Cytology meets Molecular:

Don’t throw away your microscope!

Andrew Fischer, M.D.

Director of Cytopathology

University of Massachusetts

British Association

for Cytopathology

November 4, 2017

Theme: How will molecular

testing and cytology evolve • What can molecular testing replace, using next

generation sequencing and bladder cancer

screening as an example.

• What molecular testing cannot do.

• The criteria of malignancy, when viewed from the

proper perspective, provide an irreplaceable insight

into the function of oncogenes.

• New microscopy techniques—fluorescent optical

sectioning and live cell imaging—will keep our

cytomorphologic skills in demand.

Cost in US (medicare):

~40$

Sensitivity for high

grade UC: Up to about

90%*

Sensitivity for ALL UC,

high and low grade,

<50%

High grade urothelial carcinoma, “microbiopsy” cytology sample

from catheterized ureter

Lee et al, Causes of False-Negative for High-Grade Urothelial Carcinoma in Urine Cytology.

Diagn. Cytopathol. 2016;44:994–999

Cost of Next generation sequencing: As low as 15,000 base pairs per penny

3 billion basepairs in whole

human genome

Only 1.5% is exomic

Need 30X coverage

(average 30 reads per DNA

molecule)

Costs approaching $1000

for biggest machines.

= ~15,000 bp for 1 cent

https://www.nature.com/news/technology-the-1-

000-genome-1.14901

Next generation sequencing for

urothelial carcinoma screening

• Compared to germline sequencing (two alleles, so 50%

of sequences are from one allele) NGS needs many

individual reads of the sequence to detect tumors that

are diluted with many normal gene sequences.

• Many genes are potentially mutated in cancers, so you

need to sequence many segments of DNA to have a

high sensitivity (~600 segments, ~100,000 bp). Would

cost (best case scenario) at least $150.

• A DNA methylation survey identified only ~150 DNA

segments, preferentially methylated in cancer, that may

be more suitable.

For NGS to work, need a high proportion of abnormal

cells. NGS could not be effective for this case.

In general, most molecular tests cannot work if the input material

is not assessed. Cytology is ideal for assessing input material,

because so few cells (~5000) are needed.

UroMark NGS platform for detecting high and low grade

bladder cancers

•Detected bladder cancer

with sensitivity of 98%,

specificity of 97%.

•To detect 1% tumor cells,

would need very roughly

2000 reads for each

amplicon (20 individual

tumor DNA sequences).

•Assume each amplified

sequence is 100 base

pairs.

•2000 X 150 X 100 /

15,000 = $20.00

• NGS-based testing is poised to replace at least some

urine cytology as a screening test for cancer.

• Likely to evolve through a phase of reflex testing for

cytologic atypia when percent abnormal cells is high.

• Most likely to first replace cytology for follow-up of

bladder cancer patients with known mutations.

Will Molecular take over urine cytology?

96.5 % of driver

genes for PTC have

been identified.

TCGA has not shed ANY light on why PTC

has diagnostic changes in chromatin and

nuclear shape.

Does Cytomorphology matter?

Molecular

vs.

Cytology

MAP kinase pathway, From Wikipedia, accessed 10-30-17

From Luger et al., Nature 389:251, 1997

Structure of the nucleosome, showing ~150 nucleotides of

DNA wrapped around the 4 pairs of histones. Note the

protruding histone tails.

Normal ductal cells vs pancreatic IPMN

A structure-function relation should also exist

at the cellular level

Hypothetical phylogeny of a cancer

Fischer, Young, DeLellis, J Cellular Biochem 93:28-36, 2004

“Hallmarks of cancer” are the hypotheses

for how oncogenes work.

In fact, 35 years after the discovery of oncogenes, we

still do not know how they actually work!

Hanahan and Weinberg, Cell 100:57, 2000 Hanahan and Weinberg, Cell 144:646, 2011

Phylogeny of Darwin’s Finches

Fischer, Young, DeLellis, J Cellular Biochem 93:28-36, 2004

Darwin knew that the morphologic

features that distinguish related

species…

…are ultimately caused by the heritable

elements (genes) that differ between the

species.

…relate in “the most essential yet often

hidden manner” to the mechanism of

increased fitness.

Darwin’s theory predicts there

should be a relation between the

morphologic changes diagnostic of

cancers, the genes that are active in

the cancers, and (most importantly)

the functional changes that allow

this cellular evolution to take place

ASC-sponsored Classification of

the Criteria of Malignancy

1. Tissue-level architectural changes reflecting clonal expansion.

2. Changes reflecting genetic instability

3. Sub-cellular changes, unrelated to genetic instability, conferring increased “cellular fitness”

A. Directly induced by oncogenes

B. Likely heritable, but no known genetic basis

Fischer et al., J Cellular Biochem 2010, 110:75-811

Group 1: Tissue architectural criteria Three common mechanisms for early clonal expansion of epithelial cells

“Crowding”

“Papillary formation” “True

stratification”

Fischer, Fundamentals of Cytological Diagnosis and Its Biological Basis. In: Pathobiology of

Human Disease. Published by Elsevier, 2014. p. 3311-3344

Loss of contact

inhibition,

Pseudostatification

Anchorage-independent

growth, resistance to

anoikis

Columnar

cell change

breast

Normal

ductal cells

Papillary thyroid carcinoma (Photomicrographs courtesy of Manon Auger, McGill)

Hepatocellular carcinoma, defined by true

stratification away from endothelial cells.

CD 34 staining to show endothelial cells

The development of true stratification

typically coincides with loss of (“basal-

apical”) polarity. Stratified cells can

then orienting freely toward any

surface (cribriforming).

There is increasing evidence for

involvement of oncogenes in breaking

down normal “basal-apical” polarity

Mammary carcinoma, with randomized

positioning of secretions and random nuclear

axis.

Objective markers of

basal-apical polarity

would likely be useful for

diagnosis

Group 2: Cytologic changes reflecting

genetic instability

Cell to cell variation in:

• Total DNA content (total integrated amount of

hematoxylin staining), reflecting chromosome

instability

• Cytoplasmic features (evidence of phenotypic

instability)

• Chromatin packaging patterns (?Epigenetic instability?)

DNA content variation reflecting chromosomal

instability in two HSIL pap tests

Polyploidization is a common

benign change

Benign polyploidization compared to

malignant nuclear pleomorphism

Group 3: Cell structural changes, not

related to genetic instability*

• Nuclear shape abnormalities

• Intermediate filament organization

• Other cytoplasmic diagnostic changes

• Abnormal nucleolar prominence

• Chromatin alterations

*Several of these criteria are known to be directly

induced by the cancer genes active in the tumor

E4 gene of

HPV

mediates

intermediate

filament

collapse

Group 3: Intermediate filament abnormalities

Group 3: Collapse of intermediate filaments

(keratin 20) in Merkel cell (small cell) carcinoma

Normal prostate epithelium

Prostatic intraepithelial neoplasia:

Diagnostic feature is nucleolar

prominence without reactive

cytoplasm

Fischer et al. (J Cellular Biochem, 2004)

Papillary thyroid carcinoma

Papillary thyroid carcinoma compared to normal thyroid

Normal thyroid epithelium

Follicular adenoma Papillary thyroid carcinoma

RET/PTC

TRK/PTC

B-RAF

H-RAS et al.

Thyroid model of carcinogenesis

Ret/PTC expressed in normal human thyroid cells

Fischer et al, Am J Pathol 153:1443, 1998

Normal human

thyroid epithelium

Human thyroid epithelium

expressing RET Tyrosine

kinase

Human thyroid epithelium

expressing H(V14)- RAS

Nuclear lamina

irregularity is

induced by

RET/PTC in

interphase.

Cells were micro-

injected with RET/PTC

6 hours previously and

stained for lamins

(green) and RET (red)

Fischer et al., Am J

Pathol 163:1091, 2003

Active RET/PTC

Inactive RET/PTC

(control)

Waggle dance of the

honeybee:

• A distinguishing trait from closest relative—the less social bumble bees*

• Different “dance dialects” have a genetic basis, shown by back-crossing to be defined by a single locus*

• Likely to be a key determinant of the fitness difference that allowed their evolutionary split.

• Essentially impossible to decipher this biology based on just the DNA sequence, and impossible to decipher with just snap shot images.

*R. N. Johnson, B. P. Oldroyd, A. B. Barron, and R. H. Crozier, J Heredity, 93:170-3, 2002

From Dan Schmolze et al., Arch Pathol Lab Med,

135:255, 2011.

Two photon microscopy

Right top: pseudocolored DAPI and fluoresceein to look like

H&E. Bottom right: H&E stained paraffin section.

• Living human papillary thyroid

carcinoma from thyroidectomy

specimen.

• Vibratome sectioned at 300

micron thickness.

• Stained for 5 minutes with 0.01%

acriflavine.

• 800 nm 2 photon excitation.

• Optically sectioned about 60

microns in depth.

• Viewed in just one time point.

• Diagnostic features of PTC

are seen in LIVING CELLS

Ending Comments • Molecular diagnosis will replace some cytologic tests. However, it

will mostly increase the role for cytology, through an increased use

of minimally sized biopsies that are ideal when molecular and

cytology are used together.

• Oncogenes are selected for at the cellular level. The DNA level of

biology CANNOT by itself inform us about how oncogenes actually

work.

• An increasingly refined understanding of the cytologic criteria of

malignancy brings us closer and closer to a description of the

exact functional changes in cancer cells.

• New microscopy techniques make it feasible to visualize new types

of diagnostic “cellular dynamics”, dynamics that will require our

expertise for use in diagnosis and prognostication for generations

to come.