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What the clinician needs to know about breast pathology and sample quality
Frédérique Penault-Llorca MD, PhD
Centre Jean PERRIN – 58 Rue Montalembert - 63011 CLERMONT-FERRAND CEDEX
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Breast cancer
• Classical biomarkers
• Proliferation
• Prediction of response
• Molecular classification
• Importance of quality assurance in breast cancer
• Molecular signature
• Emerging targets5t
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Basically for the pathologist, the missions did not change….
• What is it, is benign, at risk or malignant?
• If it is at risk, which follow up ?
• If it is malignant, is it aggressive or “quiet”?
• How do I treat?
• Did I remove everything ?
To try to answer to 5 major questions
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Answers
• What is it, is benign, at risk or malignant?
• If it is at risk, which follow up ?
• If it is malignant, is it aggressive or “quiet”?
• How do I treat?
• Did I remove everything ?
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Priceless Information is Given by the Pathologist, AND will Never be Given by Molecular Testing!5t
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TNM
But we also have classical prognosis and predictive factors
• Age
• Grade
• Histological subtypes
• ER/PR and HER2 status
• Ki67 +/- mitotic index
• Vascular invasion
• Tumor margins
Oldies but
goldies
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TNM parameters:
• Pros – Treatment decisions are
based on T size
– Node involvement is a major prognosis factor
– Micrometastasis can be detected in sentinel lymph node by IHC
• Cons– TNM is decreasing
because of mass screening (69%of T1 in France in 2015)
– 70% of N0 in France in 2015
– SLN: axillary dissection is debatable in case of Nano/micromets
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Clarification of the AJCC 7th edition
Staging multiple tumors• If in same breast:
– T category is based on single largest tumor focus
– Don’t include satellite foci when measuring tumor size
– If multiple foci of microinvasion, report the # of foci and the size of the largest focus (don’t combine)
– Use (m) modifier
• If bilateral: – Stage each side separately
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Clarification of the AJCC 7th edition in the 8th edition
• Correlate gross, microscopic and imaging findings to assign correct pT when necessary.
- For small tumors diagnosed by core biopsy, measuring only the residual tumor in the excision may result in understaging. • Example:
– 6 mm mass by imaging; largest focus in biopsy core – 4 mm – 2 mm focus of residual carcinoma in excision: categorize as
pT1b (not pT1a) – No residual cancer in excision: categorize as pT1a (not pTX)
• Same rule applies when tumor is present in multiple fragments: Use clinical and imaging findings to assign pT
• pTX should rarely be used 5t
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Grade I
Grade II
Grade III8,9
SBR grade modified by Elston and Ellis
• Standardization of tumor grading
• France 2010: Gr I 25%, Gr II 50% , Gr III 25%
• Genomic grade : not confirmed
SBR grade and RFS in operable BC (57% N-) treated by adjuvant therapy
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VASCULAR EMBOLI
Lymphovascular invasion and BCSS in N-operable BC treated by adjuvant therapy (from Lee) 5t
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Group 3 - Average prognosis:Medullary, classical lobular, lobular mixed
Group 1 - Excellent prognosis: Tubular, invasive cribriform, mucinous
Group 2 - Good prognosis:Tubular mixed, mixed ductal NST and special type like adenoid cystic, secretory
Group 4 - Poor prognosisDuctal NST, solid lobular, mixed ductal NST and lobular, micropapillary
18 Histological types: morphology matters!
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Special types
« The heterogeneous « Triple negative » subtype includes adenoid cystic, juvenile secretory (good prognosis), medullary (intermediate prognosis), and metaplasic (either low grade, with good prognosis; or high grade, with poor prognosis) carcinomas, for which no generalizations can be proposed) »
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Medullary features and BCSSFrom medullary
Tubular
Non tubular
Tubular carcinoma and DFS (Rakha)
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Special types
“Tubular and cribriform carcinoma may be suitable for observation without therapy or
for endocrine therapy alone”
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Special types
“rare variant of lobular carcinoma (e.g. pleomorphic) (up to 25% HER2+) and apocrine carcinoma require treatment according to their biological features in a manner analogous to that used for ductal carcinoma “
Pleiomorphic lobular carcinoma
E CADH HER2
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Breast cancer histological types and molecular alterations
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Prediction
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Biomarker Prognostic PredictiveTechnical
validation
Clinical
validation
ER ++ +++ YES LOE Ib YES
PgR +++ ++ YES LOE Ib NO
HER2 ++ +++ YES LOE Ib YES
Ki67 ++ + NO NO
Test and scoring recommendations
IHC ≥10%
IHC ≥10%
IHC ≥10% cells with complete membrane staining
ISH: number of HER2 gene copies ≥6 or the ratio HER2/chromosome
17 ≥2
IHC no final consensus on cut-off around 20%
(Ki67< 10% = low ; Ki67>30% =high)
ER
pgR
HER2
Ki67
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Ki67 why?
• Definition of luminal A and B
• Decision of CT for ER+, Grade II tumors
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Ki67 = Not standardized
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Ki67 >70%
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Ki67 >70%
Ki67<5%
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Ki67 >70%
Ki67<5%
Ki67~20%
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Ki67 15%
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HER2
Negative predictive value
(<5% chance to respond to anti-estrogens or trastuzumab)
HIGH 95%
Positive predictive value
30-50%
Breast Cancer
ER/PGR
What is the level of prediction accuracy clinically useful?
Cut off 1%
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• 240 cases• 144 high ER (>10%), 75 ER negative and 21 low-ER
(1-10%) tumors by IHC• qRT-PCR test with 6 ER related genes• ½ low-ER positive tumors ER negative group
based on the probability score• 95% of ER negative and 92% of the high ER positive
tumors classified correctly (p<0.0001). • Survival of the low-ER group was intermediate
between that of the high ER positive and ER negative groups (p<0.05).5t
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In case of weak ER (1-9%) in practise
• On biopsy: redo on surgical specimen
• On surgical specimen:take into account alsothe other parameters
• Role of Gene Expression Signatures (GES) ?
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INTRINSIC (MOLECULAR) CLASSIFICATION AND SURROGATE
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Towards a simplified taxonomy of breast cancer? « definition
of intrinsic subtypes has proven efficient in defining prognosis for breast cancer patients »
RE neg RE pos
C Perou & T Sorlie
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Biology of breast cancer is influenced by age
de Kruijf Mol Oncol 2014, Jenskins Oncologist 2014
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African (mean age = 45 y)
African American (premenopausal)
African American (postmenopausal)
White in US (premenopausal)
White in US (postmenopausal)
White in Poland (mean age = 56 y)
Japanese (median age = 54 y)
0% 20% 40% 60% 80% 100%
Molecular biology of BC is influenced by ethnies and country of residence
Basal
HER2
Luminal A
Luminal B
Non classées
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Molecular biology of BC influence the metastatic diffusion routes
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• ER+, HER2- EBC: Luminal A and B subtypes predict 10-
year outcome regardless of previous systemic treatment as
well as residual risk of distant recurrence after 5 years of
endocrine therapy.
• HER2+: the 4 main intrinsic subtypes can be found
• HER2+/HER2-enriched benefit the most from
neoadjuvant trastuzumab, or dual HER2 blockade with
trastuzumab/lapatinib, in combination with CT
• HER2+/Luminal A disease have a relative better
outcome compared to the other subtypes.
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• triple-negative breast cancer (TNBC), of 70-80%
Basal-like from a biological perspective, should be
considered a cancer-type by itself.
• Distinction between Basal-like versus non-Basal-
like within TNBC predict
• survival following (neo)adjvuvant multi-agent
chemotherapy,
• bevacizumab benefit in the neoadjuvant setting
(CALGB40603)
• docetaxel vs. carboplatin benefit in first-line
metastatic disease (TNT study).
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St. Gallen 2013
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Personalizing the treatment of women with early breast
cancer: highlights of the St Gallen International Expert
Consensus on the Primary Therapy of Early Breast Cancer
2013
Luminal A all of:ER and PR positiveHER2 negativeKi-67 ‘low’Recurrence risk ‘low’ based onmulti-gene-expression assay (if available)
Luminal B ER positiveHER2 negativeand at least one of:Ki-67 ‘high’PR ‘negative or low’Recurrence risk ‘high’ based onmulti-gene-expression assay (if available)
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Personalizing the treatment of women with early breast
cancer: highlights of the St Gallen International Expert
Consensus on the Primary Therapy of Early Breast Cancer
2013
Luminal A Endocrine therapy often used alone.Cytotoxics may be added in selected patients.
• High risk status with gene signature, if available;• Grade 3 disease;• Involvement of four or more lymph nodes;• Young age (<35 years)
Luminal B Endocrine therapy for all patientsCytotoxic therapy for most.
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Surrogate definition of intrinsic subtypes of breast cancer
«basal-like»• ER and PgR absent• HER2 negative• Approximately 80% overlap between « triple
negative » and intrinsic « basal-like »• But « triple negative » also include good
prognosis special types such as medullary and adenoid cystic carcinoma
• Staining for basal keratin is considered insufficiently reproducible for general use
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Basal-likeTriple-negative
Triple negative BC by IHC and molecular subtypes: a 80% concordance
Medullary
Adenoid cystic
Low grade squamous
Secretory carcinoma
TNBC subtypes of excellent prognosis
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TNM
Classical prognosis and predictive factors
• Age
• Grade
• Histological subtypes
• ER/PR and HER2 status
• Ki67 +/- mitotic index
• Vascular invasion
• Tumor margins
Oldies but
goldies
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FOCUS ON HER2 GUIDELINES
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Huge benefit from anti HER2 therapies for patients
with mBC and eBC
13-15% HER2+ in eBC18% in mBC if previously treated ~20-25 if naive
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2013
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Heterogeneity: Where to count?
3+
0
2+
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1. Simplification of IHC 2+ definition (moderate/weeak)
2. Re-testing on surgical specimen if a biopsy is HER2 - :
“may” in instead of “should”
3. Revision and/or definition of difficult ISH categories (monosomies, co-amplification, “equivocal”) avoid as much as possible “equivocal/eligible” cases
Act III?
Based on IHC results
2018
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Messages for HER2 ASCO/CAP new guidelines
Effet magique recoHER2 2007-2018 ?
• Simplification of HER2 2+ 2• No longer systematic re-testing• Difficult ISH categories: between 4-6 copies +/- ratio
HER2/CEP17<2• Interpretation with IHC++++• Independant (second reader for ISH) for 2+• Disparition of equivocal ISH category
• Category 2 (monosomy): rather negative• Caegory 3 3 (co-ampl): rather positive• Category 4(ex-equivocal): rather negative
• Avoid single probe ISH5t
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QUALITY ASSURANCE
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Sources of variation in biomarkers testing
Time to slicing and
fixation Method of tissue
processing
Type of
fixation
Equipment
calibration
Laboratory
procedures
Time of
fixation
Assay
validation
Staff
competenceType of antigen
retrieval
Test
reagents
Control
materials
Assay
conditions
Use of
image
analysis
Interpretation
criteria
Reporting
elements
Scoring
system
Wolff et al 2007
IHC, ISH
testing
variables
Post-analytical Pre-analytical
Analytical
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Interlaboratory discrepancies
All the HER2+ patients are not
inditentied for treatmentKey Insight
CAC
PRIVE
CHU/AP
CHG/CH
FRANCEOverexpression Rate EBC
10,7%
11,5%
15,6%
10,9%
15,0%
11,5%
13,0%
14,0%
10,3%
13,0%
14,8%
10,0%
12,0%
11,0%
23,0%
13,0%
10,8%11,0%
23,0%
18,0%
12,0%
15,0%
12,0%
13,4%
12,0%
0%
5%
10%
15%
20%
25%
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Avg
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Courtesy of Antonio Wolff
HER2 testing (dis)concordance in adjuvant trastuzumab trials
Concordance Central vs. Local Lab
N9831JNCI
2002(total n = 119)
ASCO
2004(total n = 976)
JCO 2006(total n = 2535)
IHC 3+(HercepTest)
74% 79.5% 82%(false pos 18%)
FISH +(PathVysion)
67% 85% 88%(false pos 12%)
Magnitude of false-neg HER2 testing unclear but real…5th
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The ALTTO trial
PATIENTS 10829
NO TUMOR 244
DCIS only 297
HER-2 NOT
ELIGIBLE
1792
1329 locally pos (14.6%)
463 locally equivocal
ELIGIBLE 8496
730 locally equivocal
(61.2%)
Courtesy G Viale
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Patients 3451
No tumor 15
DCIS only 34
Not assessable (core biopsy, etc.)
66
HER-2 Not eligible 338 (9.8%)
Eligible 2998
APHINITY Trial
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Quality is a multidisciplinary matter
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Specimen is viable
& biologically
reactive
Molecular composition subject to
further alteration/degradation
Factors (examples)
• Antibiotics
• Other drugs
• Type of anesthesia
• Duration of anesthesia
• Arterial clamp time
Factors (examples)
• Time at room temperature
• Temperature of the room
• Type of fixative
• Time in fixative
• Rate of freezing
• Size of aliquots
Time.0
Pre-acquisition Post-acquisition
Biospecimen lifecycle: preanalytical factors
affect molecular composition and integrity
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Rapidity of fixation
Invasive ductal carcinoma
Breast cancer
Histopronostical Annotations
Quick procedure quality of RNA but alsoof proteins
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Antigen’s degradation
→ degradation of antigens (ER, PR, HER2) after retardated fixation (from1h) and overnight à +4°C in a saline solution
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Deleterious effect of
underfixation on breast
biomarkers
Modern Pathol
2009
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Key Recommendations: Pathologists
• Should ensure that any specimen used for HER2 testing (cytological specimens, needle biopsies, or resection specimens) begins the fixation process quickly (time to fixative within 1 hour) and is fixed in 10% neutral buffered formalin for 6 to72 hours, and that routine processing, as well as staining or probing are done according to standardized analytically validated protocols
• Should ensure that the laboratory conforms to standards set for CAP accreditation, or an equivalent accreditation authority, including initial test validation, ongoing internal quality assurance, ongoing external proficiency testing, and routine periodic performance monitoring.
www.asco.org/guidelines/ © American Society of Clinical Oncology®. All rights reserved.
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Multidisciplinary sessions
Treatment initiation Oncologist afterthe multidisciplinary tumor board
Tissue biopsy
Radiologist, endoscopist, surgeons
Sampling Fixation IHC testing
Treatment
Realization of IHC tests
Technician
Fixation and tissue transportation
Nurses , OR nurses, courier
Interpretation and pathology report
Pathologist
Diagnostic
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Possibility of vaccum preservation before fixation (tissue safe system)
Inking of the breast specimen
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Her2 testing - Internal control: normal breast
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External controls
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Overfixation is less harmfull
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TAKE HOME MESSAGES
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When to question a pathology report
• PgR+, ER-
• Lobular, tubular carcinoma HER2+
• Grade 1, ER+++, PgR+++, HER2+
• Grade 3, ER-, ki67 <5%
• Grade 3 ER+++, PgR+++
• Medulary carcinoma (carcinoma with lymphocyticinfiltrate) is extremely rare (ask for a second opinion)
Redo HER2 on surgical specimen if grade 3, ER- or ER+
If ER and/or PgR is negative on a biopsy redo on surgical specimen
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Is it a breast cancer?
• Metastasis to the breast are rare 0,2 à 1,3
• Most frequent– Lymphoma
– Melanoma
– Carcinoma (lung, GYN, kidney, digestive tract, prostate …..)
– Neuroendocrine non mammary tumors
• First clinical sign of the disease in 30% of cases
• Metastasis can occur after 10 yrs (up to 22 yrs) (melanoma, ovary…).
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Is it a breast cancer?
• Clinical presentation large nodules, growing fast, welldemarcated sometimes they are supercial.
• Can mimmick benign lesions (ACR3)
• Frequently unique
• beware of triple negative lesions with an unusual presentation (mucinous for instance)
• beware of ER+ with papillary aspects and psammomas
• aspect of lobular carcinoma in a men
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CONCLUSION
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TNM
Classical prognosis and predictive factors.
• Age
• Grade
• Histological subtypes
• ER/PR and HER2 status
• Ki67
• Molecular tests
• Tumor margins
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Patient with
cancer
Biospecimen
tumor « mini-me »
Researcher
Oncologist
Patient cured
Reflects tumor
biology
Personalized medicine
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Patient with
cancer
Biospecimen
tumor « mini-me »
Researcher
Induced
molecular
changes
Oncologist
Patient not cured
Reflects tumor
biology
Personalized medicine
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MOLECULAR SIGNATURES
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TNM
Yes , we have molecular biology !
• Age• Grade
• Histological subtypes
• ER/PR and HER2 status
• Vascular invasion
• Tumor margins
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Centralized tests
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MammaPrint
(Agendia, NL)
HR+ ET HR - / HER2- , T < 5cm, N ≤ 3
Fresh frozen=> FFPEDNA array
70 GENES CELL CYCLE/ PROLIFERATION
SIGNAL TRANSDUCTION
INVASION, METASTASIS, ANGIOGENESIS
« CENTRALIZED » TEST
RECENTLY ADAPTATED TO FFPE
Group of genes (« signatures »)
EARLY RECURRENCE (Dg < 5 ans)
PROGNOSTIC GOOD SIGNATURE :
LOW RISK
POOR SIGNATURE :HIGH RISK
HR+&
HR-
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OncotypeDX
(Genomic Health, USA)
HR+ / HER2- , T1-3, N-/N+
FFPE specimens
qRT-PCR
21 GENES PROLIFERATION, OESTROGENE,
HER2, INVASION (16 GENES) + REFS (5 GENES)
« CENTRALIZED » TEST
(recurrence score) RS
Late recurrence (10 years)
Benefit from adjuvant TT
PROGNOSTIC AND PREDICTIVE
LOW RISK :+ HORMONOTHERAPY / - CHEMOTHERAPY
INTERMEDIATE RISK :DISCUSSION
HIGH RISK :+ HORMONOTHERAPY / + CHEMOTHERAPY
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<26 prognosis
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Decentralized tests
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EndoPredict
(Sividon, GE)
HR+ / HER2- , T1-2, N0
FFPEqRT-PCR
8 GENES SIGNATUREPROLIFERATION, OESTROGENES
« LOCAL » TEST(SPECIAL EQUIPMENT IS REQUIRED)
SCORE OF RECURRENCE EP SCORE
LATE AND EARLY RECURRENCES
(5 & 10 YEARS)
PROGNOSIS
LOW RISK
HIGH RISK
UBE2C
BIRC5
DHCR7
STC2
AZGP1
IL65T
RBBP8
MGP
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Prosigna (PAM50)
(NanoString Technology, USA )
IDENTIFICATION OF « MOLECULAR3 SUBTYPES »
(LumA, LumB, HER2-enrichi, Basal)
FFPEDNA ARRAY WITH BARCODES
(1 gene = 1 barcode)
50 GENES
« LOCAL » TEST (SPECIAL EQUIPMENT IS REQUIRED)
LATE AND EARLY RECURRENCES
(5 & 10 YEARS)
PROGNOSIS
LOW RISK (ROR)
Intermediate risk
HIGH RISK (ROR)5t
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Mammaprint™ Oncotype DX® Prosigna® Endopredict®Number of Genes 70 21 50 11
Method DNA-Microarray RT-PCR Nanostring RT-PCR
Sample Type Frozen/FFPE FFPE FFPE FFPE
Location Central* Central Local Local
Test Results High or low risk + SubtypeRS High/Low risk
ROR High/Intermediate/Low risk + Subtype
Calculated with T&N
EP Clin High or low riskCalculated with T&N
Clinical Indication by EGTM
Predicting prognosis and guiding decision-making
regarding chemotherapy for women with ER+/HER2- EBC
with LN- or LN+ (1-3)
Predicting prognosis and guiding decision-making
regarding chemotherapy for women with ER+/HER2- EBC
with LN- or LN+ (1-3)
Predicting prognosis and guiding decision-making
regarding chemotherapy for women with ER+/HER2- EBC
with LN- or LN+ (1-3)
Predicting prognosis and guiding decision-making
regarding chemotherapy for women with ER+/HER2- EBC
with LN- or LN+ (1-3)
Prospective Validation MINDACT (positive)TAILORx (positive)
/ RxPONDER (ongoing)OPTIMA trial (ongoing) None
Agency approval EMA, FDA EMA, FDA EMA, FDA EMA, FDA
Original Validation set
Developed in young patients (<55 years), who had not
received systemic therapy following surgery
Developed in patients who had received tamoxifen only in the NSABP B-20 and B-14
studies
Developed in postmenopausal patients,
who received ET in the TransAtac trial
Developed in postmenopausal patients,
who had received ET only in the ABCSG-6 and 8 trials
Guidelines / scientific societies that recommend the test
ASCO, EGTM, ESMO, St. Gallen Panel
ASCO, EGTM, ESMO, NCCN, St. Gallen Panel
ASCO, EGTM, ESMO, St. Gallen Panel
ASCO, EGTM, ESMO, St. Gallen Panel
Paik et al. N Engl J Med. 2004, 51:2817-26; Paik J Clin Oncol 2006, 24:3726-3734; Filipits et al. Clin Cancer Res. 2011; 4. Bueno-de-Mesquita et al. Lancet Oncol. 2007;
5. Mook et al. Breast Cancer Res Treat. 2009; 6. Sapino et al. J Mol Diagn. 2013; 7. Dowsett et al. J Clin Oncol. 2013; 8. Gnant et al. Ann Oncol. 20135th
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EMERGING BIOMARKERS
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Upcoming biomarker: quantification of stromalLymphocytes (sTILs)
• Standardized (international recommendations)
• Semi-quantitative evaluation on H&E
• Predictive of NACT response in TNBC and HER2 breast cancer
• Prognostic value (primary tumor, and residual T after NACT)
• Predictive for immunotherapy?
Salgado et al Annals of Oncol,2015
Loi et al Annals of Oncol 2014
Dieci et al Sem Can Biol 2017
Hendry et al Adv Anal Pathol 2017
Standardized, St Gallen 2019, but clinical validityto prove5t
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Somatic BRCA status
PARPi
PD-L1 Immune cells
IO
BRCA status PARPi
PIK3CA mutations
PIK3Ci
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CONCLUSION …
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TNM
Classical prognosis and predictive factors
• Age
• Grade
• Histological subtypes
• ER/PR and HER2 status
• Ki67 +/- mitotic index
• Vascular invasion
• Tumor margins
Molecular signatures
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Luminal A Luminal B Triple-negative
ER<10%
PR<10%
HER2-
Ki67 >14%
High grade
ER >10%
Ki67 >14%
PR < 20%
HER2 + possible
ER > 10%
Ki67<14%
PR > 20%
HER2 -
HER2
ER <10%
PR < 10%
HER2 3+
High
Grade
ER+ ER-
AR +
EGFR+/-
HER2 +/-
Molecular
apocrine
Tubular
Cribriform,
IDC grade 1,
Mucinous
ILC grade 1
Micropapillary
ILC grade 2 & 3
IDC grade 2 et3,
Mucinous type B
NeuroendocrineApocrine
CCI grade 3
Medullairy
metaplastic
CCI &CLI grade 3
Proliferation
Mutations of TP53 or PIK3CA
Genomic instability
Intratumor heterogeneity
BRCAness
Breast Cancer classification in practice
- Low frequency of mutations
- Mutation in numerous genes
- Most frequently mutated genes:
PIK3CA, MAP3K1, MAP2K4
CCI grade 2 & 3,
Micropapillaire
TILs
Cheang et al CCR 2008, Prat et al JCO 2013, Kennecke et al JCO 2010,
Cheang et al, JNCI 2009, Goldhirsh et al Annals of Oncol 2012 , Cirqueira et al Breast J 2015
Blows et al Plos Medicine 2010
Adenoid
cystic
Secretory
Low grade
metaplasic
ER<10%
PR<10%
HER2-
Low grade
Translocations
(ETV6; NTRK3)
(MYB; NF1B)
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• ER+, HER2- EBC: Luminal A and B subtypes predict 10-
year outcome regardless of previous systemic treatment as
well as residual risk of distant recurrence after 5 years of
endocrine therapy.
• HER2+: the 4 main intrinsic subtypes can be found
• HER2+/HER2-enriched benefit the most from
neoadjuvant trastuzumab, or dual HER2 blockade with
trastuzumab/lapatinib, in combination with CT
• HER2+/Luminal A disease have a relative better
outcome compared to the other subtypes.
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• triple-negative breast cancer (TNBC), of 70-80%
Basal-like from a biological perspective, should be
considered a cancer-type by itself.
• Distinction between Basal-like versus non-Basal-
like within TNBC predict
• survival following (neo)adjvuvant multi-agent
chemotherapy,
• bevacizumab benefit in the neoadjuvant setting
(CALGB40603)
• docetaxel vs. carboplatin benefit in first-line
metastatic disease (TNT study).
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