Impatto dei biomarcatori sulle
strategie diagnostiche e
terapeutiche del tumore del
polmone
Mauro Papotti
Dipartimento di Oncologia
Università di Torino
Impact of biomarkers
Diagnostic
-morphological
-immunophenotypic
(-genetic)
Prognostic
-immunophenotypic
-genetic
-molecular (miR, …)
Predictive
-determination of specific
targets in tissues vs blood
Impact of biomarkers
Innumerable options, most to be
validated, many not cost-effective,
some useless  the pathologist’s view:
-Provide histopathological diagnosis
-Define immunophenotype
-Maximize information on cytology/biopsy
-Test predictive markers as appropriate
-Technical issues
- type of specimen & amount of tissue
- interpretation
Histological features
Any sample  primary or mets
2015
Morphology & Immunoprofile
Easy
Squamous carcinoma
Adenocarcinoma
Difficult
Undifferentiated ca:
LCC? Sarcomatoid ca ?
Poor reproducibility of histological diagnosis of
PD SqC (k: 0.46 on H&E alone). Better after IHC
Thunnissen et al. JTO 2014;9:1354-62
DIFFICULT CASE
On bronchial biopsy,
it looks like
squamous ………
….but it also
looks like adeno
ADC
mucin
New criteria for resected ADC
-Invasive ADC
classified by
predominant pattern
2015
Grading
ADC
Staging
G1
G2
OEL
ADC
IEL
G3
Oyama et al. Prognostic Impact of
Pleural Invasion in 1488 Patients
with Surgically Resected NSCLC.
Jpn J Clin Oncol 2013;43:540-6.
LCC
2015
LCC & others
1
2
3
NO LCC diagnosis on bx
Terminology for bx
LCC
Reclassification
of LCC by IHC
LCC
Correlation of LCC with
differentiated lung cancers
Non SqC/non-NE
NSCLCs have
phenotypic (TTF1) &
genotypic (KRAS)
similarities with solid
ADC, except for mucin
loss
Refine diagnosis on cytology/biopsy
**
*
surgical
small samples
J Clin Pathol
2013;66:832-8
Refine diagnosis on cytology/biopsy
70% NSCLC are advanced: histotyping
possible on small samples only
FNA
Cell
Bronchial
smear
block
biopsy
CYTOLOGICAL MATERIAL
(sputum, FNA, BAL, effusion)
core bx
micro-HISTOLOGY
Define immunophenotype with
a tissue sparing approach
•
•
•
•
TTF-1
ADC
Napsin A
Surfactant prot
CK7
• p40 (ex p63) SqC
• HMWCKs
(i.e. CK5/6, 34bE12)
• Desmocollin-3
Noh & Shim Lung
• SOX2
Ca 2011
?
Diagnosis after a 4-marker panel
NSCLC-NOS: 36%
ADC & SqC: 64%
Cancer
Cancer
2011
2011
NSCLC-NOS: 14%
Favor ADC or
SqC: 22%
Null or
ambiguous
phenotpye
ADC & SqC: 64%
Refine diagnosis on cytology/biopsy
Correlation biopsy – surgical specimens:
TTF-1, napsin, p63/p40, CK5 were used
for IHC classification of 40 biopsy cases
 33 (82%) were concordant with the
resection specimens.
Zachara-Szczakowski S et al. Accuracy of classifying
poorly differentiated NSCLC biopsies with commonly used
lung carcinoma markers. Hum Pathol 2015;46:776-82
DIFFICULT
CASE
Both SqC &
ADC markers
PD ca with
ambiguous
phenotype
NPS A +
p40 –
«favor
ADC»
p63
CK5
TTF1
CK7
“Why to force NSCLC subtyping?”
-224 advanced
non-Sq NSCLC
-Similar OS for
morphological ADC
& “favor ADC” NSCLC
vs null phenotype
NSCLC (much poorer
outcome)
“Why to force NSCLC subtyping?”
LCC with null phenotype
have a significantly
shorter survival
compared to cancers
with an ADC-like
phenotype (p=0.007)
Impact of biomarkers
Diagnostic
-morphological
Having
-immunophenotypic
we can
(-genetic)
named the tumor,
move forward……
Prognostic
-immunophenotypic
-genetic
-molecular (miRNA, …)
Predictive
-determination of
specific targets in
tissues vs blood
Test response
to therapy on
xenografts?
-type of specimen and amount of tissue
Any, from primary or metastatic tumors,
provided tumor cells are present.
POOR CELLULARITY
Sputum
Core bx
Antibody cocktails in lung ca FNAB
Figure 2
(in the case of poor cellularity)
Napsin A + p63 (p40)
ADC
SqC
positive control
TTF1+Desmocollin-3
ADC
SqC
positive control
Cancer 2011
Cancer
Cancer
2011
2011
Sample evaluation: available and
adequate for molecular testing?
The larger
not always
the
better!!!!
OK
no
no
Accurate tumor cell selection
Which biomarkers ?
ERCC1
RRM1
TS
….
Unproven predictive
role of some targets of
chemotherapy agents
No impact of target
determination on DFS
He YW, et al. Prognostic value of ERCC1,
RRM1 &TS proteins in patients with
resected NSCLC. Cancer Chemother
Pharmacol 2015;75:861-7.
TS
pre-oper. material
and
surgical samples
Which biomarkers ?
PD-L1
Abs SP142
& 22C3
In 272 SqC pts, the response rate was
20% with nivolumab vs 9% with
docetaxel (p=0.008). PD-L1 expression
was neither prognostic nor predictive of
Brahmer J et al. Nivolumab versus Docetaxel in advanced
benefit
squamous-cell NSCLC. NEJM 2015;373:123-35.
PD-L1 expression was analyzed by IHC
(Merck Ab 22C3) in 678 st I-III NSCLC.
32.8% of cases. High PD-L1 expression
was found in 7.4% of NSCLC.
Pts with high PD-L1 expression had
significantly longer OS (p<0.05).
Cooper WA et al. PD-L1 expression is a favorable prognostic factor in
early stage non-small cell carcinoma. Lung Cancer 2015;89:181-8.
Which biomarkers ?
PD-L1
Response to PD-1 inhibitors (pembro) is
dependent on genomic profile (better with
high no of non-synonymous mutations)
Rizvi NA et al. Mutational landscape determines sensitivity
to PD-1 blockade in NSCLC. Science 2015;348:124-8.
In 495 patients receiving pembrolizumab, PDL1 IHC was scored as the % of membranous
reactivity in neoplastic cells.
Among patients with a score >50%, the
response rate to pembrolizumab was 45.2%.
Garon EB et al. Pembrolizumab for the treatment of non-smallcell lung cancer. NEJM 2015;372:2018-28.
ADC
Optimal method?
ALK
D5F3 Ab
granular
strong diffuse
membrane
ADC
Interpretation
ALK rearrangement if the two
signals are separated by a
distance >2 signal diameters)
[70% of cases] or by green signal
loss, in >15% of 50 scored nuclei.
>2 O
ALK IHC is now reliable with appropriate
antibodies.
A study is being proposed (coordinator A.
Marchetti) to validate IHC interpretation
ability in various Italian centers
…but what about other markers??
ADC
EGFR
RAS
ALK
c-MET
BRAF
ROS1
…….
Which biomarkers ?
amplification
phosphoMET
ADC
Other molecular biomarkers
miRNA 197 associated to >size, SqC,
poor outcome
Mavridis K et al. The oncomiR miR-197 is a novel prognostic
indicator for NSCLC patients. Br J Cancer 2015;112:1527-35.
-miRNAs are very stable & their expression profiling is
reproducible  ideal biomarker candidates.
-Novel committee of decision trees to derive 2-and 3-miRNA
100%-frequency rules  four 2-miRNA and three 3-miRNA
top-ranked rules. One rule is that:
If miR-98 expression >7.356 and miR-205 expression
<9.601  normal sample (no cancer) (SP & SN 100%).
-miR-98 and miR-205 have two common target genes
FZD3 and RPS6KA3 (carcinoma associated genes).
Song R et al. Rule discovery and distance separation to detect reliable miRNA
biomarkers for the diagnosis of lung SqC. BMC Genomics 2014;15 S9:S16.
ADC
Molecular biomarkers
The model of invasive mucinous ADC:
prevalent KRAS mutations (G12D/V) &
ERBB2, BRAF, PIK3CA (TP53 rare). In
addition, rearrangements involving NRG1
gene [chimeric proteins as primary
oncogenic drivers?].
NGS help to reveal the genetic landscape
of lung cancer. Role of identified mutations
& rearrangements to be verified.
Search of the optimal method (FISH vs
IHC) for screening.
Then identify optimal targets for therapy
Davies et al. Editorial JTO 2015; 10: 1129-30
Shim et al. JTO 2015;10:1156-62
Fernandez-Cuesta et al. Cancer Discov 2014; 4:415-422
Righi, Scagliotti, Novello, Papotti (submitted)
SqC
Squamous cell carcinoma
“… there are no approved targets for
advanced SqC, although the cancer
genome atlas project has detected a
significant number of somatic gene
mutations/amplifications, some
targetable, but their frequency of changes
is low (5-20%)….”
Herbst et al
Clin Cancer Res 2015
SqC
Appropriate predictive markers
Which targets
for SqC ?
Nature 2012; 489, 519-23
Marked genomic complexity
CN alterations:
SOX2, PDGFRA,
FGFR1, WHSCIL1,
CDKN2A (deletion)
mean
8.1 mutations/Mb
>> Mutations [chr. 3q]:
TP53 (81%!),
NFE2L2, KEAP1,
BAI3, DDR2, etc
therapeutic targets
SqC
Appropriate predictive markers
SqC: future molecular targets
FGFR1 +
DDR2 +
PTEN +
PI3K path
=
approx 50% of SqC have
known druggable alterations
Others include: TP53, CDKN2A, MLL2, NOTCH1, RB1,
HL-A, AKT (7%), EGFR VIII deletions in exons 2/3
(5%) [not sensitive to current EGFR inhibitors]
PD-1 (Nivolumab Mab)  active in SqC & ADC
Impact of biomarkers
along tumor progression.
Role of re-biopsy
Diagnostic
-morphological shift
Prognostic
-immunophenotypic or
-molecular markers
Predictive
-change of genetic profile
& modulation of targets
Response evaluation after biological drugs
ADC switched to SCLC after
TKI (retained mutations)
Data on repeat-biopsies (50 cases)
Genetic abnormalities & higher complexity
-in 74% bx of primary tumors, pre-tx
-in 84% re-bx after one treatment
-overall 54% had changes at re-bx
after one treatment [any CT,RT,TKI]
-Biologically, the tumor changes & adapts
-Therapy selects resistant cell population
-Single drugs activate other (survival)
pathways
-Other unknown reasons?
Data on repeat-biopsies (50 cases)
Compare different sites & sample types
Morphology & IHC  1/50 histology shift
EGFR mutated ADC  SCLC (EGFR mut)
TTF1
ORIGINAL BIOPSY
REPEAT BIOPSY
SYN
54% changed in
repeat-biopsies
Changes
in repeatbiopsies
Complexity
Figure 3A
The commonest
changes were MET
amplification and
T790M mutation
…….secondary mutations inevitably occur, leading to the
emergence of acquired resistance  role of the cMET
receptor and its ligand (HGF) …. cMET-inhibitors have been
developed (including Mabs and TKIs, as dual combinations
with an anti-EGFR TKI).
Passiglia et al. The Role of cMet in Non-Small Cell Lung Cancer Resistant to EGFR
Inhibitors: Did We Really Find the Target? Curr Drug Targets. 2014;15:1284-92.
Significance of changed
molecular profile in re-biopsy
RELEVANT!! If specific resistance
mutations or amplifications are detected,
which are associated to a specific
alternative treatment
TO BE CONFIRMED, if new mutations are
found to co-exist (no more mutually
exclusive), whose origin is not clear [ab
initio due to tumor heterogeneity vs
acquired after one or more therapies?]
LIQUID BIOPSY - Circulating tumor cells
Repeat biopsies to study genomic evolution as a result of therapy are difficult, invasive
and may be confounded by intra-tumour heterogeneity.
Genomic alterations in solid cancers can be characterized by massively parallel
sequencing of circulating cell-free tumour DNA released from cancer cells into plasma,
representing a non-invasive liquid biopsy.
Here we report sequencing of cancer exomes in serial plasma samples to track genomic
evolution of metastatic cancers in response to therapy. Six patients with advanced
breast, ovarian and lung cancers were followed over 1-2 years.
Quantification of allele fractions in plasma identified increased representation of
mutant alleles in association with emergence of therapy resistance. These included
-an activating mutation in PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase,
catalytic subunit alpha) following treatment with paclitaxel;
-a truncating mutation in RB1 (retinoblastoma 1) following treatment with cisplatin;
-a truncating mutation in MED1 (mediator complex subunit 1) following treatment with
tamoxifen and trastuzumab, and following subsequent treatment with lapatinib, a
splicing mutation in GAS6 (growth arrest-specific 6) in the same patient;
-a resistance-conferring mutation in EGFR (epidermal growth factor receptor; T790M)
following treatment with gefitinib.
These results establish proof of principle that exome-wide analysis of circulating
tumour DNA could complement current invasive biopsy approaches to identify
mutations associated with acquired drug resistance in advanced cancers.
Murtaza et al. Non-invasive analysis of acquired resistance to cancer
therapy by sequencing of plasma DNA. Nature. 2013 May 2;497:108-12.
LIQUID BIOPSY
PREVENTION
The serum levels of 4 biomarkers (ProGRP,
CEA, squamous cell carcinoma antigen [SCC],
and CK19 fragment [CYFRA21-1]) were
determined in 153 patients. This panel
increased the diagnostic specificity for highrisk subjects, especially for SCLC.
Yang DW et al. Role of a serum-based biomarker panel in the early diagnosis of
lung cancer for a cohort of high-risk patients. Cancer 2015;121 S17:3113-21.
In 152 NSCLC patients and 300 healthy controls,
serum miR-148a, miR-148b, miR-152 were
significantly downregulated, while serum miR-21
was overexpressed.
High miR-21 was correlated with large size &
advanced cancer stage.
Yang JS et al. Serum miR-152, miR-148a, miR-148b, and miR-21 as
novel biomarkers in NSCLC screening. Tumour Biol 2015;36:3035-42.
LIQUID BIOPSY
noninvasive biomarker and potential surrogate for
the entire tumor genome,
circulating tumor DNA (ctDNA) has been applied to
the detection of driver gene
mutations and epigenetic alteration and
monitoring of tumor burden, acquired
resistance, tumor heterogeneity and early
T et al. Role of circulating-tumor DNA analysis in non-small
diagnosis. MJiang
cell lung cancer. Lung Cancer. 2015. pii: S0169-5002(15)30057
Plasma-Therascreen® kit & peptide nucleic acids
(PNA)-clamp approach to detect EGFR mutations
in plasma-derived circulating-free tumor DNA from
96 NSCLC pts. Specificity: 100%, sensitivity:
65.4% (17/26 EGFR-mutant patients).
Similar results were obtained with the PNA-clamp
R et al. Assessment of high-sensitive methods for the
method. Pasquale
detection of EGFR mutations in circulating free tumor DNA from
NSCLC patients. Pharmacogenomics 2015;16:1135-48.
CONCLUSION
Oncologists are now faced with interpreting
large-scale genomic data from multiple
tumor types, possibly making it difficult to
stay current with practice standards in lung
cancer.
Although several professional societies
have incorporated biomarker testing
recommendations into clinical practice
guidelines for NSCLC dx and therapy,
health care providers still face considerable
challenges when establishing and
implementing these standards.
Levy BP et al. Molecular Testing for Treatment of Metastatic NSCLC: How to
Implement Evidence-Based Recommendations. Oncologist. 2015 Sep 1.
Thank
you!!
University of Turin Medical School
Susanna Cappia
Marco Volante
Luisella Righi
Ida Rapa
Simona Vatrano
Eleonora Duregon
Gaia Gatti
Arianna Votta
Jessica Giorcelli
Simone Busso
Federica Massa
Marco Loiacono
Valentina Monica
Massimo DiMaio
Silvia Novello
Giorgio Scagliotti
SqC
- Well differentiated keratinising SqCs
are generally easily recognized.
- Basaloid variant of SqC has specific
histological & molecular signature and a
more aggressive behavior.
Brambilla CG. Lung SqCs with basaloid histology represent a
specific molecular entity. Clin Cancer Res 2014 Sep 4 [Epub]
ADC
Molecular biomarkers
YAP1 mutant-allele carrier
frequency was 1.1% in patients
with lung adenocarcinoma
compared with 0.18% in controls.
YAP1 R331W is an allele
predisposed for lung
adenocarcinoma with high
familial penetrance
Chen HY et al. R331W Missense Mutation of Oncogene YAP1 Is a Germline Risk
Allele for Lung Adenocarcinoma With Medical Actionability. JCO 2015;33:2303-10.