Cancers of unknown primary site: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up

definition, incidence and biologyCancers of unknown primary site (CUPs) represent a heterogeneous group of metastatic tumours for which a standardised diagnostic work-up fails to identify the site of origin at the time of diagnosis. CUPs account for 3%–5% of all malignancies. The unique biology of these tumours remains almost unknown [1.Massard C. Loriot Y. Fizazi K. Carcinomas of an unknown primary origin—diagnosis and treatment.Nat Rev Clin Oncol. 2011; 8: 701-710Crossref PubMed Scopus (88) Google Scholar]. Nonetheless, current data suggest that metastatic dissemination can occur in the absence of growth of a primary tumour by virtue of inherent metastatic aggressiveness of cancer cells. Chromosomal instability was recently suggested to account for part of the uncommon clinical presentation, chemoresistance and poor outcome in patients with CUP [2.Vikeså J. Møller A.K. Kaczkowski B. et al.Cancers of unknown primary origin (CUP) are characterized by chromosomal instability (CIN) compared to metastasis of know origin.BMC Cancer. 2015; 15: 151Crossref PubMed Scopus (33) Google Scholar].diagnosis, pathology and molecular biologyDiagnosis of CUP requires pathology evaluation of a good quality tissue sample. These tumours are categorised by pathology into:•well- and moderately differentiated adenocarcinomas;•squamous cell carcinomas;•carcinomas with neuroendocrine differentiation;•poorly differentiated carcinomas (including poorly differentiated adenocarcinomas);•undifferentiated neoplasms.Immunohistochemistry should be applied meticulously [3.Oien K.A. Pathologic evaluation of unknown primary cancer.Semin Oncol. 2009; 36: 8-37Crossref PubMed Scopus (146) Google Scholar, 4.Abbruzzese J.L. Abbruzzese M.C. Lenzi R. et al.Analysis of a diagnostic strategy for patients with suspected tumors of unknown origin.J Clin Oncol. 1995; 13: 2094-2103Crossref PubMed Scopus (281) Google Scholar] in order to identify the tissue of origin and to exclude chemosensitive and potentially curable tumours (i.e. lymphomas and germ-cell tumours) (Table 1) [III, A]. If the diagnosis is carcinoma or adenocarcinoma, immunostaining for prostate-specific antigen (PSA) in male patients and for oestrogen and progesterone receptors in females with axillary node metastases is advisable to rule out hormone-sensitive tumours amenable to specific therapy. Staining for keratins CK7 and CK20 may provide indications of a possible primary site, and staining for chromogranin A and synaptophysin is needed to profile neuroendocrine differentiation (Figure 1). Examples of stainings that are rather specific include CK7+, WT-1+, PAX8+, CK20- (ovarian cancer) and RCC+, PAX8+ (renal cancer).Table 1Immunohistochemical work-up in patients with cancers of unknown primary site (CUPs)CytokeratinsPSAER, PgRCDX2+, CK20+, CK7–TTF1, NapsinA, CK7+Thyroglobulin, calcitoninNSE, chromogranin, synaptophysinAFP, OCT4, hCG, PLAPLCAS100, HMB45Vimentin, desminUndifferentiated carcinoma+-±-------±Prostate cancer++---------Breast cancer+-±-------±Colorectal cancer+--+-------Lung adenocarcinoma+---+------Thyroid cancer+---±+±----Neuroendocrine+---±±+----Germ-cell cancer+------+--±Lymphoma--------+--Melanoma---------+±Sarcoma---------±+The table shows general staining patterns but exceptions exist, especially for S100 and vimentinThyroid and neuroendocrine cancers often positive with CK7 and TTF1 but not with NapsinA.PSA, prostate specific antigen; ER, oestrogen receptor; PgR, progesterone receptor; CK, cytokeratin; TTF1, thyroid transrcription factor 1; NSE, neuron-specific enolase; AFP, alpha fetoprotein; hCG, human chorionic gonadotropin; PLAP, placental alkaline phosphatase; LCA, leukocyte common antigen. Open table in a new tab personalised medicineSeveral gene expression profiling assays have become commercially available, claiming to blindly and correctly identify a known primary cancer and a likely tissue of origin in patients with CUP in ∼80% [6.Greco F.A. Lennington W.J. Spigel D.R. Hainsworth J.D. Molecular profiling diagnosis in unknown primary cancer: accuracy and ability to complement standard pathology.J Natl Cancer Inst. 2013; 105: 782-790Crossref PubMed Scopus (73) Google Scholar, 7.Hainsworth J.D. Rubin M.S. Spigel D.R. et al.Molecular gene expression profiling to predict the tissue of origin and direct site-specific therapy in patients with carcinoma of unknown primary site: a prospective trial of the Sarah Cannon Research institute.J Clin Oncol. 2013; 31: 217-223Crossref PubMed Scopus (231) Google Scholar] [III]. These assays are based on mRNA or miRNA RT-PCR or oligonucleotide microarray technologies [8.Pillai R. Deeter R. Rigl C.T. et al.Validation and reproducibility of a microarray-based gene expression test for tumor identification in formalin-fixed, paraffin-embedded specimens.J Mol Diagn. 2011; 13: 48-56Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 9.Kerr S.E. Schnabel C.A. Sullivan P.S. et al.Multisite validation study to determine performance characteristics of a 92-gene molecular cancer classifier.Clin Cancer Res. 2012; 18: 3952-3960Crossref PubMed Scopus (59) Google Scholar, 10.Meiri E. Mueller W.C. Rosenwald S. et al.A second-generation microRNA-based assay for diagnosing tumor tissue origin.Oncologist. 2012; 17: 801-812Crossref PubMed Scopus (117) Google Scholar]. No significant differences in the tumour microRNA expression profile were evident when CUP metastases biologically assigned to a primary tissue of origin were compared with metastases from typical solid tumours of known origin [11.Pentheroudakis G. Spector Y. Krikelis D. et al.Global microRNA profiling in favorable prognosis subgroups of cancer of unknown primary (CUP) demonstrates no significant expression differences with metastases of matched known primary tumors.Clin Exp Metastasis. 2013; 30: 431-439Crossref PubMed Scopus (17) Google Scholar]. These tests may aid in the diagnosis of the putative primary tumour site in some patients [12.Gross-Goupil M. Massard C. Lesimple T. et al.Identifying the primary site using gene expression profiling in patients with carcinoma of an unknown primary (CUP): a feasibility study from the GEFCAPI.Onkologie. 2012; 35: 54-55Crossref PubMed Scopus (18) Google Scholar]. However, their impact on patient outcome via administration of primary site-specific therapy remains questionable and unproven in randomised trials [13.Hainsworth J.D. Greco F.A. Gene expression profiling in patients with carcinoma of unknown primary site: from translational research to standard of care.Virchows Arch. 2014; 464: 393-402Crossref PubMed Scopus (55) Google Scholar] [IV, C]. A large prospective non-randomised phase II study of 252 patients suggested that survival may be improved by site-specific therapy determined by a gene expression profile assay of the biopsy specimen, particularly for patients with a tissue of origin diagnosis of more responsive tumour types [7.Hainsworth J.D. Rubin M.S. Spigel D.R. et al.Molecular gene expression profiling to predict the tissue of origin and direct site-specific therapy in patients with carcinoma of unknown primary site: a prospective trial of the Sarah Cannon Research institute.J Clin Oncol. 2013; 31: 217-223Crossref PubMed Scopus (231) Google Scholar]. A prospective randomised phase III trial testing such a precision medicine strategy versus empirical chemotherapy is currently on-going in Europe (NCT01540058).staging and risk assessmentCUPs are by definition metastatic cancers, and the prognosis for patients with CUP is generally poor. However, an appropriate diagnostic work-up can help to identify a minority of CUP patients who can expect to benefit from directed therapy. The following recommendations epitomise the standard and optional assessments suggested. Diagnostic and staging guidelines for patients with an anticipatory CUP diagnosis are summarised in Table 2.Table 2Diagnostic and staging guidelines for cancers of unknown primary site (CUPs)Assessment suggestedTarget patient populationThorough medical history and physical examinationAll patientsBasic blood and biochemistry analysesAll patientsCT scans of thorax, abdomen and pelvisAll patientsMammographyFemale patientsWork-up for CUP subsets Breast MRIFemales with axillary adenocarcinoma Serum α-fetoprotein and human chorionic gonadotropinPatients with midline metastatic disease Serum prostate-specific antigenMales with adenocarcinomatous bone metastases Head and neck CT/PET scan (optional)Cervical squamous cell carcinoma EndoscopiesSign/symptom/laboratory-oriented Octreoscan and plasma chromogranin APatients with neuroendocrine tumour CUP Additional diagnostic pathologySign/symptom/laboratory-orientedCT, computed tomography; MRI, magnetic resonance imaging; PET, positron emission tomography. Open table in a new tab Thorough physical examination (including head and neck, rectal, pelvic and breast examination), basic blood and biochemical analyses, and computed tomography (CT) scans of thorax, abdomen and pelvis constitute a minimal basic work-up [IV, B].Endoscopies should be sign-, symptom- or laboratory abnormality-guided. Serum assessment of α-fetoprotein, human chorionic gonadotropin, plasma chromogranin A and PSA is suggested in male patients to exclude potentially curable extragonadal germ-cell tumours, neuroendocrine tumours and prostate cancers amenable to hormonal treatment.Whole-body 2-deoxy-2-[18F]fluoro-d-glucose-positron emission tomography (FDG–PET)/CT may contribute to the management of patients with cervical adenopathies from CUP and those with a single CUP metastasis [IV, B]. For other CUPs, the role of FDG–PET is limited [14.Moller A.K. Loft A. Berthelsen A.K. et al.A prospective comparison of 18F-FDG PET/CT and CT as diagnostic tools to identify the primary tumor site in patients with extracervical carcinoma of unknown primary site.Oncologist. 2012; 17: 1146-1154Crossref PubMed Scopus (30) Google Scholar, 15.Seve P. Billotey C. Broussolle C. et al.The role of 2-deoxy-2-[F-18]fluoro-d-glucose positron emission tomography in disseminated carcinoma of unknown primary site.Cancer. 2007; 109: 292-299Crossref PubMed Scopus (120) Google Scholar], making this imaging procedure not mandatory in the systematic work-up [III, C].For patients with predominant midline lymph node involvement, the diagnosis of lymphoma or extragonadal germ-cell tumours should be ruled out.Distinct subsets of patients with CUP have been defined based on clinical and pathological criteria [2.Vikeså J. Møller A.K. Kaczkowski B. et al.Cancers of unknown primary origin (CUP) are characterized by chromosomal instability (CIN) compared to metastasis of know origin.BMC Cancer. 2015; 15: 151Crossref PubMed Scopus (33) Google Scholar] (Table 3). An additional subset of CUP with a colorectal IHC or molecular profile also seems to have a better prognosis, likely thanks to more active systemic treatments developed over the last two decades for colon cancer [16.Hainsworth J.D. Schnabel C.A. Erlander M.G. et al.A retrospective study of treatment outcomes in patients with carcinoma of unknown primary site and a colorectal cancer molecular profile.Clin Colorectal Cancer. 2012; 11: 112-118Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar]. A minority of patients (15%–20%) belong to clinico-pathological subsets with a more favourable prognosis. These favourable-risk CUP patients harbour chemosensitive and potentially curable tumours and may experience long-term disease control with appropriate multidisciplinary management.Table 3Therapy for patients with favourable-risk cancers of unknown primary site (CUPs)CUP subtypeProposed treatmentPotential equivalent tumourPoorly differentiated neuroendocrine carcinomas of an unknown primaryPlatinum + etoposide combination chemotherapyPoorly differentiated neuroendocrine carcinomas with a known primaryWell-differentiated neuroendocrine tumour of unknown primarySomatostatin analogues, streptozocin+5-FU, sunitinib, everolimusWell-differentiated neuroendocrine tumour of a known primary sitePeritoneal adenocarcinomatosis of a serous papillary histological type in femalesOptimal surgical debulking followed by platinum–taxane-based chemotherapyOvarian cancerIsolated axillary nodal metastases in femalesAxillary nodal dissection, mastectomy or breast irradiation and adjuvant chemohormonotherapyBreast cancer (found in 50%–70% when breast MRI is performed)Squamous cell carcinoma involving non-supraclavicular cervical lymph nodesNeck dissection and/or irradiation of bilateral neck and head–neck axis. For advanced stages induction chemotherapy with platinum-based combination or chemoradiationHead and neck squamous cell cancerCUP with a colorectal IHC (CK20+ CDX2+ CK7-) or molecular profileSystemic treatment used for colorectal cancerMetastatic colorectal cancerSingle metastatic deposit from unknown primaryResection and/or RT ± systemic therapySingle metastasisMen with blastic bone metastases or IHC/serum PSA expressionAndrogen deprivation therapy ± RTProstate cancer5-FU, 5-fluorouracil; MRI, magnetic resonance imaging; IHC, immunohistochemistry; PSA, prostate-specific antigen; RT, radiotherapy; CK, cytokeratin. Open table in a new tab The majority of patients (80%–85%) do not belong to specific subsets. Sensitivity to therapy is only modest and median overall survival is generally <1 year (6–10 months). Two prognostic groups can be identified among patients with CUP:•those with a good performance status (0–1) and a normal lactate dehydrogenase (LDH) value, with a median life expectancy of 1 year, and•those with either one or both of these prognostic factors (poor performance status and elevated serum LDH), with a median overall survival of only ∼4 months [17.Culine S. Kramar A. Saghatchian M. et al.Development and validation of a prognostic model to predict the length of survival in patients with carcinomas of an unknown primary site.J Clin Oncol. 2002; 20: 4679-4683Crossref PubMed Scopus (161) Google Scholar].A proposal for the practical management of patients with CUP, including recognition of specific subsets, exclusion of non-CUP neoplasms and the use of prognostic parameters in clinical practice, is summarised in Figure 2.Figure 2Clinical management of patients presenting with CUPs. IHC, immunohistochemistry; PS, performance status; LDH, lactate dehydrogenase; OS, overall survival.View Large Image Figure ViewerDownload (PPT)treatmentTherapy should be individually tailored according to the clinico-pathological subset with a distinct prognosis to which the patient belongs [III, B]. Referral to specialised centres is strongly encouraged. The 10%–15% of CUP patients in the favourable-risk subsets should be treated similarly to patients with equivalent known primary tumours with metastatic dissemination [IV, B]. These patients achieve long-term disease control in 30%–60% of cases, and optimal management is pivotal for long-term survival (Table 3). Retrospective analyses confirm that the clinical behaviour, biology, response to treatment and outcome of patients with favourable-risk CUP are not different from similar factors relative to metastatic tumours from a known primary site [18.Spigel D.R. Hainsworth J.D. Greco F.A. Neuroendocrine carcinoma of unknown primary site.Semin Oncol. 2009; 36: 52-59Crossref PubMed Scopus (71) Google Scholar, 19.Pentheroudakis G. Pavlidis N. Serous papillary peritoneal carcinoma: unknown primary tumour, ovarian cancer counterpart or a distinct entity? A systematic review.Crit Rev Oncol Hematol. 2010; 75: 27-42Crossref PubMed Scopus (63) Google Scholar, 20.Pentheroudakis G. Lazaridis G. Pavlidis N. Axillary nodal metastases from carcinoma of unknown primary (CUPAx): a systematic review of published evidence.Breast Cancer Res Treat. 2010; 119: 1-11Crossref PubMed Scopus (67) Google Scholar, 21.Pavlidis N. Pentheroudakis G. Plataniotis G. Cervical lymph node metastases of squamous cell carcinoma from an unknown primary site: a favourable prognosis subset of patients with CUP.Clin Transl Oncol. 2009; 11: 340-348Crossref PubMed Scopus (47) Google Scholar, 22.Hainsworth J.D. Fizazi K. Treatment for patients with unknown primary cancer and favorable prognostic factors.Semin Oncol. 2009; 36: 44-51Crossref PubMed Scopus (58) Google Scholar].Patients with poor-risk CUP have a dismal prognosis despite management with a variety of chemotherapeutic combinations in small clinical studies [23.Bugat R. Bataillard A. Lesimple T. et al.Summary of the standards, options and recommendations for the management of patients with carcinoma of unknown primary site (2002).Br J Cancer. 2003; 89: S59-S66Crossref PubMed Scopus (108) Google Scholar]. A review conducted in the 2000s showed no evidence of superior efficacy of any of the administered regimens comprising platinum salts, taxanes or new-generation cytotoxic compounds (gemcitabine, vinca alkaloids or irinotecan) [24.Golfinopoulos V. Pentheroudakis G. Salanti G. et al.Comparative survival with diverse chemotherapy regimens for cancer of unknown primary site: multiple-treatments meta-analysis.Cancer Treat Rev. 2009; 35: 570-573Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar]. A randomised prospective phase III study of 198 patients compared gemcitabine/irinotecan with paclitaxel/carboplatin/oral etoposide in fit poor-risk patients and reported significantly less toxicity with the two-drug regimen and equal survival rates [II, A] [25.Hainsworth J.D. Spigel D.R. Clark B.L. et al.Paclitaxel/carboplatin/etopside versus gemcitabine/irinotecan in the first-line treatment of patients with carcinoma of unknown primary site: a randomised phase III Sarah Cannon Research Consortium Trial.Cancer J. 2010; 16: 70-75Crossref PubMed Scopus (47) Google Scholar]. On the other hand, the efficacy/toxicity ratio of the cisplatin–gemcitabine combination was found to be better than that of the cisplatin–irinotecan regimen in a randomised phase II trial [I, A] [26.Culine S. Lortholary A. Voigt J.J. et al.Cisplatin in combination with either gemcitabine or irinotecan in carcinomas of unknown primary site: results of a randomised phase II study—Trial for the French Study Group on Carcinomas of Unknown Primary (GEFCAPI 01).J Clin Oncol. 2003; 21: 3479-3482Crossref PubMed Scopus (124) Google Scholar]. Finally, better outcome was reported with the two-drug cisplatin–gemcitabine regimen when compared with cisplatin alone, although this was not assessed in a large and adequately powered randomised phase III trial [27.Gross-Goupil M. Fourcade A. Blot E. et al.Cisplatin alone or combined with gemcitabine in carcinomas of unknown primary: results of the randomized GEFCAPI 02 trial.Eur J Cancer. 2012; 48: 721-727Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar] (II). Modest survival prolongation and symptom palliation with preservation of quality of life are currently the only realistic aims of therapy for these patients [II, A], although rare cases of cure have been reported [28.Levy A. Massard C. Gross-Goupil M. Fizazi K. Carcinomas of an unknown primary site: a curable disease?.Ann Oncol. 2008; 19: 1657-1658Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar]. Consequently, low-toxicity patient-convenient chemotherapy regimens should be administered to reasonably fit poor-risk CUP patients (Table 4). If evaluation of patient demographics, metastatic pattern, results of clinical and laboratory tests, imaging data, pathological evaluations and gene expression is relatively unambiguous, a site-specific treatment may be considered, though prospective evidence that this is better than empirical chemotherapy is lacking so far.Table 4Commonly used low-toxicity palliative chemotherapy regimens for poor-risk patients with cancers of unknown primary site (CUPs)Chemotherapy (mg/m2)TimeIntervalCommentsCisplatin 60–75 +Gemcitabine 1000Day 1Day 1+8Q3 weeksFit patients, adequate hydrationCisplatin 75 +Etoposide 100Day 1Day 1–3Q3 weeksFit patients with neuroendocrine-feature CUP, adequate hydrationPaclitaxel 175 +Carboplatin AUC 5Day 1Q3 weeksConvenient outpatient regimen, monitor neurotoxicityDocetaxel 75 +Carboplatin AUC 5Day 1Q3 weeksConvenient outpatient regimen, monitor neurotoxicityIrinotecan 160 +Oxaliplatin 80Day 1Q3 weeksOutpatient regimen, monitor for neurotoxicity and diarrhoeaOral capecitabine 2000 ±Oxaliplatin 85–130Days 1–14Day 1Q3 weeksOutpatient regimen, risk for diarrhoea and neurotoxicityGemcitabine 1000/irinotecan 100Day 1+8Q3 weeksConvenient outpatient regimen, monitor diarrhoeaAUC, area under the curve. Open table in a new tab Whether targeted agents should be used or not in patients with CUPs is still unknown [29.Massard C. Voigt J.J. Laplanche A. et al.Carcinoma of an unknown primary: are EGF receptor, Her-2/neu, and c-Kit tyrosine kinases potential targets for therapy?.Br J Cancer. 2007; 97: 857-861Crossref PubMed Scopus (39) Google Scholar]. Although only a few non-chemotherapy drugs have been tested in patients with CUP, belinostat was randomly assessed and it did not improve the results of the carboplatin-paclitaxel regimen [30.Hainsworth J.D. Daugaard G. Lesimple T. et al.Paclitaxel/carboplatin with or without belinostat as empiric first-line treatment for patients with carcinoma of unknown primary site: a randomized, phase 2 trial.Cancer. 2015; 121: 1654-1661Crossref PubMed Scopus (28) Google Scholar]. Preliminary retrospective data suggest that CUP patients with immunohistochemical and/or molecular profile assay diagnoses of ‘colorectal’ carcinomas have response rates and survival after colorectal site-specific therapies (i.e. FOLFOX or FOLFIRI) that are similar to those of patients with known advanced colorectal carcinomas [16.Hainsworth J.D. Schnabel C.A. Erlander M.G. et al.A retrospective study of treatment outcomes in patients with carcinoma of unknown primary site and a colorectal cancer molecular profile.Clin Colorectal Cancer. 2012; 11: 112-118Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 31.Varadhachary G.R. Raber M.N. Matamorous A. Abbruzzese J.L. Carcinoma of unknown primary with colon-cancer profile-changing paradigm and emerging definitions.Lancet Oncol. 2008; 9: 596-599Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar] [IV, B]. These data are from small numbers of patients, and additional prospective validation is necessary to substantiate these preliminary findings.Participation in clinical trials evaluating combinations of cytotoxic compounds with targeted agents or site-specific therapy in patients with putative primary tumour sites highly suspected from immunohistochemical or microarray studies should be strongly encouraged.response evaluationResponse evaluation is recommended after two or three chemotherapy cycles by individually adequate tests. Quality-of-life issues are particularly relevant for patients with poor-risk CUP for whom excessive treatment-related toxicity is not justified [IV, B].follow-up and long-term implicationsThere is no evidence that follow-up of asymptomatic patients is needed. Specific examinations as clinically indicated.methodologyThese clinical practice guidelines were developed in accordance with the ESMO standard operating procedures for clinical practice guidelines development. The relevant literature has been selected by the expert authors. Levels of evidence and grades of recommendation have been applied using the system shown in Table 5. Statements without grading were considered justified standard clinical practice by the experts and the ESMO faculty. This manuscript has been subjected to an anonymous peer review process.Table 5Levels of evidence and grades of recommendation(adapted from the Infectious Diseases Society of America-United States Public Health Service Grading Systema)Levels of evidenceIEvidence from at least one large randomised, controlled trial of good methodological quality (low potential for bias) or meta-analyses of well-conducted randomised trials without heterogeneityIISmall randomised trials or large randomised trials with a suspicion of bias (lower methodological quality) or meta-analyses of such trials or of trials with demonstrated heterogeneityIIIProspective cohort studiesIVRetrospective cohort studies or case–control studiesVStudies without control group, case reports, experts opinionsGrades of recommendationAStrong evidence for efficacy with a substantial clinical benefit, strongly recommendedBStrong or moderate evidence for efficacy but with a limited clinical benefit, generally recommendedCInsufficient evidence for efficacy or benefit does not outweigh the risk or the disadvantages (adverse events, costs, … ), optionalDModerate evidence against efficacy or for adverse outcome, generally not recommendedEStrong evidence against efficacy or for adverse outcome, never recommendedaBy permission of the Infectious Diseases Society of America [32.Dykewicz C.A. Summary of the guidelines for preventing opportunistic infections among hematopoietic stem cell transplant recipients.Clin Infect Dis. 2001; 33: 139-144Crossref PubMed Scopus (410) Google Scholar]. Open table in a new tab definition, incidence and biologyCancers of unknown primary site (CUPs) represent a heterogeneous group of metastatic tumours for which a standardised diagnostic work-up fails to identify the site of origin at the time of diagnosis. CUPs account for 3%–5% of all malignancies. The unique biology of these tumours remains almost unknown [1.Massard C. Loriot Y. Fizazi K. Carcinomas of an unknown primary origin—diagnosis and treatment.Nat Rev Clin Oncol. 2011; 8: 701-710Crossref PubMed Scopus (88) Google Scholar]. Nonetheless, current data suggest that metastatic dissemination can occur in the absence of growth of a primary tumour by virtue of inherent metastatic aggressiveness of cancer cells. Chromosomal instability was recently suggested to account for part of the uncommon clinical presentation, chemoresistance and poor outcome in patients with CUP [2.Vikeså J. Møller A.K. Kaczkowski B. et al.Cancers of unknown primary origin (CUP) are characterized by chromosomal instability (CIN) compared to metastasis of know origin.BMC Cancer. 2015; 15: 151Crossref PubMed Scopus (33) Google Scholar]. Cancers of unknown primary site (CUPs) represent a heterogeneous group of metastatic tumours for which a standardised diagnostic work-up fails to identify the site of origin at the time of diagnosis. CUPs account for 3%–5% of all malignancies. The unique biology of these tumours remains almost unknown [1.Massard C. Loriot Y. Fizazi K. Carcinomas of an unknown primary origin—diagnosis and treatment.Nat Rev Clin Oncol. 2011; 8: 701-710Crossref PubMed Scopus (88) Google Scholar]. Nonetheless, current data suggest that metastatic dissemination can occur in the absence of growth of a primary tumour by virtue of inherent metastatic aggressiveness of cancer cells. Chromosomal instability was recently suggested to account for part of the uncommon clinical presentation, chemoresistance and poor outcome in patients with CUP [2.Vikeså J. Møller A.K. Kaczkowski B. et al.Cancers of unknown primary origin (CUP) are characterized by chromosomal instability (CIN) compared to metastasis of know origin.BMC Cancer. 2015; 15: 151Crossref PubMed Scopus (33) Google Scholar]. diagnosis, pathology and molecular biologyDiagnosis of CUP requires pathology evaluation of a good quality tissue sample. These tumours are categorised by pathology into:•well- and moderately differentiated adenocarcinomas;•squamous cell carcinomas;•carcinomas with neuroendocrine differentiation;•poorly differentiated carcinomas (including poorly differentiated adenocarcinomas);•undifferentiated neoplasms.Immunohistochemistry should be applied meticulously [3.Oien K.A. Pathologic evaluation of unknown primary cancer.Semin Oncol. 2009; 36: 8-37Crossref PubMed Scopus (146) Google Scholar, 4.Abbruzzese J.L. Abbruzzese M.C. Lenzi R. et al.Analysis of a diagnostic strategy for patients with suspected tumors of unknown origin.J Clin Oncol. 1995; 13: 2094-2103Crossref PubMed Scopus (281) Google Scholar] in order to identify the tissue of origin and to exclude chemosensitive and potentially curable tumours (i.e. lymphomas and germ-cell tumours) (Table 1) [III, A]. If the diagnosis is carcinoma or adenocarcinoma, immunostaining for prostate-specific antigen (PSA) in male patients and for oestrogen and progesterone receptors in females with axillary node metastases is advisable to rule out hormone-sensitive tumours amenable to specific therapy. Staining for keratins CK7 and CK20 may provide indications of a possible primary site, and staining for chromogranin A and synaptophysin is needed to profile neuroendocrine differentiation (Figure 1). Examples of stainings that are rather specific include CK7+, WT-1+, PAX8+, CK20- (ovarian cancer) and RCC+, PAX8+ (renal cancer).Table 1Immunohistochemical work-up in patients with cancers of unknown primary site (CUPs)CytokeratinsPSAER, PgRCDX2+, CK20+, CK7–TTF1, NapsinA, CK7+Thyroglobulin, calcitoninNSE, chromogranin, synaptophysinAFP, OCT4, hCG, PLAPLCAS100, HMB45Vimentin, desminUndifferentiated carcinoma+-±-------±Prostate cancer++---------Breast cancer+-±-------±Colorectal cancer+--+-------Lung adenocarcinoma+---+------Thyroid cancer+---±+±----Neuroendocrine+---±±+----Germ-cell cancer+------+--±Lymphoma--------+--Melanoma---------+±Sarcoma---------±+The table shows general staining patterns but exceptions exist, especially for S100 and vimentinThyroid and neuroendocrine cancers o