Cancer Unknown Primary Sarah W. Gordon, DO Assistant Professor Virginia Commonwealth University

What is cancer of unknown primary? Clinical syndrome representing many different types of cancer Heterogeneous group: many primary sites with varying biology Goal remains: improved diagnosis so that site-specific treatments can be applied

Frequency Pavlidis, N, et al. Eur J Cancer. 2003.

Pathologic Evaluation Histology examination remains gold standard for initial evaluation Five major light microscopy histologic diagnoses: Poorly differentiated neoplasm Poorly differentiated carcinoma (± feature of adenoca) Well/moderately well-differentiated adenoca Squamous cell carcinoma Neuroendocrine carcinoma

Other histologies? Sarcoma Melanoma Treat as per established guidelines

Poorly differentiated neoplasm 35-65% found to be lymphoma Primarily Non-Hodgkin’s Remaining tend to be carcinomas Includes poorly differentiated neuroendocrine Melanoma & sarcoma < 15%

Poorly differentiated neoplasm To determine differential diagnosis: IHC, electron microscopy, genetic analysis Most common cause of nonspecific light microscopy diagnosis is inadequate or poorly handled biopsy specimen Avoid FNA: histologic pattern is not preserved and limited ability to perform special studies

Poorly differentiated carcinoma ~ 30% of carcinomas of unknown primary ~ 1/3 have features of poorly differentiated adenocarcinomas Need IHC staining Electron microscopy can be useful to identify by subcellular features Lymphoma Occasionally: sarcoma, melanoma, mesothelioma, neuroendocrine tumors

Poorly differentiated carcinoma Consider histologically atypical germ cell tumors Cytogenetics: abnormalities of chromosome 12 may be diagnostic of germ cell tumors At autopsy, primary site only found in 40% of patients

Adenocarcinoma Most common tumors identified by light microscopy ~ 60% of CUP diagnoses in US Typically elderly patients with multiple sites of metastatic tumors Certain histologic features associated with particular tumor type, but still not specific Papillary features in ovarian ca Signet cells with gastric cancer

Adenocarcinoma Autopsy series of patients with adenocarcinoma of unknown primary site (884 patients, 1944 – 2000), most common sites: Lung Pancreas Hepatobiliary tree Kidney 2/3 of cases Pentheroudakis G, et al. Eur J Cancer. 2007.

Squamous Carcinoma ~ 5% of patients with CUP Diagnosis usually made by histology Can consider IHC or molecular studies if poorly differentiated squamous carcinoma, especially if clinical presentation atypical

Squamous Carcinoma Upper & midcervical lymphadenopathy: H&N primary Consider ENT referral, DL, possible tonsillectomy Inguinal lymphadenopathy: often detectable primary site in genital or anorectal area Adenopathy other sites (including lower cervical + supraclav): usually primary lung cancer

Neuroendocrine Carcinoma ~ 3% of call CUP Three histologic subgroups: Well-differentiated/low-grade neuroendocrine: histology similar to carcinoids/islet cell tumors, frequently secrete bioactive substances Small-cell carcinoma/atypical carcinoid/poorly differentiated neuroendocrine carcinoma: typical neuroendocrine features and aggressive histology Poorly differentiated neoplasm/poorly differentiated carcinoma

Extragonadal Germ Cell Tumors Poorly differentiated carcinoma or adenocarcinoma of unknown primary site with some, but not all characteristics: Young age Male gender Predominant tumor location in mediastinum or retroperitoneum Elevated of hCG or AFP Presence of 12p chromosomal gain (isochromosome 12p) IHC staining for octamer binding transcription factor 4 (aka POU domain class 5 transcription factor 1) Excellent response to chemotherapy & some are cured with cisplatin-based regimens Treat like poor prognosis germ cell tumors

Immunohistochemistry Conner, JR, et al. Adv Anat Pathol. 2015.

Immunohistochemistry Oien, K. Semin Oncol. 2009.

Immunohistochemistry Oien, K. Semin Oncol. 2009.

Immunohistochemistry Oien, K. Semin Oncol. 2009.

Immunohistochemistry Fizazi, K, etc al. Ann Oncol. 2015.

Figure 1. Basic immunohistochemical work-up of carcinomas of unknown primary. Reproduced with permission: Varadhachary GR. Carcinoma of unknown primary origin, Gastrointest Cancer Res 2007; 1: 229–235. From: Cancers of unknown primary site: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up Ann Oncol. 2011;22(suppl_6):vi64-vi68. doi:10.1093/annonc/mdr389 Ann Oncol | © The Author 2011. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com 21

Immunohistochemistry Problems: Technical expertise of performing tests accurately and reproducibly Proper interpretation by experienced pathologist False-positives, false-negatives Classic staining patterns often overlap among primary sites

Electron Microscopy Reliable in undifferentiated sarcoma Ultrastructural features: Neurosecretory granules: neuroendocrine tumors Premelanosomes: melanoma Absence of cannot be used to rule out specific diagnosis Should not be used for adenoca of unknown primary site

Karyotypic or Cytogenetic Analysis Specific chromosomal abnormalities are well characterized in several hematopoietic neoplasms Limit to patients with histologic diagnosis of poorly differentiated neoplasm or poorly differentiated carcinoma No aid in evaluation of adenocarcinomas

Gene Expression Profiling Cancer cells retain some normal cell-type specific functional characteristics in their gene expression profile Usually origin can be predicted regardless of neoplastic differentiation Molecular profiling assays designed to determine type of cancer do not look at tumor-specific markers, but rather gene expression dynamics in relation to cell lineage

DNA array technology. DNA array technology. Tumor cDNA and normal cDNA are labeled with different fluorescent dyes. The labeled cDNAs are hybridized to DNA arrays containing oligonucleotides representing known genes. The fluorescence intensity is analyzed using statistical algorithms, and color intensities are assigned to the ratios of gene expression. David M. Mintzer et al. The Oncologist 2004;9:330-338 ©2004 by AlphaMed Press

Histologic Dx: poorly differentiated carcinoma with pleomorphic features. Comment: Immunohistochemical staining performed on the needle core biopsy (1A) shows that the malignant cells are positive for CK7, GATA3, Uroplakin 2/3 (patchy), CDX2, p63 (focal, LungSquamous 2 multiplex), CD56 (patchy), while negative for CK20, TTF1, synaptophysin, CK5 (LungSquamous 2 Multiplex), and OCT 3/4, . Control sections stain appropriately. The malignant cells are poorly differentiated and have ambiguous features. The immunophenotype, with positivity of CK7, GATA3, Uroplakin 2/3 (patchy) and p63 (focal), suggests a urothelial origin. CDX2 is also positive, which can be seen in a subset of poorly differentiated urothelial cell carcinoma. However, a lung primary squamous cell carcinoma cannot be definitively excluded. Clinical and radiologic correlation is necessary.

Fizazi, K, etc al. Ann Oncol. 2015.

Focused Diagnostic Eval Intial Evaluation Additional Evaluation Women with features of breast cancer (bone, lung, liver mets; CK7+) Breast MRI ER, GCDFP-15, HER2 stains Women with features of ovarian cancer (pelvic/peritoneal mets; CK7+) Pelvic/vaginal US WT-1 stain Mediastinal/retroperitoneal mass Testicular US Serum HCG, AFP PLAP, OCT4 stains; FISH for i(12p) Features of lung cancer (hilar/mediastinal adenopathy; TTF-1 +) Bronchoscopy Features of colon cancer (liver/peritoneal metastases; CK20+/CK7–, CDX2+) Colonoscopy Poorly differentiated carcinoma, with or without clear cell features Stains for chromogranin, synaptophysin, RCC, Hepar-1, HMB-45 (If Hepar-1+, obtain serum AFP; if neuroendocrine stains +, obtain octreotide scan) Adapted from DaVita, et al. Cancer. 2011.

Clinical Management Algorithm Fizazi, K, etc al. Ann Oncol. 2015.

Fizazi, K, etc al. Ann Oncol. 2015.

Study Site Contact: Alison Ryan (SIIT Team) SWOG – 1609 Local PI: Sarah Gordon Study Site Contact: Alison Ryan (SIIT Team)

NCI-Molecular Analysis for Therapy Choice (NCI-MATCH / EAY131) A phase II precision medicine cancer trial Co-developed by the ECOG-ACRIN Cancer Research Group and the National Cancer Institute Version Date: 11/27/2016

What is NCI-MATCH? Now 6000 **Screening about 500 pts/month! 11/27/2016

NCI-MATCH Tumor Gene Testing aMOI: actionable mutation of interest 11/27/2016

NCI-MATCH Testing Expectations One in every four to five (23%) 11/27/2016

NCI-MATCH Objective To determine whether matching certain drugs or drug combinations in adults whose tumors have specific gene abnormalities will effectively treat their cancer, regardless of the cancer type This is a signal-finding trial—treatments that show promise can advance to larger, more definitive trials 11/27/2016

NCI-MATCH Updates! NCI will no longer be doing screening No more on-trial biopsies – NCI site reimbursement will end Deadline to consent patients has passed 6 new arms to open (June 2017) May be more treatment arms to come… If patients go on treatment, biopsy at time of progression is strongly encouraged. Funding for this expected to continue

NCI-MATCH Updates! Outside assays will now be accepted for screening from designated labs Foundation Medicine (live now) CARIS MDACC institutional assay MSKCC institutional assay Any samples sent to these labs from a CIRB approved trial site will be screened for eligibility to some arms of MATCH Start with rare gene variants (< 1.5%) 19 arms Delay before opening other arms (2-3 months)

Patients do NOT need fresh biopsy! Archived tissue allowed Other Changes… Patients do NOT need fresh biopsy! Archived tissue allowed Treatment after biopsy is allowed No more off treatment waiting period between biopsy & genetic results

Other Changes… If patient has a MATCH by the outside lab, tissue will be requested to be sent to NCI-MATCH for verification Study team will need to coordinate sending archived specimens If MATCH  patient gets treated If no confirmation by NCI-MATCH of outside assay results, then patient will be assessed for toxicity only, not response New version of genetic assay being introduced Increasing # of gene fusions from 271 to 762 Also adding new genes

Cases?