

## Validation Checkpoints:

- Loaded: 6 imaging files (H&E, ER, CD8, Ki67, HER2, PR)
- H&E: Invasive ductal carcinoma morphology
- ER: Strongly positive (85-95%)
- Ki67: Low-moderate (10-20%) — Luminal A
- CD8: Moderate infiltration (15-30 cells/mm2)
- HER2: Negative (confirms clinical HER2- status)
