PATHOLOGY AND HISTOGENESIS OF SALIVARY GLAND TUMOURS

With Immunohistochemistry of Each Lesion

INTRODUCTION

PART I: HISTOGENESIS OF SALIVARY GLAND NEOPLASMS

A. Normal Architecture of Salivary Glands - The Foundation

• Luminal (inner) cells: Acinar cells (serous/mucous) and ductal epithelial cells
• Abluminal (outer) cells: Myoepithelial cells (wrapping acini and intercalated ducts) and basal cells (supporting striated and excretory ducts)

B. Histogenetic Theories

1. Basal Reserve Cell Theory (Eversole, 1971)

• Also called the Progenitor Cell Theory
• States that the basal cells of both the excretory ducts AND the intercalated ducts function as reserve (stem) cells for all more highly differentiated components of the salivary gland
• Based on histological observations of embryonic palatal minor salivary glands and bilayered major ducts in the human fetal salivary gland
• Tumours arising from this theory: Pleomorphic adenoma, basal cell adenoma, canalicular adenoma

2. Pluripotent Unicellular Reserve Cell Theory (Batsakis, 1977)

• States that only the basal cells of the excretory duct are responsible for the development of ALL other salivary gland units
• Eliminates any role for intercalated duct reserve cells
• A single undifferentiated cell type with pluripotency gives rise to all neoplasms
• This theory is now largely discredited as it oversimplifies the biology

3. Semi-Pluripotent Bicellular Reserve Cell Theory (Dardick, 1981) - MOST WIDELY ACCEPTED

• A refined version: Two distinct reserve cell populations are implicated:
  • Excretory duct reserve cells - give rise to squamous cell carcinoma, mucoepidermoid carcinoma, and salivary duct carcinoma
  • Intercalated duct reserve cells - give rise to all other salivary gland tumours (pleomorphic adenoma, adenoid cystic carcinoma, acinic cell carcinoma, polymorphous adenocarcinoma, basal cell adenocarcinoma)
• Myoepithelial cells are recognized as important contributors to the morphologic diversity of neoplasms
• The separation of two cells of origin explains why MEC and SCC differ so fundamentally from adenoid cystic carcinoma

4. Multicellular Theory (Dardick et al., 1989)

• Proposes that all mature, differentiated cell types - including acinar cells, ductal cells, and basal cells - are capable of cell division and neoplastic transformation
• Supported by autoradiographic studies showing tritiated thymidine uptake in:
  • Duct basal cells
  • Luminal cells at all levels of the duct system
  • Even acinar cells
• In adult rat salivary gland hyperplasia studies, more acinar cells than intercalated duct cells were found in the S-phase of the cell cycle
• This theory challenges the exclusive role of reserve cells and supports that any differentiated salivary cell can undergo neoplastic induction
• Epithelial-myoepithelial carcinoma, intercalated duct adenoma, and striated duct adenoma are most appropriately explained by this theory

C. Stem Cell Hypothesis

PART II: MORPHOGENESIS OF SALIVARY GLAND NEOPLASMS

A. The Morphogenic Concept

• What the tumour cell differentiates into
• How cells are arranged within the tumour
• What extracellular matrix (ECM) material is produced

B. The Ducto-Acinar (Bicellular) Concept in Morphogenesis

• Luminal/acinar cell differentiation
• Abluminal (myoepithelial/basal) cell differentiation
• The presence or absence of extracellular matrix (proteoglycans, basal lamina, collagen, elastin)

C. Three Basic Cell Differentiation Models in SGN

D. Taxonomic (Morphogenetic) Classification of SGN

E. Role of Myoepithelial Cells in Morphogenesis

• Are stellate-shaped around acini and spindle-shaped around intercalated ducts
• Possess a dual epithelial-smooth muscle phenotype
• Produce extracellular matrix - basement membrane materials and myxoid substances
• May exert an anti-invasive effect by secreting proteinase inhibitors and suppressing angiogenesis

• Spindle cells
• Plasmacytoid (hyaline) cells
• Epithelioid cells
• Clear cells (glycogen-rich)

PART III: PATHOLOGY AND IHC OF INDIVIDUAL SALIVARY GLAND TUMOURS

1. PLEOMORPHIC ADENOMA (PA)

• Most common salivary gland tumour (parotid >> submandibular >> minor)
• Encapsulated but the capsule is often incomplete
• Histology shows: ductal/tubular structures (luminal cells), myoepithelial cells in various forms, myxoid stroma, chondroid areas, and occasionally ossification
• Neoplastic myoepithelial cells produce fibrous, mucinous, chondroid, and osseous tissue - accounting for histomorphological diversity
• Genetic markers: PLAG1 (chromosome 8q12) and HMGA2 (chromosome 12q14) alterations

• Luminal cells: CK7, CK8, CK13, CK18, CEA, EMA - positive
• Myoepithelial cells: SMA (smooth muscle actin), MSA (muscle-specific actin), calponin, CK14, p63, S-100, vimentin, GFAP, SOX10 - positive
• PLAG1: Strongly positive (highly sensitive and specific for PA; overexpression helps differentiate from ACC and other SGN)
• HMGA2: Highly specific marker for PA compared to histologically mimicking tumours (Mito et al., 2017)
• WT1 (Wilms Tumour 1): Sensitive marker for neoplastic myoepithelial cells in PA; not expressed in normal myoepithelium
• GFAP: Intensely positive - most advantageous in PA and myoepitheliomas; helps differentiate from polymorphous adenocarcinoma (GFAP negative) and ACC (GFAP negative)
• Ki-67: Low, generally <5% (supports benign nature)
• HER2/neu: Negative to weakly positive in normal ductal cells; overexpression in salivary duct carcinoma helps exclude PA

2. WARTHIN'S TUMOUR (Papillary Cystadenoma Lymphomatosum)

• Second most common salivary gland tumour
• Almost exclusively in the parotid gland; bilateral in ~10% cases
• Cystic spaces lined by characteristic double-layered oncocytic epithelium (inner tall columnar with palisaded nuclei, outer basaloid) in a background of lymphoid tissue with germinal centres
• Bilayered epithelium reflects differentiation of both luminal and basal cells

• Anti-mitochondria antibodies (AMA): Strongly positive in oncocytic cells
• Pan-cytokeratin: Positive in epithelial component
• CD20/CD3: Positive in lymphoid component
• Ki-67: Low (<5%)
• S100: Variable

3. ADENOID CYSTIC CARCINOMA (ACC)

• Cribriform (most common): Balance between luminal ductal structures and basal/myoepithelial cells; pseudocysts contain glycosaminoglycans (ECM)
• Tubular: Luminal cells forming ducts surrounded by few layers of basal/myoepithelial cells without significant intercellular material accumulation
• Solid (worst prognosis): Exuberant proliferation of neoplastic basal/myoepithelial cells with minimal luminal formation

• Slow-growing, highly recurrent malignant tumour
• Classic "Swiss cheese" cribriform pattern on histology
• Perineural invasion is characteristic and diagnostically significant
• Poor prognosis despite slow growth due to high recurrence and late metastases (especially lung)
• Small, uniform, angulated cells with hyperchromatic nuclei
• Pseudocystic spaces contain basophilic glycosaminoglycans

• CD117 (c-Kit): Strongly positive in the inner (luminal) cell layer - >50% positivity; this is a KEY differentiating marker from PAC (which shows <50% or none)
• CK7: Positive in luminal cells
• Calponin, SMA, SMMHC, p63, SOX10, S100: Positive - highlighting myoepithelial differentiation around pseudocysts
• Type IV collagen and Laminin: Positive within pseudocyst lumens (basement membrane components)
• GFAP: Usually NEGATIVE (distinguishes from PA, where GFAP is strongly positive)
• PLAG1: Not overexpressed (does not play a role in ACC tumorigenesis; helps differentiate from PA)
• MYB overexpression: Detected by IHC but not entirely specific to ACC
• Ki-67: >10%, particularly high in solid variant (indicates poorer prognosis)
• p53: Increased expression indicates poor prognosis

4. MUCOEPIDERMOID CARCINOMA (MEC)

• Most common malignant salivary gland tumour; most common in children
• Three cell types: mucous cells, epidermoid (squamoid) cells, intermediate cells
• Graded as low, intermediate, or high grade
• Genetic alteration: CRTC1-MAML2 or CRTC3-MAML2 chromosomal fusion (chromosome 9-11 translocation) - activates CREB target genes
• High-grade tumours may lack obvious mucous cells

• CK7: Positive in mucinous cells
• CK14, CK5/6, p63: Positive in intermediate, squamoid, and basaloid cells - KEY feature
• p63: Strongly expressed in epidermoid component of MEC (unlike acinic cell carcinoma which is p63-negative - very useful differential!)
• MUC1, MUC4, MUC5AC, MUC5B: Expressed in varying proportions; high MUC1 = high grade, increased recurrence; MUC5AC helps differentiate high-grade MEC from SCC
• EGFR and AREG (amphiregulin): Overexpression detected by IHC, contributing to tumour growth and survival (CRTC1-MAML2 fusion is downstream target of EGFR ligand)
• PCNA, p53, TNF-alpha: Strong expression seen
• pAKT: Positive
• Ki-67: Low grade ~2.2%; High grade ~8% (useful in grading)
• p63 positivity distinguishes MEC from retention cysts and papillary cystadenomas (where p63 is limited to basal layers only)

5. ACINIC CELL CARCINOMA (AciCC)

• Third most common salivary gland malignancy; predominantly parotid
• Low-grade malignancy with slow progression but capacity for local and distant metastasis
• Cells show serous acinar differentiation with basophilic, granular (zymogen-containing) cytoplasm
• Growth patterns: solid, microcystic, papillary-cystic, follicular
• Genetic alteration: NR4A3 rearrangements (SCPP-NR4A3 translocation; NR4A3 on chromosome 9q31)
• Rare variant: MSANTD3-HTN3 translocation (more serous nature)

• DOG1 (Discovered On GAIST 1): Diffuse, strong positivity on the luminal aspect of acini - KEY MARKER; membrane channel protein highly specific for AciCC
• SOX10: Diffuse strong positivity
• alpha-Amylase: Positive (low sensitivity; not always detected)
• NR4A3 (nuclear immunostaining): Specific and sensitive novel marker - IHC has proven more specific than FISH for NR4A3 expression
• S100, Mammaglobin: Largely NEGATIVE (distinguishes AciCC from secretory carcinoma, which is strongly S100+ and mammaglobin+)
• p63: NEGATIVE - very useful! All MEC are p63 strongly positive, while AciCC is negative
• Maspin: Negative
• MSANTD3: Relatively high specificity but requires further validation

6. POLYMORPHOUS ADENOCARCINOMA (PAC)

• Renamed from "polymorphous low-grade adenocarcinoma" (WHO 2017) due to unpredictable behaviour
• Characteristic: cytological uniformity but architectural diversity (hence "polymorphous")
• Prominent perineural invasion
• Infiltrative growth pattern
• Genetic alteration: PRKD1 mutation (especially E710D hotspot mutation on chromosome 14)

• S100: Positive (97%) - helpful but not specific
• Vimentin: Positive (92.5%)
• Pan-CK: Positive (97.3%)
• CK7: Positive (96.8%)
• E-cadherin: Positive (90%)
• p63: Positive (91.7%) - however, p40 (isotype of p63) is uniformly NEGATIVE, indicating lack of true myoepithelial differentiation
• SOX10: 100% positive
• GFAP: Largely NEGATIVE (5% only) - differentiates from PA (GFAP strongly positive) and helps differentiate from ACC
• CK20: Negative (0%)
• C-kit (CD117): Negative or <50% positivity - differentiates from ADCC (which shows >50%)
• Ki-67: Usually <10%; elevated >10% in 10-20% of cases
• Immunophenotype summary for PAC: CK7+/CK20-, p63+/p40-, S100+, Vimentin+, GFAP-

7. MYOEPITHELIOMA

• Benign; composed exclusively or predominantly of myoepithelial cells
• Cell forms: spindle, plasmacytoid (hyaline), epithelioid, clear
• Lacks ductal structures (differentiates from PA, which always has ductal elements)
• No myxochondroid matrix (unlike PA)

• Calponin: High specificity; very useful diagnostic marker
• SMA (smooth muscle actin): High specificity; very useful
• p63: Positive (myoepithelial + basal cells)
• S100, SOX10, GFAP: Positive
• Vimentin: Positive (good for screening, low specificity)
• Luminal markers (CEA, EMA): NEGATIVE - key distinction from PA (which has positive luminal cells)
• Pan-CK: Positive (confirms epithelial nature; differentiates from non-epithelial spindle cell tumours)
• Ki-67: Low

8. EPITHELIAL-MYOEPITHELIAL CARCINOMA (EMC)

• Rare, biphasic, low malignant potential
• Inner layer: small cuboidal eosinophilic epithelial cells (ductal)
• Outer layer: clear cells (myoepithelial, rich in glycogen)
• Solid, tubular, cribriform, papillary architectural variants
• HRAS mutations are characteristic

• Inner luminal cells: CK AE1/AE3, CK7 - positive
• Outer myoepithelial cells: p63, SMA, vimentin, S-100 - strongly positive
• PAS stain: Highlights glycogen in clear cells
• The biphasic IHC pattern (inner CK7+, outer p63+/SMA+) is diagnostically characteristic

9. SALIVARY DUCT CARCINOMA (SDC)

• High-grade malignant neoplasm; most aggressive SGN
• Almost exclusively in the parotid gland
• Mimics ductal breast carcinoma: intraductal and invasive components with comedo necrosis
• Locoregional metastasis is common
• Genetic: AR (androgen receptor) gene on Xq11-12; ERBB2 amplification; TP53, PTEN, HRAS mutations

• Androgen Receptor (AR): Strongly positive (similar to ductal breast carcinoma and prostate cancer) - diagnostic and predictive marker; guides androgen deprivation therapy
• HER2/neu (ERBB2): >20% show diffuse strong membranous staining - diagnostic, prognostic; guides targeted therapy
• Gross Cystic Disease Fluid Protein-15 (GCDFP-15): Positive in luminal cells (low specificity)
• ER, PR (estrogen receptor, progesterone receptor): Usually negative - helps differentiate from breast metastasis
• TP53: Positive with IHC; associated with poor prognosis
• Ki-67: >10%; often significantly elevated
• PSA (Prostate Specific Antigen): Can be positive (related to AR expression)

10. BASAL CELL ADENOMA

• Benign; monotonous small basaloid cells with dense chromatin, scant cytoplasm
• Distinct basement membrane-like structure
• Lacks the myxochondroid stroma of PA
• Patterns: solid, tubular, trabecular, membranous

• Basal-myoepithelial cells: CK14, p63, SOX10 - positive
• Ductules: CK7, EMA - positive
• S100: Positive spindle-shaped stromal cells
• Ki-67: <2% (distinguishes from basal cell adenocarcinoma)

11. ONCOCYTOMA / ONCOCYTIC ADENOCARCINOMA

• Oncocytes: large epithelial cells with abundant granular eosinophilic cytoplasm, central pyknotic nucleus
• Ultrastructurally: increased number of abnormal mitochondria

• Anti-mitochondria antibodies (AMA): Strongly positive
• CD117/c-Kit: Strongly positive in striated duct cells
• E-cadherin: Positive
• S100: Positive
• PAX8: Positive
• Pancytokeratin: Positive

PART IV: GENERAL IHC MARKERS IN SGN - SUMMARY

A. Markers for Cellular Differentiation

• Calponin and SMA = most specific for myoepithelial cells. Spindle cells stain diffusely; epithelioid and clear cells stain focally; plasmacytoid cells are calponin-positive but SMA-negative
• GFAP = low sensitivity overall but intensely positive in PA and myoepitheliomas; negative in PAC and ACC (very useful discriminator)
• CD117/c-Kit = negative in normal salivary gland; positive in luminal cells of various SGN; >50% in ACC (key differential from PAC)
• WT1 = sensitive marker for neoplastic myoepithelial cells in PA; negative in normal myoepithelium

B. Markers for Benign vs. Malignant Distinction

PART V: ROLE OF IHC IN SPECIFIC DIFFERENTIAL DIAGNOSES

CONCLUSION