Pathology 430/827 Bladder cancer Etiology, classification, and diversity David M. Berman, MD, PhD Pathology and Molecular Medicine Queen’s Cancer Research Institute bermand@queensu.ca bermanlab.org

Objectives Recognize who gets bladder cancer and why

Objectives Recognize who gets bladder cancer and why Recognize different types of bladder cancers Two genomic pathways cause bladder cancer Bladder cancers can change (“progress”) over time Grade Stage

Objectives Recognize who gets bladder cancer and why Recognize different types of bladder cancers Two genomic pathways cause bladder cancer Bladder cancers can change (“progress”) over time Grade Stage Understand concept of epithelial differentiation and how it produces different types of bladder cancer cells

Epidemiology 4th most common cancer in men, Affecting ~3% of men in Western countries 3x less frequent in women Peak age 75yrs

Epidemiology In 2013 in US & Canada, ~80,000 new cases ~16,000 deaths

Recurrence Up to 70% of cases recur. Estimated $3 billion annually U.S., Biggest expense = surveillance (cystoscopies).

Urinary bladder: A urine storage system Lumen (optional)

Epithelial architecture Intermediate Cell (basal) (niche?) 9

Uroplakins form permeability barrier The 3D structure of the 16 nm mouse uroplakin particle at 10 Å resolution. (A) The top view of the 3D density map of a mouse 16 nm uroplakin particle that is contoured at the 1.5σ level. The boundary of a `subunit' consisting of an inner and an outer subdomain is outlined in blue. (B) The particles as seen in a hexagonal crystalline array with one unit cell illustrated. (C) The side view of the 16 nm particle showing, from top to bottom, the joint (J), trunk region (TK), transmembrane domain (TM) and cytoplasmic domain (C). (D) One of the six inverted U-shaped subunits of the 16 nm particle, consisting of an inner subdomain (left) connected to an outer (right) subdomain via a (top) horizontal joint (j; outlined in blue). This inverted U-shaped subunit presumably represents a fundamental building block of the 16 nm particle (Staehelin et al., 1972; Hicks et al., 1974). (E) The inner subdomains of two neighboring subunits are connected via a minimal contact between them (double arrowhead). In A, the stellate-shaped particle has a maximal diameter of 17.5 nm and has a large lipid-filled, central hole (∼6 nm in diameter); in C the particle is about 12 nm tall with a 6.5 nm extracellular and a 0.5 nm cytoplasmic region [from atomic force microscopy data (Min et al., 2002)]. All panels except B are to the same scale; bar (in A) 2 nm. The unit cell in B is 16.5 nm in length. Min G et al. J Cell Sci 2003;116:4087-4094 ©2003 by The Company of Biologists Ltd

Three cell layers of benign urothelium Differentiation in urothelial carcinoma Uroplakins Basal cells repopulate Intermediate cells mature Superficial cells protect He X et al., 2009 Stem Cells, 27:1487 11

Epidemiology Older male 336,000 cases/yr ~3,000 cases/yr *

Presentation Jemal A et al. Cancer statistics, 2007. CA Cancer J Clin. 2007 *

5-year survival Jemal A et al. Cancer statistics, 2007. CA Cancer J Clin. 2007 *

Recurrence $$$$$$$$$ *

Symptoms > Hematuria (>75%) Dysuria (~10%) *

RISK Smoking Strong (~50%) Occupation Strong (~25%) similar to lung cancer in environmental risk factor. Rarely familial. Even Smoking Strong (~50%) Occupation Strong (~25%) *

RISK Iatrogenic Schistosomes similar to lung cancer in environmental risk factor. Rarely familial. Even *

RISK Arsenic Muir Torre Syndrome Family History similar to lung cancer in environmental risk factor. Rarely familial. Even *

Common (75%) Recur (50%) Local treatment urologist jargon vs. pathologist *

Uncommon (25%) Usually progress urologist jargon vs. pathologist *

urologist jargon vs. pathologist Local treatment Radical treatment *

Progression and classification of urothelial carcinoma Grade Low or High High 85% Grade High High

Bladder Cancer Staging STAGE Primary tumour (T) Regional Lymph Nodes (N) Distant Metastasis (M) Ta or Tis I T1 II T2 III T3 or T4a IV T4b AND/OR N1-N3 AND/OR M1

Survival rates by stage Relative 5-year  Survival Rate 98% I 88% II 63% III 46% IV 15%

Non-invasive papillary urothelial carcinoma

Papillary urothelial neoplasia Fibrovascular cores Urothelium

Low grade non-invasive papillary urothelial carcinoma

High grade non-invasive papillary urothelial carcinoma

Invasive urothelial carcinoma

Flat/invasive urothelial carcinoma Benign Carcinoma in situ (CIS)

Invasive urothelial carcinoma

H-RAS/FGFR3 Activation Urothelial Hyperplasia Two Pathways H-RAS/FGFR3 Activation Papillary/Noninvasive PUNLMP LG Papillary UrCa Urothelial Hyperplasia Loss of CDKN2A Normal Urothelium P53/RB Inactivation Flat/Invasive Flat CIS Superficial inv. Muscle inv. UrCa

HRAS/FGFR3+ P53/RB- urologist jargon vs. pathologist *

Studies describing molecular subtypes Sweden (Lund) Texas (MD Anderson TCGA (U.S.) Bladder cancer has had a terrific explosion of important research since 2013.

Molecular Subtypes Non-invasive Invasive Urobasal A (Lund) ~ Luminal (Texas) ~ Group B (TCGA) Urobasal B (Sweden) Squamous-like (Sweden) , Basal (Texas)

Different cell types within a bladder cancer

Hypothetical link between injury and cancer

Activation of Sox9 by bladder injury Ling et al., 2009 Cancer Research

Autocrine loop linking injury to bladder cancer Ling et al., 2009 Cancer Research

Epithelial differentiation in cancer Epigenetic changes Intermediate Cell Epigenetic changes (Basal cell) 41

Urothelial differentiation in Urothelial Carcinoma Xenografts Benign Urothelium SW780 Cell Line Xenograft 42

67 Kd Laminin Receptor: Surface marker of tumor “basal cells” Ki67!!!!!!!! But not in vitro He X et al., 2009 Stem Cells, 27:1487 43

67LR expression in vivo identifies basal-like bladder cancer cells CK17 FACS SW780 Human Bladder cancer xenograft Single cell digest 67LR- (87%) 67LR+ (13%) Inject 10 sites, 2-20k cells each He X et al., 2009 Stem Cells, 27:1487 44

“Basal” cells form tumors. More differentiated cells do not 67 LR bright (Basal) Unfractionated 67 LR dim (Differentiated) He X et al., 2009 Stem Cells, 27:1487 45

Gene Expression Programs in Basal-like Urothelial Cancer Cells Migration Angiogenesis Apoptosis   Proliferation   67LR+ 67LR_ CK17 Signaling pathways Jak-STAT Notch FAK mTOR EGFR Wnt TGF beta He X et al., 2009 Stem Cells, 27:1487 46

Conclusions Two pathways of bladder cancer Genomic differences between cancers Epithelial injury is a risk factor Molecular links exist between injury repair and cancer Bladder cancers differentiate in a manner similar to benign urothelium Genomic differences within cancers