Presentation by Fabio Grizzi at Pathology Horizons 2016 conference in Galway, entitled: "Spectral analysis for tumour diagnosis and classification in surgical pathology: what is missing?

1. Spectral analysis for tumour
diagnosis and classification in
surgical pathology and
cytopathology: what is missing?
Fabio Grizzi
Humanitas Clinical and Research Center
E-mail: fabio.grizzi@humanitasresearch.it
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2. • Pathology Suites are digital pathology
platform for imaging, scoring and reporting of
quantitative brightfield and fluorescent
samples.
• After image capture of the ROIs, Pathology
Suites automatically analyze and score the
sample. Within seconds, pathologists obtain
accurate and objective statistical analyses.
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Pathology Horizons 2016

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Pathology Horizons 2016
A Spectral Pathology Laboratory is focused on
developing new label free-imaging approaches that
allow pathologists to “identify” additional information
embedded within tissues. The technology is based on
the Fourier Transform Infrared spectroscopy and other
spectral imaging approaches to address translational
research questions, including the prediction of early
organ failure.

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Dr. Michael Walsh Laboratory, Chicago
spectral-pathology.uic.edu

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12. Pathology Horizons 2016
DIGITAL PATHOLOGY
IMAGE STORING
TELEPATHOLOGY
QUANTITATIVE PATHOLOGY
ELEMENTARY
RAPID
RELIABLE
BASIC AND SIMPLE
MOVING QUICKLY
REPRODUCIBLE
Able to be trusted to do or provide what is needed

13. TECHNOLOGY
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SCIENCE
Pathology Horizons 2016
IS NOT

14. The human body as a complex system
The concept of spatial conformation or organization in general
inorganic, organic and particularly biochemistry has a fundamental role
in the study of the various properties of biological macro-molecules
(i.e. nucleic acids, proteins, carbohydrates, lipids).
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15. The human body as a complex system
• The biological polymers that have been most widely studied in
structural and functional terms are proteins and nucleic acids (DNA
and RNA).
• The information needed to determine the three-dimensional
structure of a protein is entirely contained in its linear amino acid
sequence.
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16. The human body as a complex system
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• DNA exists in many possible conformations that include A-
DNA, B-DNA, and Z-DNA sub-forms, although, only B-DNA and
Z-DNA have been directly observed in functional organisms.
• The conformation that DNA adopts depends on the hydration
level, DNA sequence, the amount and direction of
supercoiling, chemical modifications of the bases, the type
and concentration of metal ions.

17. The human body as a complex system
Changes in environmental conditions (i.e. pH, temperature,
pressure) may reversibly or irreversibly alter the tri-dimensional
structure of a biological molecule, and thus change its specific
function.
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18. The human body as a complex system
The definition of the spatial
conformation of either a
microscopic or a
macroscopic “anatomical
structure”, and the definition of
a change in its shape, are still
debated by contemporary
morphologists.
D'Arcy Thompson (1829–1902)
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19. The human body as a complex system
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20. The human body as a complex system
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21. The human body as a complex system
The term structure defines the property resulting from the
configurations of the parts (i.e. cells) that form a Whole and their
relationships to each other and to the Whole itself (i.e. tissue).
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22. The human body as a complex system
Two properties of all anatomical systems can be highlighted:
A. every anatomical structure is capable of expressing a
particular function in a particular environment;
B. the different configurations and functions of an anatomical
entity emerge from structures organised in overlapping
hierarchical levels.
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23. The human body as a complex system
Human cells differ in their shapes, dimensions and sizes.
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24. The human body as a complex system
Some cells have specific, particular and consequently invariable
characteristic shapes, regardless of whether they are isolated or
grouped to form more complex anatomical entities known as
tissues.
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25. The human body as a complex system
Other cells are subject to conformational changes that depend
particularly on:
A. the action exerted by their environment;
B. the compression induced by contiguous cells;
C. the relationships between the cells and the intra- and extra-
forces.
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26. The human body as a complex system
The action exerted by their environment
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27. The human body as a complex system
The compression induced by contiguous cells
Cells resemble normal cells but are increased in numbers. Cells may also be increased
in size (hypertrophy). Hyperplasia is different from hypertrophy in that the cell change
in hypertrophy is an increase in the size of cells, whereas hyperplasia involves an
increase in the number of cells.
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28. The human body as a complex system
The relationships among the intra-cellular components
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29. The human body as a complex system
The relationships between the cells and the abnormal extra-
cellular matrix
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30. The human body as a complex system
The fact that all anatomical systems can be classified on the
basis of their appearance is an important indication that each has
a specific form. The morphological criterion is therefore of
considerable importance in identifying and classifying anatomical
entities.
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31. The human body as a complex system
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32. The human body as a complex system
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33. The human body as a complex system
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34. The human body as a complex system
The different configurations and functions of an anatomical
entity emerge from structures organised in overlapping
hierarchical levels.
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35. The human body as a complex system
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36. The human body as a complex system
The change in shape, and therefore function, can be considered
as a dynamic process that advances through states that are
qualitatively different.
TIME (t)
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37. The human body as a complex system
The word “state” indicates the pattern configuration of a
system at a particular instant, which is specified by a large
number of dynamic variables. A dynamic system can be
characterised by a set of different states (x) and a number of
transitions from one state to another during a certain time
interval (t).
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38. The human body as a complex system
The speed of change is the time necessary for a change in
shape to occur or for the development of a perceptible
difference between the modified entity and its unchanged
reference system.
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39. The human body as a complex system
However, the parameter time depends on a large number of
variables that are interconnected in a complex number of
pathways. This makes it extremely difficult to predict the
exact time interval between two successive states.
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40. The human body as a complex system
DYNAMICAL DISEASE: In many different diseases, the natural
organization breaks down and is replaced by an abnormal
spatial pattern. These diseases, characterized by abnormal
temporal organization are called “dynamical diseases” (i.e.
cancer).
Leon Glass & Michael C. Mackey. From clocks to chaos (1988)
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41. The human body as a complex system
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42. The human body under the microscope
A HISTOLOGICAL SECTION (i.e. IMAGE) IS A SNAPSHOT OF A
DYNAMICAL DISEASE
TIME (t)
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43. The human body under the microscope
The main questions are: how can you get a histological image? and
what is behind a histological picture?
THE PRE-ANALYTICAL PHASE
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44. The human body under the microscope
A. SAMPLING;
B. FIXATION;
C. PROCESSING;
D. EMBEDDING;
E. CUTTING;
F. STAINING
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The human body under the microscope
SAMPLING
In medical sciences, sampling is gathering of matter from the
body to aid in the process of a medical diagnosis and/or
evaluation of an indication for treatment, further medical tests
or other procedures (i.e. digital pathology).

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The human body under the microscope
FIXATION
Process that inhibits autolysis and putrefaction, hardens tissue
and allows easy manipulation of soft tissues such as friable
tumours and brain.

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The human body under the microscope
FIXATION
A. Formalin fixation will affect every subsequent step that the
tissue has to endure.
B. It maintains the relationship between cells and extracellular
components, thereby protecting the tissue from the
denaturing effects of the damages of subsequent processing.

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The human body under the microscope
FIXATION
If fixation is inadequate or unsuitable, then the subsequent
staining methods will be impaired. Delays in fixation, variations
in the duration of fixation (normally, 24 hours) or changes in
the concentration of the fixative may result in poor overall
fixation with tissue loss occurring.

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The human body under the microscope
ABNORMAL FIXATION DETERMINES ARTIFACTS THAT CAN
AFFECT THE IMAGE ANALYSIS PROCEDURE

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The human body under the microscope
PROCESSING
It defines the diffusion of various substances into and out of
porous tissues. This movement requires no energy since it
always progresses down the concentration gradient.

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The human body under the microscope
EMBEDDING
Paraffin embedding is the standard method used in histology
laboratories to produce blocks of tissue for section cutting
(i.e. microtomy). This process usually involves surrounding the
tissues by a medium such as paraffin wax which when cooled
and solidified will provide sufficient support for section cutting.

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The human body under the microscope
CUTTING
A microtome is used to cut extremely thin slices or sections (2-4
micrometers) of tissue for optical microscopy studies. Microtomes
use steel, glass, or diamond blades depending upon the
specimen and thickness of the section required.

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The human body under the microscope
CUTTING
Longitudinal section
Trasversal section

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The human body under the microscope
CUTTING

55. The human body under the microscope
A. Intra-sample variability;
B. Inter-sample variability;
C. Inter-patients variability;
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The human body under the microscope
STAINING
Actually, there are four sub-types of investigators:
• Chemistry-oriented but not concerned with morphology.
• Those with contemporaneous interests in physiologic
chemistry, histology, and technology;
• Diagnostic histochemists (i.e. histopathologists);
• Morphologists equipped with computer-aided technologies.

57. The human body under the microscope
A. STUDY DESIGN;
B. PRE-ANALYTIC STANDARDIZATION;
C. DATA COLLECTION;
D. ANALYSIS;
E. RESULTS INTERPRETATION.
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The human body under the microscope
TISSUE MICROARRAY – ARE SMALL TISSUE FRACTION
REPRESENTATIVE OF A PATCHED DISEASE?

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Kinzler and Vogelstain,
CELL, 1996
BIOLOGICAL ASPECTS - CANCER CONSISTS OF CELLS THAT
ACCUMULATE GENETIC ABNORMALITIES

60. The human body under the microscope
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Kinzler and Vogelstain,
CELL, 1996

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NATURE REVIEW
IMMUNOLOGY, 2015

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Turley et al. NATURE
REVIEW IMMUNOLOGY,
2015

63. The human body under the microscope
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• The definition of the “measure of complexity” as “the number of
species or connections in the environment” designates the
“ecosystem” as the first level of anatomical complexity.
• The complexity of any anatomical system can be geometrical
when it regards the architecture of the system, or behavioural
when it concerns the intricate relationships of the system’s
components.

64. The human body under the microscope
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• The data must reflect a hypothesis;
• Sample size required for a particular study depends on the
wthin-group variation relative to differences between groups;
• Measurement error always exists in any collection of data, but it
doesn’t matter if it is substantially less than the differences you
want to measure.
• THE REAL MEASURE OF A NATURAL OBJECT DOES NOT EXIST.
QUANTITATIVE PATHOLOGY- SOME BASIC RULES

65. The human body under the microscope
65
• The number of specimens to be analysed depends on the
question being addressed;
• Depends on the error in your data;
• You need more specimens when the differences you want to
measure are small compared to the variation within your group
(natural or due to error).
QUANTITATIVE PATHOLOGY- SOME BASIC RULES

66. The human body under the microscope
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Despite the fact that all anatomical forms are characterised by non-
polyhedral volumes, rough surfaces and irregular outlines, it has
been suggested that sophisticated computer-aided analytical
systems based on the Euclidean principles of regularity, smoothness
and linearity can be used in human quantitative anatomy.
ALL PARAMETERS USUALLY APPLIED IN THE FIELD OF
QUANTITATIVE PATHOLOGY ARE BASED ON THE EUCLIDEAN
GEOMETRY.
The data must represent the “shape” adequately

67. The human body under the microscope
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However, the Fractal Geometry is a more powerful means of
quantifying the spatial complexity of real objects.
THE GEOMETRY OF ANATOMICAL SYSTEMS
Mandelbrot, SCIENCE,
1967

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Recently, a group of students in the School of Physics, Trinity
College Dublin, under the supervision of Prof. Stefan Hutzler,
studied Ireland’s coastline. Unsurprisingly, the ragged Atlantic
shore has a higher fractal dimension (D = 1.26) than the
relatively smooth east coast (D = 1.10).
The human body under the microscope
Hutzler S. (2013). Fractal
Ireland. Science Spin, 58, 19-
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69. The human body under the microscope
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Anatomical entities cannot be properly described by Euclidean
Geometry. Fractal geometrically entities are mainly characterized by
four properties:
A. irregularity of their shape;
B. self-similarity of their structures;
C. non-integer or fractional (fractal) dimension;
D. scaling, which means that measured properties depend on the
scale at which they are measured.
THE GEOMETRY OF ANATOMICAL SYSTEMS

70. The human body under the microscope
70
The most important property of fractal objects is that the schemes
that characterize them are similarly found again and again at
descending orders of magnitude so that their component parts, in all
dimensions, have a form similar to the whole (Self-similarity).
Self-similarity can be defined geometrically or statistically. An
object is geometrically self-similar when every smaller piece of the
object is an exact, or nearly exact, duplicate of the whole object.
THE GEOMETRY OF ANATOMICAL SYSTEMS

71. The human body under the microscope
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Di Ieva…Grizzi, THE
NEUROSCIENTIST, 2014

72. The human body under the microscope
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Statistical self-similarity, also indicated with the term “self-affinity,”
concerns anatomical objects. Small pieces that constitute anatomic
systems are rarely identical copies of the whole system. If we
consider a portion of tree branches or vascular vessels, they are not
a copy of the whole tree but represent the same self-similarity and
structural “complexity” (i.e., roughness and spatial pattern).
THE GEOMETRY OF ANATOMICAL SYSTEMS

73. The human body under the microscope
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Di Ieva…Grizzi, THE
NEUROSCIENTIST, 2014

74. The human body under the microscope
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A fundamental concept for the evaluation of geometric objects is
that of dimension, which is a characteristic value of the system. Two
main definitions of dimension have been proposed.
The first, named “topological dimension” It assigns an integer
number to every point in Euclidean space, indicated with the
symbol E3.
THE GEOMETRY OF ANATOMICAL SYSTEMS

75. The human body under the microscope
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The second definition of dimension came from Hausdorff and
Besicovitch. They attribute a real number to every natural object in
E3, lying between the topological dimension and 3. Benoit
Mandelbrot indicates the dimension of Menger with the symbol Dγ
and that of Hausdorff and Besicovitch with the symbol D.
THE GEOMETRY OF ANATOMICAL SYSTEMS

76. The human body under the microscope
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E = Dg = 0
0 2 31
E = Dg = 1 E = Dg = 2 E = Dg = 3
Euclidean topological
dimension (E)

77. The human body under the microscope
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DIGITAL PATHOLOGY AND MODELLING - NEOVASCULARITY

78. The human body under the microscope
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DIGITAL PATHOLOGY AND MODELLING
Grizzi et al, BMC Cancer,
2006

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Grizzi et al, BMC Cancer,
2006

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Grizzi et al, BMC Cancer,
2006

81. The human body under the microscope
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Grizzi et al, BMC Cancer,
2006

82. The human body under the microscope
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Grizzi et al, BMC Cancer,
2006

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84. The human body under the microscope
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85. The human body under the microscope
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86. The human body under the microscope
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QUANTITATIVE PATHOLOGY AND MODELLING – LIVER
STEATOSIS
a
Hepatic steatosis
Micro-
vesicular
Medio-
vesicular
Macro-
vesicular
bc

87. The human body under the microscope
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AS ASL ASMM ASM
0
5
10
15
Area(%)
* *
*
*
*
b
a
DS DSL DSMM DSM
0.0
0.5
1.0
1.5
2.0
SurfaceFractalDimension(D) *
*
*
*
*
*
c
d e
QUANTITATIVE PATHOLOGY – LIVER STEATOSIS

88. The human body under the microscope
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ASL ASMM ASM
****
****
****
b c d
e

89. The human body under the microscope
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QUANTITATIVE PATHOLOGY – HEPATIC INFLAMMATION

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QUANTITATIVE PATHOLOGY – HEPATIC FIBROSIS

92. The human body under the microscope
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94. The human body under the microscope
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95. The human body under the microscope
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QUANTITATIVE PATHOLOGY– PANCREATIC DESMOPLASIA
d e
N
A
TU
R
A
L
PA
N
C
R
EA
TITIS
PD
A
C
0
10
20
30
Siriusredstainedsurface(%)
f
N
A
TU
R
A
L
PA
N
C
R
EA
TITIS
PD
A
C
0
50
100
150
SpatialHeterogeneity(CV,%)
***
***
*** ***
a b c

96. The human body under the microscope
96
d e
a b c
N
A
TU
R
A
L
PA
N
C
R
EA
TIS
PD
A
C
0
10
20
30
40
Cycles
***
***
N
A
TU
R
A
L
PA
N
C
R
EA
TTIS
PD
AC
0.0
0.2
0.4
0.6
0.8
1.0
Degradation(%/Cycles)
*** ***

97. The human body under the microscope
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QUANTITATIVE PATHOLOGY AND PROGNOSIS

98. The human body under the microscope
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QUANTITATIVE PATHOLOGY AND PROGNOSIS

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DIGITAL PATHOLOGY AND PROGNOSIS

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QUANTITATIVE PATHOLOGY WORLWIDE STANDARDIZATION

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QUANTITATIVE PATHOLOGY AND WORLWIDE PROCEDURE
STANDARDIZATION

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Laghi…Grizzi et al, THE
LANCET ONCOLOGY, 2009

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DIGITAL PATHOLOGY AND WORLWIDE PROCEDURE
STANDARDIZATION
Laghi…Grizzi et al, THE
LANCET ONCOLOGY, 2009

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The human body under the microscope
CONCLUDING REMARKS
Complexity is so pervasive in the anatomical world that it has
come to be considered as a basic characteristic of anatomical
systems;
Anatomical entities, when viewed at microscopic as well as
macroscopic level of observation, show a different degree of
complexity;

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The human body under the microscope
CONCLUDING REMARKS
Complex system admits many descriptions (ways of looking at
the system) each of which is only partially true. Each way of
looking at a complex system requires its own description and own
mode of analysis;

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The human body under the microscope
CONCLUDING REMARKS
All the anatomical entities display hierarchical forms: their
component structures at different spatial scales or their process at
different time scales are related to each other.

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The human body under the microscope
CONCLUSIONS
Altrock et al., NATURE
REVIEWS CANCER, 2015

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The human body under the microscope
CONCLUDING REMARKS
Modelling the complexity of living beings should be take into
account the 10-12 order-of-magnitude span of timescales for
events in the biological system, whether molecules (ion channel
gating: 10-6 seconds), cellular (mitosis: 103 seconds), or
physiological (cancer progression, aging: 105 seconds)

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The human body under the microscope
CONCLUDING REMARKS
It is compulsory to underline that observed morphological
patterns can often be conceptualised as macro-scale
manifestations of micro-scales processes;
However, observed patterns or system states are created or
influenced by multiple processes and controls.

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The human body under the microscope
CONCLUDING REMARKS
Further, multiple processes operate at multiple spatial and
temporal scales, both larger and smaller than the scale of
observation (EMERGENT PROPERTIES);
The application of these concepts promise to be useful to
analyse and model the real significance of an observed
anatomical system at a given scale of observation.

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The human body under the microscope

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The human body under the microscope

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The human body under the microscope
TAKE-HOME MESSAGES
A. Technology is not science;
B. Human beings are complex and hierarchical systems;
C. Human diseases are dynamical processes complex in time
and space;
D. Histology represents any investigation at tissue level;
E. A histological section is ONLY a snapshot state of the
process.

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TAKE-HOME MESSAGES
F. The “interpretation of a histological image” is fundamental
to comprehend the disease’s behaviour;
G. Understanding “morphological changes” occurring at the
“tissue level” might open new questions on the complex
nature of human system.
H. Morphometrics does not tell you what “shape” mean.

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The human body under the microscope
Scientific knowledge develops through the evolution of new
concepts, and this process is usually driven by new methodologies
that provide previously unavailable observation.
The potential broad applicability of our study makes it possible to
increase, not only the knowledge in the prostate cancer research,
but also in the development of new therapeutic strategies.
“We cannot solve problems by using the same kind of thinking we
used when we created them”
Albert Einstein

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The human body under the microscope
This way of thinking may help to clarify concepts, interpret
new and old experimental data, indicate alternative
experiments and categorize the acquired knowledge.

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THANK YOU FOR YOUR ATTENTION
Pathology Horizons 2016