cancers of unknown primary origin ( cup ) pose a significant challenge for clinicians in terms of identifying the primary cancer and designing an effective treatment strategy for the patients ( 1 ) . cups exhibit an aggressive biological behavior and the prognosis of patients with cups is typically significantly poorer compared with that of patients with cancers of known origin , with a median survival of 34 months after initial diagnosis ( 2 ) . while cups only comprise ~35% of new cancer cases annually , they constitute the fourth most common cause of cancer - related mortality in western countries , highlighting the significance of this diagnostic challenge ( 3,4 ) . thus , there has been a surge in the development of advanced diagnostic tools for primary tumor identification using molecular testing . notably , microrna expression assays have demonstrated a remarkable ability to identify the origin of cups ( 5 ) . micrornas are small , non - coding rnas that regulate gene expression by rna degradation or translational inhibition ( 6 ) . aberrant micrornas play a critical role in the development and progression of cancer , making them ideal biomarkers for cancer diagnosis . multiple studies have reported high sensitivity and accuracy for microrna assays in diagnosing cases with cancers of known and unknown primary , allowing for their use as a key tool for cancer diagnosis . the case presented herein is a representative example of the possible clinical impact of microrna assays . a 54 year - old male patient was presented to his orthopedist with low back pain in june , 2013 . the patient had no significant past medical history other than mild hypertension and a history of smoking ( 35 pack - years ) . an initial magnetic resonance imaging ( mri ) examination of the lumbar spine revealed two findings : a mass in the right kidney and a mass in the t9-t10 vertebrae that was impinging on the spinal cord . initial pathology identified a metastatic low - grade carcinoma of epithelial origin and the differential diagnosis included renal carcinoma , neuroendocrine carcinoma , melanoma and testicular carcinoma . on immunohistochemistry ( ihc ) , the lesion was strongly positive for cytokeratin ae1/ae3 , cam5.2 , epithelial membrane antigen , e - cadherin , melan - a , synaptophysin and placental alkaline phosphatase . additionally , there was mild and/or focal staining for renal cell carcinoma antigen , cd10 , neuron - specific enolase , s100 and human melanoma black 45 . the immunostaining for cytokeratin ( ck)7 , ck20 , inhibin , thyroid transcription factor 1 , ck5/6 , gross cystic disease fluid protein 15 , vimentin and cd117 , was negative . however , the serum prostate - specific antigen ( psa ) levels were within the normal range and ihc for psa was negative in the tissue sample . initial evaluation with computed tomography ( ct ) of the brain , chest , abdomen and pelvis and a bone scan revealed multiple bone lesions , two enlarged mediastinal lymph nodes , a block of enlarged para - aortic lymph nodes and a mass located in the upper pole of the right kidney , sized 1113.512.5 cm . the kidney mass exhibited no significant contrast uptake ; following resection , it was found to be a benign cyst . a positron emission tomography ( pet)-ct , ordered in late september , revealed a relatively mild radioactive uptake in the subcutaneous area of the right shoulder girdle [ standardized uptake value ( suv ) = 3.1 ] , and a significantly higher uptake in several bones ( suvmax = 9.3 ) . physical examination of the patient revealed a soft , hairy , pigmented skin lesion on the right anterior axillary line . an mri scan of the right shoulder was ordered on the 18th of october , and the mass appeared as a subcutaneous lesion with homogeneous contrast uptake with an associated group of lymph nodes . as the primary cancer remained unknown , the microrna rosetta cancer origin test ( rosetta genomics ltd . , philadelphia , pa , usa ) was ordered 16 weeks after presentation . the test results were received 18 weeks after presentation and identified the tissue as breast cancer , with a sensitivity of 90% . the axillary mass was then resected ( 20 weeks after presentation ) and pathology identified that mass as a malignant neoplasm similar to the mass removed from the lumbar area . ihc was 100% positive for estrogen receptor ( er ) and progesterone receptor ( pr ) and positive for human epidermal growth factor receptor 2 ( her2 ) ( figs . the patient was treated for breast cancer with capecitabine and trastuzumab as the first - line regimen ; following relapse on left iliac and right pubic bone , the regimen was changed to vinorelbine and trastuzumab . the patient responded well and proceeded to maintenance trastuzumab and tamoxifen . on december 30 , 2014 , treatment was switched to letrozole and trastuzumab , due to an increase in serum marker levels ( carcinoembryonic antigen and carbohydrate antigen 15 - 3 ) that was followed by the appearance of a skin nodule right by the scar of the axillary mass resection . the patient is completely asymptomatic and continues on letrozole and trastuzumab with stable bone disease ever since , while the relevant tumor markers are within normal limits . the patient currently remains on maintenance letrozole and trastuzumab , has stable disease ( bone lesions alone ) and a good overall clinical status . identifying the primary cancer is crucial in selecting the optimal therapeutic strategy for the patients . when clinical and pathological methods fail to identify the primary tumor , treatment is often delayed and inaccurately targeted , contributing to the generally very poor prognosis of patients with cups . ihc is the most widely used tool for the diagnosis of several types of cancer , but fails to identify the tissue of origin in > 30% of the cases ( 7 ) . furthermore , ihc may be biased based on the patient 's presentation and past medical history and the interpretation of the results may be subjective , further signifying the need for alternative cancer diagnostic tests . a number of studies have demonstrated that microrna profiling may be useful for solving the diagnostic problem posed by cups , with agreement to final diagnosis for microrna testing ranging from 84% to as high as 92% , depending on the study ( 810 ) . micrornas are small , non - coding rnas of 1725 nucleotides in length that play an essential regulatory role in protein translation and expression ( 6 ) . since their discovery in 1993 , micrornas have been implicated in a number of major cell processes and have been mapped to areas of the genome prone to deletions , mutations and amplifications ( 6 ) . their ability to act as oncogenes or tumor suppressors , combined with their unique tissue pattern expression in malignancies , makes micrornas ideal cancer biomarkers and provides the basis for their use as a diagnostic tool ( 11 ) . in the present case , microrna testing was crucial , as the clinical and pathological findings did not lead to a diagnosis of breast cancer for a number of reasons . first , the pathological examination of the vertebral mass did not raise the suspicion of breast cancer , so this was not included in the differential diagnosis . second , while the mri and pet - ct imaging found the axillary mass to be suspicious , clinical evidence regarding the origin of the mass pointed towards the skin rather than the breast . the clinical evaluation of the skin lesion was also more consistent with skin cancer , taking into consideration the appearance and coloration of the mass ( fig . 4 ) . finally , the patient was male , with no family history of breast cancer . this is significant , as male breast cancer ( mbc ) is a rare entity , constituting < 1% of all breast cancers and < 1% of cancers in men ( 12 ) . male patients tend to be older than female patients at diagnosis , and have higher - stage disease , with more extensive lymph node involvement , contributing to the high mortality rate of mbc ( 13,14 ) . microrna testing proved to be key in identifying the origin of the primary cancer , when the clinicopathological picture was leading towards a misdiagnosis . further examination revealed that the tumor was er+ , pr+ and her2 + , indicating that the patient had a particularly good prognosis and could be treated with trastuzumab , as well as several specific targeted therapies , which would not have been applied had it not been for the microrna test . microrna profiling as a diagnostic test offers several advantages , the most important being that the assay is objective and unbiased . samples may be profiled using quantitative polymerase chain reaction technology or , more recently , a custom microrna array , and classification of tumor types relies on specific bioinformatics algorithms . the microarray is both powerful and flexible , it is able to profile thousands of transcripts and newly identified micrornas may easily be incorporated into an updated test design . furthermore , in terms of the feasibility of testing , samples of micrornas may be safely stored for significant periods of time in formalin - fixed paraffin - embedded tissues and , when extracted , still exhibit similar profiles to microrna samples from fresh tissue ( 15 ) . multiple studies have repeatedly demonstrated high test sensitivity , specificity and reproducibility , and the turnaround time for profiles is between 7 and 10 days , rendering this a reliable and practical diagnostic test . furthermore , microrna screening for early cancer diagnosis shows promising potential with changes in microrna profiles implicated in early or pre - tumor development ( 6 ) . first , the accuracy of testing can not be directly validated , as there is no primary cancer identified to be used as reference ; thus , cancers with similar profiles may be easily confused as being of the same origin ( 5,8 ) . in addition , microrna assessment may not always agree with the clinical and pathological diagnosis , with a previous study reporting a disagreement rate of 16% ( 8) . furthermore , the assay itself may produce two different diagnoses , which poses a potential problem for clinicians if the diagnoses differ regarding the optimal course of treatment ( 8) . on the other hand , the test may not be able to predict a result at all if the expression patterns do not closely correspond to those included in the panel . it is of great importance to determine the potential benefit that patients may gain from assigning cup to a primary tissue of origin . we would expect that assigning a cup to a primary would be of great benefit for such patients ; however , evidence thus far does not strongly support this hypothesis . a recent prospective study by hainsworth et al ( 16 ) , in which molecular techniques were used to identify the primary , demonstrated a median survival of 12.5 months for the group of patients in whom the cup was assigned a primary tissue of origin , in contrast to the group of patients who were treated as cup , in whom the median survival was 10.8 months . under the light of the currently available data , the european society for medical oncology and national comprehensive cancer network guidelines are quite conservative regarding the use of molecular tools in the differential diagnosis of cup , and they advise careful selection of the population considered as candidate for such diagnostic tools ( level of evidence , 2b ) . finally , while most recent tests may screen for 42 different primaries , they do not screen for all cancer origins ( 8) . microrna profiling may be quite costly , although the cost - benefit ratio may ultimately be favorable , as several unnecessary tests may be avoided . for all the abovementioned reasons , clinicians must consider all data relative to each individual case , in order to determine the optimal course of treatment for each patient . the case presented herein highlights the efficacy of using microrna analysis of tumor tissue samples to diagnose cups . as a diagnostic tool , microrna profiles may be very useful in identifying the primary tissue of origin and , thus , improving treatment and outcome for cup patients .