Importancia del patólogo en los

tumores de origen desconocido

Dr. Federico Rojo

Fundación Jiménez Díaz, Madrid IMIM-Hospital del Mar, Barcelona

• 3-5 % of diagnosed cancers

• Variability depending of centers

• 60% adenocarcinomas

• Immunohistochemistry helps in 30-85%

• Metastases to liver (25%) and bone (25%)

• Prognosis depends of identification of primary site or actionable genomic alterations.

Cancer unknown primary

• Adenocarcinoma (50%)

• Poorly Differentiated Carcinomas (35%)

• Squamous Cell Carcinoma (5%)

• Neuroendocrine Carcinoma (5%)

• Undifferentiated Carcinoma (5%)

Morphology of carcinomas of unknown primary site

Oien, KA & Dennis, JL. Ann Oncol 2012

Primary sites determined at autopsy in 884 patients with unknown primary cancer

Pentheroudakis, G et al. Eur J Cancer 2007

Greco, FA. JNCI 2013

Clinical landscape of cancer unknown primary over decades

• Cytokeratins

• Organo-specific markers (PSA, Thyroglobuline, GCDFP-15, Hepatocite)

• Non-organ-specific markers (CEA, p63, ER/PR, HMB45, Melan-A)

IHC for CUP: performance and practice

Screening IHC markers for the diagnosis of CUP

Oien, KA & Dennis, JL. Ann Oncol 2012

Massard, C. et al. Nat. Rev. Clin. Oncol. 2011

CK profile of frequently occurring primary cancers

(70-90%)

(70-90%)

(90%)

(90%)

Mesothelioma

Neuroendocrine cancer Squamous cell cancer

• GCDFP-15 (sensitivity, 55%; highest in lobular and apocrine; independent of grade, ER status, mitotic index; also expressed in vulva, eyelid; never expressed in lung, colon, ovary)

• Mamaglobin (sensitivity 46,6%,

combined with GCDFP-15,

sensitivity 69%)

• ER, PR

Mamoglobin

Cancer of unknown origin Breast cancer markers

GCDFP-15

• Villin (sensitive for colon ca; 5% lung adenoca)

• CDX-2 (sensitive for colon ca; occasionally positive in mucinous ovarian or bladder adenoca)

• CK7 / CK20

Cancer of unknown origin GI cancer markers

Villin

CDX-2

• 38 kd member of the NKx-2 family of transcription factors

• Thyroid, respiratory epithelium and diencephalon

• Lung tumors (highest in neuroendocrine and bronchioloalveolar; lowest in squamous and mucinous)

• Occasional rectal and ovarian adenoc.

• Positive in thyroid and neuroendocrine tumors

TTF-1

Molecular profiling for CUP

• One gene

• Multiple genes

Strategy #1.

Molecular alterations of tumor sites

• 63 year old woman

• Disseminated cancer (liver and lung) with pleural effusion

• Pleural cytology: Positive for malignancy, consistent with adenocarcinoma, CK7 positive, CK20 and TTF-1 negatives

Targetable single gene analysis in CUP

Targetable single gene analysis in CUP: EGFR mutation

L858R

Control

Targetable single gene analysis in CUP: EGFR mutation

Pleural metastasis from pulmonary adenocarcinoma with EGFR mutation

Treatment with EGFR TKI

Quest-Lab Corp

CUPPrint Pathworks Gene Search

Epitype CancerTYPE miRview mets

Provider Arcturus Bioscience

Agendia Pathworks diagnostics

Veridex bioThera-nostics

Rosetta Genomics

Number of genes

92 495 >1500 10 23 CpG 92 64

Cancer types

39 48 15 6 13 54 42

Method PCR, gene expression

Microarray, gene

expression

Microarray, gene

expression

PCR, gene expression

Methylation array

PCR, gene expression

PCR, miRNA expression

Material FFPE FFPE Fresh frozen /

FFPE

FFPE FFPE FFPE FFPE

Sensitivity (%)

85 83 84 75 NA 87 92

Commercial Tests for Cancer of Unknown Origin

• Identifies the tissue-of-origin for 42 different types of tumors from seven combined classes (sarcoma, kidney, thyroid, neuroendocrine lung, germ cell, astrocytic or oligodendroglial, and pancreatico-biliary adenocarcinoma) which represent over 92% of all cancer types (a dataset that contains 1282 tumours)

• Leverages measure the expression level of 64 miRNA using microarray platform

• Performance characteristics bsed on blinded validation set: sensitivity of 85%, specificity of 99.3%, sensitivity for single answer of 90%

MicroRNA based test to identify primary origin of metastasis

Varadhachary, GR et al. Clin Cancer Res 2011

Prospective miRNA signature study to identify tissue of origin with CUP

• Database of 2140 tumors of 58 types and subtypes, grouped into 15 classes: breast, bladder, colorectal, gastric, testicular germ cell, hepatocellular, kidney, non-small-cell lung, non-Hodgkin’s lymphoma, melanoma, ovarian, pancreatic, prostate, sarcoma and thyroid

• Microarray-based test that measures the expression of >1600

genes

• Pathwork reports similarity scores (SS) compared with each of the 15 tumour classes: SS > 60 agrees with 90% of reference diagnoses

Pathwork tissue of origin test

Pathwork tissue of origin test

• 64 year old woman

• Bilateral ovarian tumors

Courtesy by Dr Matias-Guiu

CK 7 negative; CK 20 positive

• Gastroscopy negative

• Gastric biopsies: Adenocarcinoma with signed ring cells

Gastric carcinoma metastatic to the ovaries

• 50 year old woman.

• Vaginal Bleeding

• Endometrial Biopsy

• Bilateral Ovarian tumors

Courtesy by Dr Matias-Guiu

AEI-AE3 + CA125 – CK7 – CA19.9 – CK 20 + ER, PR – Vimentin – CDX-2 + Villin +

Metastatic adenocarcinoma (signed-ring features) of unknown origin involving the

endometrium and the ovaries

CancerTYPE gene expression signature for molecular cancer classification

• Microarray development and validation on 92 selected genes by RT-PCR • Database containing 2206 tumours of 30 main types and 54 subtypes • Training in 466 frozen, 112 FFPE samples of primary and mx • Validation in 481 frozen, 119 FFPE samples of primary and mx

Ma, XJ et al. Arch Pathol Lab Med 2006

CancerTYPE gene expression signature for molecular cancer classification

Blinded comparation of CancerTYPE with IHC analysis in the diagnosis of primary site

Weiss, LM et al. J Mol Diag 2013

IHC sensitivity: 61% IHC specificity: 99% CancerTYPE sensitivity: 72% CancerTYPE specificity: 99%

• 62 year old woman

• Breast Cancer, 8 years ago (phenotype not available, probably ER+)

• Peritoneal Carcinomatosis

CK7

Mamoglobin, GCFDP-15

ER, PR

High grade carcinoma, CK7 focally +, ER/PR-, HER2-, Mammoglobin-, GCFDP-

15-, WT-, Inhibin-, CA125-

Peritoneal metastasis of breast cancer, TN phenotype

“For some adenocarcinomas, origins are especially difficult to establish because their morphology, IHC and molecular signatures are suggestive but not specific, and diagnostic dilemmas about the primary site are often pairwise, including pancreatic, colonic and gastrooesophageal cancer and their separation, and ovary and lung cancer.”

Erlander MG et al. J Mol Diag 2011 Kerr, SE et al. Clin Cancer Res 2012

Performance of CancerTYPE by tumor: limitations in certains types

Two questions to be addressed: 1. What difference might these molecular tests make to diagnosis and management? 2. What is the impact of molecular tests on the patient outcome?

Molecular profiling for CUP: clinical impact

Difference of molecular tests for CUP in diagnosis and management

Nystrom, SJ et al. Oncotarget 2012

Impact of molecular tests on the patient outcome: Treatment outcomes in patients with CUP and colorectal cancer molecular profile

CUP patients whose molecular profiles suggested colorectal tumour, and who then received colorectal-specific therapy, had survival times longer than historical CUP controls but similar to patients with known metastatic colorectal cancer

Hainsworth, JD et al. Clin Colorectal Cancer 2011

N=42, retrospective

Impact of molecular tests on the patient outcome: Prospective treatment outcomes in patients with CUP and specific molecular profile

Hainsworth, JD et al. J Clin Oncol 2013

Hainsworth, JD et al. J Clin Oncol 2013

Direct site-specific therapy in CUP patients, yielding a median overall survival (12.2 mo) better than survival for historical controls receiving empirical CUP therapy

Impact of molecular tests on the patient outcome: Prospective treatment outcomes in patients with CUP and specific molecular profile

Strategy #2.

Actionable genomic alteration rather than site of origin for personalized therapy

• 61 year old man

• Disseminated bilateral lung nodules cancer

• EBUS cytology: Positive for malignancy, consistent with adenocarcinoma, CK7, CK20 and TTF-1 negatives, EGFR WT, ALK WT

Targetable single gene analysis in CUP

Targetable single gene analysis in CUP: HER2 mutation

T C G T C A

HER2 V842I

Pleural metastasis from probably GI adenocarcinoma with HER2 mutation

Recruited in phase 2 clinical trial of Neratinib in

Patients With Solid Tumors With Somatic Human Epidermal Growth Factor Receptor (EGFR, HER2,

HER3) Mutations or EGFR Gene Amplification

Targeted next generation sequencing of adenocarcinoma of unknown primary site reveals frequent actionable genomic abnormalities and new routes to targeted therapies. J Ross, K Wang, GO Otto, PG Palmer, R Yelensky, D Lipson, J Chmielecki, SM Ali, D Morosini, VA Miller, PJ Stephens USCAP 2014, San Diego, abstract # 2163

236 cancer-related genes (3,769 exons) and 47 introns of 19 genes commonly rearranged. N=127 cancers of unknown origin: liver (24%) lymph nodes (23%), peritoneum (16%), pleura (6%), bone (5%), brain (4%) Next generation sequencing for Actionable Mutations

Genomic Alteration Categories

Highly Actionable

Actionable in Principle

Prognostic

Biologically Significant

Category A: Approved / standard alterations

that predict sensitivity or resistance to

approved / standard therapies

Category B: Alterations that are inclusion or

exclusion criteria for specific experimental

therapies

Category C: Alterations with limited evidence

that predict sensitivity or resistance to

standard or experimental therapies

Category D: Alterations with prognostic or

diagnostic utility

Category E: Alterations with clear biological

significance in cancer (i.e. driver mutations)

without clear clinical implications

• 484 alterations (3.8 per tumor) • 115 cases (91%) showed at least one actionable mutation • The most common actionable mutations were: KRAS (52%), EGFR (21%), STK11 (11%),

PIK3CA (7%), EGFR (7%), NF1 (6%), BRAF (4%) and others • 69% of tumors has an actionable mutation in RTK/RAS pathway.

0%

10%

20%

30%

40%

50%

60%

KR

AS

TP5

3

EGFR

STK

11

LRP

1B

PIK

3C

A

CTN

NB

1

NF1

MD

M2

JAK

2

DN

MT3

A

CD

KN

2A

ATM

TSC

1

CC

NE1

BR

AF

SMA

RC

A4

SMA

D4

RU

NX

1

RB

1

PTP

RD

NO

TCH

1

MYC

MSH

6

MA

P2

K1

MLH

1

MC

L1

GN

AS

FGFR

2

CD

KN

2B

CD

K4

BR

CA

1

AP

C

Pe

rce

nta

ge o

f C

ase

s w

ith

Mu

tati

on

Tumor Type

Genes with Actionable Alterations Genes with Alterations, Actionability Unknown

• Cancer of unknown primary site is common (3%) in cancer diagnoses • Historic treatment of CUP has generally been with empiric

chemotherapy with poor outcome • Diagnostic technology has improved, particularly IHC and molecular

tumor profiling, enabling a tissue-of-origin diagnosis • Conventional pathology and IHC solves 85% of cases • Combination of pathology, IHC and gene expression assays solves

more than 95% of cases • Gene expression tests should be interpreted in the appropriate

pathological context • Patients with CUP who received site-specific treatment directed by

molecular assay seem to improve survival • Next generation sequencing offers identification of actionable

genomic alterations regardless the site of origin

Conclusions