Importancia del patólogo en los tumores de origen desconocido Dr. Federico Rojo Fundación Jiménez Díaz, Madrid IMIM-Hospital del Mar, Barcelona
Cancer unknown primary 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.
Morphology of carcinomas of unknown primary site Adenocarcinoma (50%) Poorly Differentiated Carcinomas (35%) Squamous Cell Carcinoma (5%) Neuroendocrine Carcinoma (5%) Undifferentiated Carcinoma (5%) 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
Clinical landscape of cancer unknown primary over decades Greco, FA. JNCI 2013
IHC for CUP: performance and practice Cytokeratins Organo-specific markers (PSA, Thyroglobuline, GCDFP-15, Hepatocite) Non-organ-specific markers (CEA, p63, ER/PR, HMB45, Melan-A)
Screening IHC markers for the diagnosis of CUP Oien, KA & Dennis, JL. Ann Oncol 2012
CK profile of frequently occurring primary cancers (90%) (90%) Mesothelioma (70-90%) (70-90%) Squamous cell cancer Neuroendocrine cancer Massard, C. et al. Nat. Rev. Clin. Oncol. 2011
Cancer of unknown origin Breast cancer markers 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) Mamoglobin Mamaglobin (sensitivity 46,6%, combined with GCDFP-15, sensitivity 69%) ER, PR GCDFP-15
Cancer of unknown origin GI cancer markers Villin (sensitive for colon ca; 5% lung adenoca) CDX-2 (sensitive for colon ca; occasionally positive in mucinous ovarian or bladder adenoca) Villin CK7 / CK20 CDX-2
TTF-1 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
Molecular profiling for CUP One gene Multiple genes
Strategy #1. Molecular alterations of tumor sites
Targetable single gene analysis in CUP 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: EGFR mutation Control L858R
Targetable single gene analysis in CUP: EGFR mutation Pleural metastasis from pulmonary adenocarcinoma with EGFR mutation Treatment with EGFR TKI
Commercial Tests for Cancer of Unknown Origin Quest-Lab Corp CUPPrint Pathworks Gene Search Epitype CancerTYPE mirview mets Provider Arcturus Bioscience Agendia Pathworks diagnostics Veridex biotheranostics Rosetta Genomics Number of genes Cancer types 92 495 >1500 10 23 CpG 92 64 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 Sensitivity (%) FFPE FFPE FFPE FFPE 85 83 84 75 NA 87 92
MicroRNA based test to identify primary origin of metastasis 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 pancreaticobiliary 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%
Prospective mirna signature study to identify tissue of origin with CUP Varadhachary, GR et al. Clin Cancer Res 2011
Pathwork tissue of origin test 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
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.
Performance of CancerTYPE by tumor: limitations in certains types Erlander MG et al. J Mol Diag 2011 Kerr, SE et al. Clin Cancer Res 2012
Molecular profiling for CUP: clinical impact 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?
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 N=42, retrospective 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
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
Impact of molecular tests on the patient outcome: Prospective treatment outcomes in patients with CUP and specific molecular profile 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 Hainsworth, JD et al. J Clin Oncol 2013
Strategy #2. Actionable genomic alteration rather than site of origin for personalized therapy
Targetable single gene analysis in CUP 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: 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 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 Highly Actionable Actionable in Principle Prognostic Biologically Significant
KRAS TP53 EGFR STK11 LRP1B PIK3CA CTNNB1 NF1 MDM2 JAK2 DNMT3A CDKN2A ATM TSC1 CCNE1 BRAF SMARCA4 SMAD4 RUNX1 RB1 PTPRD NOTCH1 MYC MSH6 MAP2K1 MLH1 MCL1 GNAS FGFR2 CDKN2B CDK4 BRCA1 APC Percentage of Cases with Mutation 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. 60% Genes with Actionable Alterations Genes with Alterations, Actionability Unknown 50% 40% 30% 20% 10% 0%
Conclusions 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