Barrett's Esophagus and Esophageal Adenocarcinoma-Recent

Developments and Challenges

Dipen Maru, MDAssociate Professor

Department of Pathology

Objectives

• Definition of Barrett's esophagus (BE)

• Morphologic approach for dysplasia and intramucosal carcinoma

• Review of histologic associated with BE progression

• Discuss the non-histology biomarkers

• Brief review of prognostic markers of esophageal adenocarcinoma

Esophageal adenocarcinoma

• Rapidly rising incidence (>40% since 1990) due to increase in disease burden

• Poor survival outcome (15% 5 year survival)

Esophageal adenocarcinoma

• Apprx. 44% of adults in US have GERD at least once a month

• Estimated BE prevalence in US: More than 6 million

• Obesity-GERD-BE-Dysplasia-Adenocarcinoma sequence

• Histology is the gold standard for diagnosis and assessing the risk of adenocarcinoma in clinical practice

Barrett's esophagus

• Definition:

– Columnar epithelial metaplasia of squamous mucosa

– Gastric or intestinal type mucosa with >3 cm from GE junction long segment

– Intestinal (specialized or distinctive ) type of mucosa for ≤3 cm long segment

Different Definitions

• American College of Gastroenterology/German Society of Pathology/French Society of Digestive diseases/Amsterdam working group

Columnar mucosa in tubular esophagus with intestinal metaplasia (goblet cell metaplasia) demonstrated by histology

Different Definitions

• British Society of Gastroenterology

– Does not require intestinal metaplasia

• Japanese Groups

– Columnar lined epithelium synonymous with BE

– Columnar lined epithelium is identified by distal limit of the lower esophageal palisade vessels

– Distal 5mm excluded

Intestinal Metaplasia

• Goblet cells

• Brush border

• Villiform architecture

• Multilayering of the columnar epithelium

• Alcian Blue PAS stain

Alcian Blue PAS

Goblet CellsGoblet Cells

NonNon--Goblet Cell Columnar Goblet Cell Columnar Epithelium: Epithelium:

Cardia type mucosaCardia type mucosa

Pseudogoblet cellsPseudogoblet cells

Mild architectural changesMild architectural changes

CDXCDX--2/VILLIN/MUC2/VILLIN/MUC--22

Aneuploid Aneuploid

NonNon--Goblet Cell Goblet Cell Columnar Epithelium Columnar Epithelium with Dysplasiawith Dysplasia

?? Unsampled Intestinal?? Unsampled IntestinalMetaplasiaMetaplasia

Barrett's mucosa vs. Cardia intestinal metaplasia

Barrett's Esophagus Cardia Intestinal Metaplasia

Etiology:GERD ? H.pylori or Atrophic Gastritis

Squamous mucosa overlying intestinal metaplasia-Present

Squamous mucosa overlying intestinal metaplasia-Absent

Intestinal metaplasia with esophageal ducts or submucosal glands-Present

Intestinal metaplasia with esophageal ducts or submucosal glands-Absent

Diffuse intestinal metaplasia-Present Diffuse intestinal metaplasia-Absent

Incomplete intestinal metaplasia-Present

Incomplete intestinal metaplasia-Absent

Focal intestinal metaplasia with predominant cardia type glands-Absent

Predominant cardia type glands with focal intestinal metaplasia-Present

From Shrivastsva et al American Journal of From Shrivastsva et al American Journal of Surgical Pathology, 2007Surgical Pathology, 2007

Barrett's mucosa vs. Cardia intestinal metaplasia-controversial

• No uniform criteria

• Need endoscopic correlation

• Not accepted by all gastroenterologists and pathologists

Biopsy reporting recommendation

Specifying the type of columnar mucosa with specific mention of absence or presence of intestinal metaplasia should be reported in a pathology report

Dysplasia-Classification• IBD study groupNegativeIndefiniteLow-grade dysplasiaHigh-grade dysplasiaAdenocarcinoma

a Intramucosal carcinomab Invasive adenoca

• Vienna classificationNegative for neoplasia/dysplasiaIndefinite for neoplasia/dysplasiaNon-invasive low-grade neoplasiaNon-invasive high-grade neoplasia

a. High-grade dysplasiab. Non-invasive carcinoma (carcinoma in situ)c. Suspicious for invasive

carcinomaInvasive neoplasia

a. Intramucosal carcinomab. Sub mucosal carcinoma or

beyond•WHO classification

Dysplasia-Diagnostic approach

• Appropriate orientation of biopsy

• Sqaumocolumnar junction-Higher threshold

• Ulceration or acute inflammation-Higher threshold

Dysplasia-Diagnostic approach

• Surface maturation:

a)Progressive increase in amount of cytoplasmic mucin

b)Intact polarity

c)Decrease in nuclear size and hyperchromaticity

d)Absence of surface mitosis

pHH3 (surrogate pHH3 (surrogate marker of mitosis) marker of mitosis) in in

BE (A and D)BE (A and D)IND (B and E)IND (B and E)LGD (C and F)LGD (C and F)

Goodarzi et. alGoodarzi et. alModern Pathology, 2009Modern Pathology, 2009

pHH3 in pHH3 in

HGD (A and C)HGD (A and C)

Intramucosal ca. Intramucosal ca. (B and D)(B and D)

Goodarzi et. al. Goodarzi et. al. Modern Pathology, Modern Pathology, 20092009

pHH3 in BEpHH3 in BE--LGDLGD--HGDHGD--AdenocarcinomaAdenocarcinoma

Dysplasia-Diagnostic approach

• Architectural abnormalities:

– Crypt branching, budding, villiform transformation, back to back arrangement

– Glands to lamina propria ratio increase

– Abrupt transition from atypical/non-atypical epithelium

Dysplasia-Diagnostic approach

• Cytologic features:

– Mucin depletion, loss/dystrophic goblet cells

– Nuclear stratification, nucleomegaly, nucleolar prominence

– Loss of polarity, increase N/C ratio, irregular nuclear membrane, hyperchromasia, full thickness nuclear stratification

Maru D. Annals of Diagnostic Pathology, 2009Maru D. Annals of Diagnostic Pathology, 2009

LGDLGD

LGDLGD

High Grade dysplasiaHigh Grade dysplasia

HGDHGD

HGDHGD

Acute Inflammation with Regenerative Acute Inflammation with Regenerative AtypiaAtypia

LGD with acute Inflammation LGD with acute Inflammation HGD with acute Inflammation HGD with acute Inflammation

Basal crypt Basal crypt dysplasiadysplasia

Basal Crypt DysplasiaBasal Crypt DysplasiaBasal Crypt DysplasiaBasal Crypt Dysplasia

Interobserver variations in diagnosis of dysplasia

• Good reproducibility for diagnosis of HGD (k=0.65)

• Poor reproducibility for LGD (k=0.32) and indefinite for dysplasia (k=0.15) categories

• Poor consensus for HGD vs. Intramucosal carcinoma

Montgomery et al, Human Pathology 2001 120(7)

Guidelines for Surveillance (American Gastroenterology Association

2011)• No Dysplasia

Repeat endoscopy in 3-5 years

• Low-grade dysplasia

Confirmed by additional pathologist preferably one who is expert in esophageal histopathology

6-12 months

AGA guidelines (contd.)

• High-grade dysplasia

Confirmed by additional pathologist preferably one who is expert in esophageal histopathology

In absence of eradication- surveillance at 3-months interval

Multifocal HGD-More aggressive treatment

Mucosal abnormalities-endoscopic ultrasound and mucosal resection to exclude cancer

High-grade dysplasia vs. Intramucosal adenocarcinoma

• Confluent glandular proliferation with back to back arrangement expanding and overriding the lamina propria

• Haphazardly arranged highly dysplastic glands in lamina propria or muscularis mucosa

• Single cell infiltration or incomplete glands in a well oriented non ulcerated biopsy

• Dirty necrosis in the glandular lumen

• Ulceration and true desmoplastic stroma

• Reverse maturation

Risk for progression

• Presence of high-grade dysplasia

• Extent of high-grade dysplasia

• High-grade dysplasia with certain endoscopic abnormalities

• Low-grade dysplasia with consensus diagnosis of two or three gastrointestinal pathologists

High-grade dysplasia-Risk for progression

• Extensive/focal.– Mayo clinic definition: More than 5 crypts in

one biopsy or HGD in more than one biopsy at a time

– Cleveland clinic definition: HGD in more than one biopsy at a time

• Presence of endoscopic abnormalities associated with dysplasia– Ulcer– Stricture– Nodule/mass/polyp

• Clinical features

BE-Progression

• Biomarkers:

– Ploidy

– P53 (LOH for chromosome 17p)

– P16 (LOH for chromosome 9p)`

– Aberrant DNA methylation

– Sialyl Lewisa, Lewisx, Aspergillus oryzae lectin (AOL)

DNA Ploidy

• Presence of aneuploid population and increase in tetraploid population associated with progression of BE to HGD

• Flow cytometry, FISH, and laser scanning cytometry

• Need validation in multicenter trial

• Unavailability of flow cytometry at smaller health care centers

Aberrant p53 expression by Immunohistochemistry

• Mutation leads to abnormal p53 protein with prolong half-life leading to increase number of p53 positive cells

• Truncating mutation or epigenetic silencing leads to loss of p53 protein

• Limitations:

– 10% biopsies for negative for dysplasia are p53+

– Reproducible, easily applicable interpretation guidelines not available

Aberrant DNA Methylation

• P16, HPP1, RUNX3 genes methylation

• Combination with presence of LGD on histology and length of BE increase likelihood to predict progression to HGD

• Three tier (low-intermediate-high) risk system recommended

• Need validation in large trial

Early Detection Research Network Early Detection Research Network (National Cancer Institute) 2008 report(National Cancer Institute) 2008 report

Markers for Esophageal Adenocarcinoma (EAC)

• Histologic markers

• Tissue based molecular markers-

– Her2-Neu overexpression/amplification in Gastric and GE junction adenocarcinoma

Early (T1) EAC- Histologic marker

• Lymphovascular invasion in early EAC associated with poor outcome

• Duplicated muscularis mucosae can overstage pT in EAC

• Invasion in to the space between the duplicated muscularis mucosae has similar risk of lymph node metastasis as intramucosal carcinoma

Liu et al. American Journal of Surgical Pathology, 29(8):2005

Estrella J et al. American Journal of Surgical Pathology, 29(8):2011

Duplicated Muscularis MucosaeH&E and Trichrome Stains

T1 EAC invading into the space between the duplicated muscularis mucosaeH&E and Trichrome Stains

T1 EAC with lymphovascular invasion

Univariate AnalysisMultivariate

Analysis

POdds Ratio

POdds Ratio

Depth of Invasion (n=99) 0.028 1.03 0.85 1.00Tumor configuration

Ulcerative/Flat (n=67) 1.00Exophytic (n=32) 0.57 0.72

Tumor differentiation 0.009 0.39Well (reference) (n=37) 1.00 1.00Moderate (n=47) 0.70 0.74 0.48 0.57Poor (n=15) 0.01 5.37 0.52 1.75

Lymphovascular InvasionNo (reference) (n=76) 1.00 1.00Yes (n=23) 0.002 6.38 0.001 6.38

Pathologic tumor stageMucosa and duplicated MM# (reference) (n=69) 1.00 1.00Submucosa (n=30) 0.022 3.83 0.62 1.52

Pathologic tumor stage

Duplicated MM/submucosa (reference) (n=59)1.000

Mucosa (n=40) 0.70 0.78

RFS in patients with EAC invading duplicated MM is similar to intramucosal EAC

Patients with T1 EAC with LVI have shorter RFS then pts. with T1 EAC without LVI

EAC-Advanced Loco regional disease-Histologic marker

Neuroendocrine differentiation in pretreated biopsy is associated with poor outcome

Maru D et al. American Journal of Surgical Pathology, August 2008

Clinical Stage Median Overall Survival (months)

Loco regional disease (n=17) 28 ± 13 (Range 8-49)

Systemic disease (n=21) 11 ± 9 (Range 3-33)

p=0.006

SCNEC

LCNEC MIX ADENOCA/NEC

Histology Median Overall Survival (months)

Pure Neuroendocrine ca. (n=16) 15 ± 10 (Range 2-43)

Mix Ca. with Neuroendocrine component (n=24)

28 ± 13 (Range 3-49)

p=0.03

EAC-Advanced Loco regional disease-Histologic marker

In advanced loco-regional disease complete pathologic response and tumor downstaging are associated with better outcome

Chirieac et al. Cancer. 2005

Chemoradiation-Pathologic response.

Chirieac et al. Cancer.103 (7); 2005

Her2-Neu amplification

• Trastuzumab approved by FDA for stage IV gastric and gastroesophageal junction adenocarcinoma

• ToGA trial (Bang et al, Lancet 2010;376:687-97)

showing benefit in OS in combination with chemotherpay for inoperable locally advanced or metastatic adenocarcinoma

Issues specific to the Upper GI Adenocarcinoma

• Guidelines are somewhat different than breast cancer

• For IHC no requirement of percentage of cells in biopsy specimen and 10% positive cells cut of point in resection specimen

• For FISH cut off ratio of Her2-Neu/CEP17 is 2.0

• Heterogeneity within same tumor and between primary and mets are seen not infrequently

Staining Score Interpretation

No membranous staining of the invasive tumor cells

0 Negative

Faint or barely perceptible membranous staining of the invasive tumor cells

1 Negative

Weak or moderate membranous staining of the invasive tumor cells

2 Equivocal

Strong complete membranous staining of the invasive tumor cells

3 Positive

Her2-Neu Immunohistochemistry guidelines for biopsy specimens Staining Score Interpretation

No membranous staining of the invasive tumor cells 0 Negative

Faint or barely perceptible membranous staining of < 10% of the invasive tumor cells

0 Negative

Faint or barely perceptible membranous staining of > 10% of the invasive tumor cells

1 Negative

Weak or moderate membranous staining of < 10% the invasive tumor cells

1 Negative

Weak or moderate membranous staining of > 10% the invasive tumor cells

2 Equivoval

Strong complete membranous staining of < 10% invasive tumor cells

2 Equivocal

Strong complete membranous staining of > 10% invasive tumor cells

3 Positive

Her2-Neu Immunohistochemistry guidelines for resection specimens

Heterogeneous Her2/Neu expression/amplification in same tumor

Her2-Neu interpretation inUpper GI Adenocarcinoma

• Focal overexpression/amplification• Difficulty identifying the tumor cells•Technical issues with tissue fixation and processing•Multiple Her2/Neu FISH signals closely packed•Lower expression in poorly differentiated and signet ring cell carcinoma•False negative IHC (~5%)

Conclusions

1. Histologic interpretation by a proficient pathologist-Best predictor of progression of BE to EAC

2. Except for Her2-Neu no biomarkers is ready for routine clinical use in EAC

3. Meticulous gross and microscopic interpretation is critical in identifying predictive and prognostic factors for EAC