МОРФОЛОГИЧНИ АНАЛИЗИ на канцерогенезата при

колоректалния карцином------------

Carcinogenesis in colorectal cancer - morphological analysis

07 – 11 - 2009

СБАЛО – ст.н.с ІІст. Ангел МилевУМБАЛ “Св. Анна” София АД – Стефан Петров

--------SBALO – Angel MILEV

UMBAL St. Ann – Stefan Petrov

Era Uma Vez...Основни понятия “CA for clinicians” 1983… (важат и днес)

The New Era - Hamamatsu, Japan…

and…what happens later ?

Kubota & Kino say:

Аденом с умбиликация в средата (1)Взаимоотношения на честотите на трите вида (3)

Аденоматозните жлези формират неравна повърхност (2a) и в част от тях жлезите са атипични (2b)

2a

2b

1

3

15 %

THE NEXT STEP: ABERRANT CRYPT FOCI

AND…

ACF & AGE

The American Concept (1999) – C M Fenoglio-Preiser & all.

1

2The American Concept (1999) – C M Fenoglio-Preiser

& all.

3The American Concept (1999) – C M Fenoglio-Preiser

& all.

methylene blue ( x 5 ) HE ( x 32 )

HE ( x 50 )Ki 67 ( x 50 )

>

>

>

>> >

>

>

>

>

4The American Concept (1999) – C M Fenoglio-Preiser

& all.

5The American Concept (1999) – C M Fenoglio-Preiser

& all.

Pit-Saw Blade Like Pit-Saw BladeSurface

For the first time used the term

"serrated" type of polyps

The American Concept (1999) – C M Fenoglio-Preiser & all.

5

ACF& possiblerelationships betweenhyperplastic (HP) & adenomatous (AP) polyps

ABC OF COLORECTAL CANCER (2001) - 1Molecular basis for risk factors - Robert G Hardy,

Stephen J Meltzer, Janusz A Jankowski

>

Edited by: D J KERR Professor &

ANNIE M YOUNG, Institute

for Cancer Studies, University of Birmingham

&F D RICHARD HOBBS

Molecular basis for risk factors - Robert G Hardy, Stephen

J Meltzer, Janusz A Jankowski

ABC OF COLORECTAL CANCER (2001) - 2

Ki-ras; KRAS; Kras – WHO IS IT?The ras family of oncogenes is constituted of three principal

members - K-ras, H-ras and N-ras - all of which have been implicated in the development of human malignancies. The K-ras oncogene resides on chromosome 12p12, and encodes a 21-kD protein (p21ras) involved in the G-protein signal transduction pathway, modulating cellularproliferation and differentiation. Actually V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog also known as KRAS is a protein which in humans is encoded by the KRAS gene. Like other members of the Ras family, the KRAS protein is a GTPase and is an early player in many signal transduction pathways.

Mutations of the K-ras oncogene result in constitutive activation of this signal transduction pathway and consequently, unregulated proliferation and impaired differentiation.

K-ras abnormalities are one of the earliest events in the stepwise progression of

colorectal neoplasms, being detectable even in histologically unremarkable

epithelium and aberrant crypt foci adjacentto cancers.

(as shown in the figure > left)

Biomarkers Predicting Clinical Outcome of Epidermal Growth Factor Receptor - Targeted Therapy in Metastatic Colorectal Cancer (S Siena , A Sartore-Bianchi , F Di Nicolantonio , JBalfour & A Bardelli) - Milan, Italy 2009Overview of

interlinked cellular

signaling pathways

involved inthe proliferation and progression

of colorectal cancer.

Agents targetingsignaling proteins

that have been evaluated or are currently

being evaluatedin phase II, III, or IV

clinical trials for colorectal

cancer are shown. The

epidermal growth factor receptor

(EGFR) – related family

of receptortyrosine kinases includes human

epidermal growth factor

receptor(HER1), EGFR, or c-

erbB1; HER2 or c-erbB2; HER3

or c-erbB3; and HER4

or c-erbB4.

C-MET = mesench

ymal – epithelial transition factor;

EGF =epiderm

al growth factor;

HDAC = histone

deacetylases;

HGF = hepatocy

tegrowth factor;

IGF-1 = insulin-

like growth

factor-I; IGF-1R =

insulin-like

growth factor-I

receptor; IR =

insulin receptor;

VEGF = vascular

endothelial

growth factor;

VEGF-R =

vascular endothelial growth

factor receptor.

KRAS Mutation Testing – Identify metastatic colorectal patients who are non-responsive to EGFR therapy. -1 The Some of the previous Figure on 3D:

KRAS Mutation Testing – Identify metastatic colorectal patients who are non-responsive to EGFR therapy. -2

The Basis of the Modern Therapy of CRC - 1

KRAS Mutation Testing – Identify metastatic colorectal patients who are non-responsive to EGFR therapy. -3 The Basis of the Modern Therapy of CRC - 2

KRAS Mutation Testing – Identify metastatic colorectal patients who are non-responsive to EGFR therapy. -4The Visual Image of K-RAS – LUNG ADENO-CA - FLU

WHERE IS IT?

KRAS Mutation Testing – Identify metastatic colorectal patients who are non-responsive to EGFR therapy. - 51

Molecular Pathology Laboratory Network, Inc. (MPLN) offers KRAS mutation testing to assist with the identification of colorectal cancer (CRC) patients who are most likely to show limited clinical response to anti-epidermal growth factor receptor (anti-EGFR) therapies such as cetuximab (Erbitux®) and panitumumab (Vectibix®).

In January 2009, the American Society of Clinical Oncology released its first Provisional Clinical Opinion recommending that all patients with CRC have tumors tested for KRAS gene mutations prior to the initiation of therapy.

Study Forecasts Savings Using KRAS TestBased on annual incidence of new metastatic CRC cases, a savings of more than

$604 million for cetuximab alone would result through treatment drug stratification based upon KRAS status.

KRAS Mutations TestedThe incidence of KRAS gene mutations in CRC is approximately 35-45%.

Identifying patients who will benefit from treatment is vital to ensuring better clinical outcomes as well as determining the best treatment strategies for each patient.

The KRAS mutation test performed at MPLN detects 12 somatic mutations in codons 12 and 13 of the KRAS oncogene.

METHODOLOGYTumor tissue is microdissected and DNA extracted from

fivemicron sections of formalin-fixed paraffin-embedded tissue

blocks.

KRAS Mutation Testing – Identify metastatic colorectal patients who are non-responsive to EGFR therapy. - 6

Practical: Test Screen – D-r S. Alexov Private Laboratory( sertificated in SWEDEN )

HaggittclassificationLevel 0:Carcinoma n

situ Notinvasive.Level 1:Carcinoma

Invadingthrough

muscularismucosa-limited

to theHead.Level 2:

Carcinomaneck of the

adenoma. Level 3:

Carcinomainvading any

part ofThe stalk. Level 4:

Carcinomainvading into

the thesubmucosa of

bowel.

PREDICTING CA RISK IN CR ADENOMA (2009) – 1 (Stavanger, Norway)

PREDICTING CA RISK IN CR ADENOMA (2009) – 2 (Stavanger, Norway)

Metachronous and synchronous cancer detection. Time from index adenoma detection to cancer detection in synchronous versus metachronous cancer.

Synchronous cancer is usually defined as detection at the time

of, or within 6 months from, index adenoma detection.

Metachronous cancer detection is defined from 6 to 24 months after index

detection, but definitions vary

considerably in the literature. No

universal definitions exist.

Risk of cancer in adenomas according to type and size.

Derived and modified from WHO

PREDICTING CA RISK IN CR ADENOMA (2009) – 3 (Stavanger, Norway)

PREDICTING CA RISK IN CR ADENOMA (2009) – 4 (Stavanger, Norway)

Morphology versus morphometry for adenoma assessment. Morphometric measurements performed by quantitative, digitalized assessment of histopathology slides compared with regular pathology assessment through the microscope.

PREDICTING CA RISK IN CR ADENOMA (2009) – 5 (Stavanger, Norway)Main pathwaysincolorectalcarcinogenesisrepresent themajor partin 75-80% ofpatients.These pathwayshavedistinct clinical,pathological andgeneticcharacteristics,whichcan be used formolecularclassificationfor improveddiagnostics,prognosis andtreatment.

PREDICTING CA RISK IN CR ADENOMA (2009) – 5 (Stavanger, Norway)

Bias in biomarker research. Bias may

develop at

any level, as illustrated in the figure – ranging from

clinical, biological

or technical

standpoints.

AIM: Which of the Polyp is MOST OFTEN SERATED ?

MATHERIAL: Pinch-biopsy of 241 patients with 404 total lesions, with polyps of the colon, of which 203 single and 201 combined (194 double and 7 triple) were investigated routinely (HE).

METHODS: In addition to all GIT histochemycal methods, a pilot study of a part of hyperplastic polyps with controls (KRAS) and other was carried out and intestinal adeno-Ca as well – a total of 10 patients. The study was retrospective and the results were processed, using EXCEL 2003 (only %). Graphs were created with the same program.

OWN STUDIES – AIM AND M. & M. - 1 - (St.Ann)

OWN STUDIES RESULTS – POLYPS – 1 - (St.Ann)

BASIS OF THE POLYP’S SCIENCE…

Hyperplastic Polyps, HE x 32 Tubular Adenoma, HE x 32

Villous Adenoma, HE x 32 Tubulo-Villous Adenoma, HE x 50

OWN STUDIES RESULTS – POLYPS – 2 - (St.Ann)BASIS OF THE POLYP’S SCIENCE.

Serated Adenomas, HE x 32 Lymphomatoid Polyp, HE x 32

Inflamatory Fibrous Polyp, HE x 32 Melanosis in Peutz-Jeghers Polyp, HE x 32 (arrow)

>

DYSPLASIAS - LOW & MIDDLE GRADE ( I & II )

OWN STUDIES RESULTS – POLYPS – 3 - (St.Ann)

Dyspl. Gr. I in Hyperplastic Polyp, HE x 32 (arrow)

>

Dyspl. Gr. II in Hyperplastic Polyp, HE x 32 (arrow)

>

Dyspl. Gr. II in Serated Polyp, HE x 32 (arrow)

>

Dyspl. Gr. III in Serated Polyp, HE x 32 (arrow)

>

OWN STUDIES RESULTS – POLYPS – 4 - (St.Ann)DYSPLASIAS - HIGH GRADE ( III ) & FOCAL CA.,

ADENOCA.

Dyspl. Gr. III (arrow) &Focal Ca. (2 arrows) in Peutz-Jeghers Polyp, HE x 50

>

>

>

High Differentiation (G1) in INTESTINAL ADENOCA, HE x 32

Middle Differentiation (G2) in

INTESTINAL ADENOCA, HE x 50 (arrow)

>

Low Grade Differentiation (G3) in INTESTINAL ADENOCA, HE x 160

OWN STUDIES RESULTS – POLYPS – 5 - (St.Ann)K-Ras TEST – PILOT STUDY (10 patients) – only Hyperplastic

Polyps

K-Ras ( - ) in Hyperplastic Polyp, HE x 50

K-Ras ( + ) FOCALLY in Hyperplastic Polyp, HE x 50 (arrow)

>

K-Ras ( +++ ) FOCALLY in Hyperplastic Polyp, HE x 50 (arrows)

>

>

>

K-Ras ( +++ ) FOCALLY in Hyperplastic Polyp, HE x 160 (arrows)

>

OWN STUDIES RESULTS – POLYPS – 6 - (St.Ann)K-Ras TEST – PILOT STUDY (10 patients) – CONTROLS

K-Ras ( +++ ) in Focal Ca.- Peutz- Jeghers Polyp, x 160

K-Ras ( +++ ) in G3 INTESTINAL ADENO Ca. , x 160

OWN STUDIES RESULTS – POLYPS –7 - (St.Ann) (BG)R E S U L T S – STANDARD METHODS 1.

Fig. 1. Разпределение на полипозни лезии (контролна група) без отчитане на сератни

полипи - м. април, 2009 (гастробиопсии) (бр.)

0

2

4

6

8

10

12

14

16

18

20

20 - 50 50 - 70 > 70 ж м ц к.а к.т к.д с р

( ж / м )

възраст пол локализация

показател

бр

.

хиперплазиогенен полип без дисплазия

хиперплазиогенен полип дисплазия І ст.

хиперплазиогенен полип дисплазия ІІ ст.

хиперплазиогенен полип дисплазия ІІІ ст.

хиперплазиогенен полип с фокален Са.

тубуларен аденом без дисплазия

тубуларен аденом дисплазия І ст.

тубуларен аденом дисплазия ІІ ст.

тубуларен аденом дисплазия ІІІ ст.

тубуларен аденом с фокален Са.

вилозен аденом без дисплазия

вилозен аденом дисплазия І ст.

вилозен аденом дисплазия ІІ ст.

вилозен аденом дисплазия ІІІ ст.

вилозен аденом с фокален Са.

тубуло - вилозен аденом без дисплазия

тубуло - вилозен аденом дисплазия І ст.

тубуло - вилозен аденом дисплазия ІІ ст.

тубуло - вилозен аденом дисплазия ІІІ ст.

тубуло - вилозен аденом с фокален Са.

други фибром

други възпалителен фиброзен полип

други лимфоматоиден полип

други Пьотц - Йегерс

*

*

*

*

изследвани 38 пациенти с общо 115 полипозни лезии ( 63 единични, 52 комбинирани )

OWN STUDIES RESULTS – POLYPS –8 - (St.Ann) (BG)R E S U L T S – STANDARD METHODS 2.

Fig. 2. Разпределение на полипозни лезии за 6-месечен период

(април- септември '2009) - дебело черво (гастробиопсии) (бр.)

0

5

10

15

20

25

30

20 - 50 50 - 70 > 70 ж м ц к.а к.т к.д с р

( ж / м )

въз раст пол локализ ация

показ ател

бр

.

сератен полип без дисплаз ия

сератен полип дисплаз ия І ст.

сератен полип дисплаз ия ІІ ст.

сератен полип дисплаз ия ІІІ ст.

сератен полип с фокален Са.

хиперплаз иогенен полип без дисплаз ия

хиперплаз иогенен полип дисплаз ия І ст.

хиперплаз иогенен полип дисплаз ия ІІ ст.

хиперплаз иогенен полип дисплаз ия ІІІ ст.

хиперплаз иогенен полип с фокален Са.

тубуларен аденом без дисплаз ия

тубуларен аденом дисплаз ия І ст.

тубуларен аденом дисплаз ия ІІ ст.

тубуларен аденом дисплаз ия ІІІ ст.

тубуларен аденом с фокален Са.

вилоз ен аденом без дисплаз ия

вилоз ен аденом дисплаз ия І ст.

вилоз ен аденом дисплаз ия ІІ ст.

вилоз ен аденом дисплаз ия ІІІ ст.

вилоз ен аденом с фокален Са.

тубуло - вилоз ен аденом без дисплаз ия

тубуло - вилоз ен аденом дисплаз ия І ст.

тубуло - вилоз ен аденом дисплаз ия ІІ ст.

тубуло - вилоз ен аденом дисплаз ия ІІІ ст.

тубуло - вилоз ен аденом с фокален Са.

други фибром

други въз палителен фиброз ен полип

други лимфоматоиден полип

други Пьотц - Йегерс

**

*

*

*

** *

изследвани 203 пациенти с общо 289 полипозни лезии (140 единични, 149 комбинирани)

сератни полипи – от общ брой полипи 5,19 %сератни полипи – от общ брой пациенти 7,39 %

OWN STUDIES RESULTS – POLYPS –9 - (St.Ann) (BG)

Fig.3. Комбинирани: полипозни лезии & интестинален карцином.

1 1 1 1 111

1 1

2

1 11

2

1

11

11

2

11

2

1

2

1

111

2

1

1

1

4

1

3

1 1

1

1

1

1

1

1

0

5

10

15

20

25

30

бдІ ІІ ІІІ ф

кбд

І ІІ ІІІ фк

бдІ ІІ ІІІ ф

кбд

І ІІ ІІІ фк

бдІ ІІ ІІІ ф

к a пG

1G

2G

3

сер пол хип пол туб ад вил ад т-в ад ХУХК И АК

тип лезия

% /

бр

. ко

мб

ин

аци

и

др П-Й

др лп

др вфп

др ф

ад фк

ад ІІІ

ад ІІ

ад І

т-в бд

ад фк

ад ІІІ

ад ІІ

ад І

вил бд

ад фк

ад ІІІ

ад ІІ

ад І

туб бд

пол фк

пол ІІІ

пол ІІ

пол І

хип бд

пол фк

пол ІІІ

пол ІІ

пол І

R E S U L T S – STANDARD METHODS – COMBINED LESIONS 3.

*

**

*

* *

изследвани общо 201 случая с комбинирани полипозни лезии, от които само 7 са тройни

OWN STUDIES RESULTS – POLYPS –10 - (St.Ann) (BG)R E S U L T S – K-Ras - PILOT STUDY 4.

10

0 0 0 0

10

0

10

0 0

10

0 0

10

0

0

1

2

3

4

5

6

7

8

9

10

% K

-RA

S (

+)

%

изследвани лезии при 10 случая

Fig. 4. Разпределение на положителните за K-RAS изследвани

лезии ( в %)

1 61 м Фокален Са. при Пьотц-Йегерс . полип +

2 66 м 1. Хип. полип –ДІІ-IIІ ст -

2 66 м 2. АденоСа(G3) -

3 72 м Хип. полип –ДII ст. -

4 66 ж Хип. полип -

5 50 м Хип. полип –ДIIІ ст +

6 58 м 1. вил. аденом -

6 58 м 2. АденоСа(Г2) +

6 58 м 3. вил. аденом -

7 62 м 1. Активен УХК -

7 62 м 2. Хип.полип ДІІІст. +++

8 60 м 1. Фокален Са. лри Хип. полип -

8 60 м 2. туб. аденом -

9 68 м Туб. аденом ++

10 29 ж Фокален Са. при Хип. полип -

Ф.к

при

-P-J

( +

)

Хип.

П. +

Дис

пл. І

І І с

т. (

+ )

Аде

но-С

а –

G2

( + )

Туб.

Аде

ном

( ++

+ )

Хип.

П. +

Дис

пл. І

І І с

т. (

+ ++

)

CONCLUSIONS

1. The results clearly indicate a reduction of the percentage of tubular adenomas without dysplasia at the expense of the serated polyps, which corresponds somewhat with the available data of other authors

(CM Fenoglio-Preiser & all.)

2. As for the K-Ras test, the only thing that can be assumed about that limited amount of material is, that the rate of the K-Ras (+) cases did not exceed 10%. Part of the surveyed patient samples were with focal expression, unlike other sites (breast, stomach, etc..), which have yet to be specified.

The ENDMaybe the Key to the understanding of the colorectal carcinoginesis is here:

Maybe the solution to the problem is so close, or not… who knows?

But we ought to find it !