Role of molecular diagnostics in determination of tissue of origin in Cancer of Unknown Primary (CUP)

Institution of tumor-specific therapy

Evidence suggests that patients who receive a primary tumor diagnosis have longer survival rates compared to those never get a definite identification of the primary site.^[5] It is this expectation of improved outcome with tumor-specific therapy that motivates the search for the primary site. The search has recently intensified with availability of new targeted drugs introduced as therapy for specifically indicated tumor tissue types. Furthermore, a definitive primary site relieves patient’s anxiety over uncertain diagnosis. Hence, it is important to diagnose the cancer type in patients with difficult to diagnose cancers to further guide the choice of site-specific therapy.^[6]

IHC

Numerous panels of immunohistochemical stains are used by histopathologists to identify the primary site of origin for metastatic cancers, particularly when limited diagnostic information is obtained with morphologic characteristics. IHC has been proven to be tool for differential diagnosis and to effectively rule out one or the other candidate tissues.^[8]

MCCAs

MCCA is an emerging diagnostic method that empowers prediction of the tissue of tumor origin by identifying site- specific gene expression profiles.^[7]

MCCA in combination with histopathology and IHC empowers clinical evaluation of tissue-of-origin diagnosis in >90% of difficult to diagnose patients.^[9] The gene expression tests have been developed as an adjunct to morphological evaluation and IHC analysis in the assessment of patients with dubious primary cancer.

These molecular profiling assays either use microarrays, reverse transcription polymerase chain reaction (RT-PCR), or epigenetic to quantify mRNA or microRNA. The microarray- based assays can measure the expression levels of thousands of gene markers, whereas the RT-PCR-based assays focus on a smaller subset of 10–100 gene markers.^[9]

TOO test

The TOO test by cancer genetics incorporated (CGI, USA) is a microarray-based gene expression test that uses the expression levels of 2000 genes to classify tumors by similarity scores (SS) into 15 sites of origin.^[4] The US FDA for the 1^st time approved the TOO test for GEP of tumors in June 2010.^[10]

The TOO test includes 15 tumors in the panel such as tumors of prostate, ovary, thyroid, breast, lung (non-small cell), gastric, colorectal, pancreatic, liver, urinary bladder, kidney as well as melanoma, non-Hodgkin’s lymphoma, sarcoma, and testicular germ cell tumor.^[11]

Benefits of using TOO test

1. It can help in diagnosis of cases in which the location, histology, or IHC results make it difficult to dependably diagnose the primary site.^[4]

2. In patients with unidentified primary sites, the recommended first-line chemotherapy for most patients is likely to change with the utilization of the TOO test results.^[12]

3. Decreased requirement for repeated testing, examinations, and imaging/biopsy procedures.^[13]

4. Prospects to enter appropriate clinical trials.^[13]

5. Vital information in evaluating one’s familial risks for cancer^[13]

6. Knowing the primary tissue type with greater certainty helps physicians choose the most appropriate treatment regimens.^[13]

TOO test workflow is below:^[9]

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Epigenetic test to identify tumors of unknown origin

Epigenetics refers to heritable changes in gene expression without an alteration in the DNA sequence.^[14] DNA methylation, of the carbon-5 of cytosine moieties, is the most recognized epigenetic mark in the human genome.^[15] Cancer-specific signatures have been identified by DNA-methylation profiling of hypermethylated CpG islands which enable differentiation of tumor types.^[16,17] A clear advantage of an epigenetic-based approach is that DNA remains stable overtime.

EPICUP is a test based on microarray technology and identifies the tumor-specific methylation profiles. Bisulfite-converted DNA from FFPE samples will be processed on Infinium MethylationEPIC BeadChip (850K DNA methylation microarray) for the analysis of DNA methylation.^[15]

It can identify 38 cancer types covering 90% of the most frequent solid tumors. The test has a sensitivity of 99.7%, specificity of 99.6%, and a positive predictive value of 88.6%.^[15]

The test was validated using an initial training set of 2790 samples of known origin (primary tumors or metastases) from 38 cancer types [Table 1] to interrogate more than 485,577 CpG sites and a classifier based on cancer type was established.^[17] This classification was validated in 7691 samples.^[2,15]

Implications on survival

The administration of treatment based on the diagnosis provided by the validated molecular and GEP platforms has shown survival benefits in the patients with CUP.

Conditions where TOO test and EPICUP test are useful

The molecular cancer classifier assays are useful in identification of the primary site in patients with CUP are as follows:

1. The tumor is poorly differentiated or undifferentiated.

2. There is an unresolved differential diagnosis of two or more cancer types

3. The specimen is small, constraining the diagnostic workup and limiting prognostic studies

4. The patient has a history of multiple cancers

5. IHC is inconclusive or conflicting after the first round

6. Clinical history and histology differ on the diagnosis

7. There is atypical distribution of metastases

8. Oncology and pathology differ on the diagnosis

9. The diagnosis is questioned when the patient fails to respond to treatment.

Clinical guidelines on molecular diagnostics to identify primary site in CUP

Guidelines recommend that patients with CUP undergo a thorough evaluation, including a complete history, physical examination, complete blood count, urine analysis, pathological evaluation, histologic evaluation, chest radiograph, computed tomography, magnetic resonance imaging, and IHC studies.

The Ad-Hoc Committee on IHC Standardization 2007 highlighted some potential “deficiencies” in the consistency, reproducibility, quality assurance, concordance, validation, and results reporting of IHC studies. Although the recommendations are being adopted, full characterization of the tumor-site origin by IHC may not be precisely possible.^[10]

The recent NCCN guidelines agree that GEP is comparable to the accuracy of IHC for poorly differentiated/undifferentiated carcinomas. NCCN guidelines suggest the collaboration of pathologists and oncologists on the judicious use of GEP profiling on a case to case basis with the best possible individualized patient outcome in mind.^[18]

As per the recent ESMO guidelines on CUP, GEP tests may aid in the diagnosis of the putative primary tumor site in some patients. The guidelines also highlight 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 TOO diagnosis of more responsive tumor types.^[2] There are few tests available that could be of use in CUP, a summary of the tests is mentioned in Table 2.

The recent Spanish Society of Pathology and the Spanish Society of Medical Oncology consensus statement on the diagnosis and treatment of CUP recognize that the molecular diagnostics and gene expression platforms are helpful when they are used to complement the IHC, allowing for more accurate diagnosis of tumor origin. It recommends that, based on clinical and histopathological features, a limited number of basic and specific advanced IHC tests should be performed initially to ensure the availability of sufficient quantity of tissue for molecular platforms.^[19]