Benefit of Gene Expression Profiling in Gastrointestinal Neuroendocrine Tumors of Unknown Primary Origin.

BACKGROUND/AIM: Cancer of unknown primary (CUP), representing 3-5% of all newly diagnosed cancers in the United States, is a presumptive, non-definitive diagnosis rendered when a primary tumor site cannot be identified after exhaustive diagnostic evaluation, including cases of neuroendocrine neoplasms (NENs). CUPs are characterized by findings that are challenging to reconcile, including inconclusive immunohistochemical (IHC) stains, an undifferentiated morphologic phenotype, history of multiple cancers, a clinical presentation that is discordant from histologic findings, an atypical distribution of metastases, or lack of expected response to treatment. For a significant subset of NENs (10%), traditional diagnostic evaluation is unable to determine a primary tumor site using histomorphology and IHC stains. Gene expression profiling (GEP) of either mRNA or microRNA is the technique utilized in the three commercially available platforms that provide a prediction of tumor type in cases of diagnostic uncertainty of CUPs, including those with neuroendocrine differentiation. Case Series Report: Here we present four cases of NENs, where the diagnosis based upon histomorphological and IHC features presented a unique challenge that ultimately benefited from the integration of molecular tumor classification using the validated assay. CancerTYPE ID by Biotheranostics is based on a quantitative RT-PCR assay that uses a computational algorithm to measure the collective expression of 92 genes (87 cancer-related genes and 5 control genes). This case series reports five appropriate clinical scenarios that highlight the utility of a GEP-based assay to effectively provide a molecular tumor classification to identify NEN subtypes and tumor primary site of origin. CONCLUSION: These cases demonstrated that the CancerTYPE ID test was able to resolve challenging diagnoses for primary and metastatic NENs. These cases emphasize the clinical need of utilizing a GEP-based assay for determining the anatomic site of origin and NEN subtyping, both essential for the appropriate clinical management of NENs.

[2018 Consensus statement by the Spanish Society of Pathology and the Spanish Society of Medical Oncology on the diagnosis and treatment of cancer of unknown primary]. / Consenso 2018 de la Sociedad Española de Anatomía Patológica y la Sociedad Española de Oncología Médica sobre el diagnóstico y tratamiento del cáncer de origen desconocido.

Cancer of unknown primary is defined as a heterogeneous group of tumours that present with metastasis, and in which attempts to identify the original site have failed. They differ from other primary tumours in their biological features and how they spread, which means they can be considered a separate entity. There are several hypotheses regarding their origin, but the most plausible explanation for their aggressiveness and chemoresistance seems to involve chromosomal instability. Depending on the type of study done, cancer of unknown primary can account for 2-9% of all cancer patients, mostly 60-75 years old. This article reviews the main clinical, pathological and molecular studies conducted to analyse and determine the origin of cancer of unknown primary. The main strategies for patient management and treatment, by both clinicians and pathologists, are also addressed.

Liquid biopsy: a new diagnostic, predictive and prognostic window in cancers of unknown primary.

Cancer of unknown primary (CUP) is a heterogeneous group of metastatic cancers characterised by early dissemination of metastases in the absence of any identifiable primary site. Most patients with CUP have poor prognosis with the traditional diagnostic and treatment modalities. Recognising the putative primary tumour is hypothesised to ameliorate the prognosis of patients with CUP by guiding treatment decisions. The active efforts in molecular oncology have shown that gene expression profiling is able to identify the primary tumour site and to determine targetable mutations. In this regard, liquid biopsy opens a new diagnostic, predictive and prognostic window in CUP that may lead to substantial improvement in the management of patients with CUP.

[MRI findings and pathological features of occult breast cancer].

Objective: To investigate the magnetic resonance imaging (MRI) findings and clinicopathological features of primary lesions in patients with occult breast cancer (OBC). Methods: The imaging reports from the Breast Imaging Reporting and Data System in 2013 were retrospectively analyzed to investigate the morphology and the time signal intensity curve (TIC) of breast lesions in patients with OBC. The clinical and pathological characteristics of these patients were also included. Results: A total of 34 patients were enrolled. Among these patients, 24 patients underwent modified radical mastectomy and 18 of them had primary breast carcinoma in pathological sections. MRI detected 17 cases of primary lesions, including six masse lesions with a diameter of 0.6-1.2 cm (average 0.9 cm), and 11 non-mass lesions with four linear distributions, three segmental distributions, three focal distributions, and one regions distribution. Five patients had TIC typeⅠprimary lesions, ten had TIC type Ⅱ primary lesions, and two had TIC type Ⅲ primary lesions. Among all 34 cases, 23 of them had complete results of immunohistochemistry: 11 estrogen receptor (ER) positive lesions (47.8%), tenprogesterone receptor (PR) positive lesions (43.5%), seven human epidermal growth factor receptor 2 (HER-2) positive lesions (30.4%), and 20high expression(>14%) of Ki-67 (87.0%). The proportion of type luminal A was 4.3%, type luminal B was 43.5%, triple negative breast cancer (TNBC) was 30.4%, and HER-2 over expression accounted for 21.7%. Conclusions: The primary lesions of OBC usually manifested as small mass lesions, or focal, linear or segmental distribution of non-mass lesions. The positive rate of ER and PR was low, but the positive rate of HER-2 and the proliferation index of Ki-67 was high. Type luminal B is the most common molecular subtype.

Utility of Sox10 labeling in metastatic breast carcinomas.

Sox10 labeling by immunohistochemistry has been primarily reported in tumors of neural crest origin, such as nerve sheath tumors and melanoma. However, Sox10 also labels primary breast carcinomas, particularly those with the basal-like, triple-negative phenotype. However, the utility of Sox10 labeling in metastatic breast carcinomas has not been reported. Here, we prospectively evaluated Sox10 labeling in surgically resected metastatic breast carcinomas from 26 patients sampled on tissue microarrays. In this cohort, Sox10 labeling was seen in 3 metastatic breast carcinomas (12%), all of which were grade III, triple-negative ductal carcinomas metastatic to the brain (n=2) or lung (n=1). Overall, 38% of triple-negative metastases were Sox10 positive, compared to 0% of estrogen receptor (ER)+ or human epidermal growth factor 2 (HER-2) + metastases (P=.045). In addition, we retrospectively reviewed the use of Sox10 immunohistochemistry in metastatic carcinomas in our clinical practice. We identified 21 cases from January 2012-July 2017 in which Sox10 immunohistochemistry was ordered on clinical sign-out in the work-up of a metastatic carcinoma as being of possible breast origin. Overall, Sox10 labeled 57% (n=12) of all evaluated metastatic carcinomas. All of the Sox10+ tumors were ER-, such that 71% of ER- carcinomas were Sox10+ in comparison to 0% of ER+ carcinomas (P=0.049). In conclusion, the differential diagnosis of a Sox10+ malignancy of unknown origin should not be limited to metastatic melanoma. Sox10 labeling is seen in a subset of metastatic triple-negative breast carcinomas, supporting its use as a marker of breast origin in this setting.

Lung Cancer Detected 5 Years after Resection of Cancer of Unknown Primary in a Mediastinal Lymph Node: A Case Report and Review of Relevant Cases from the Literature.

We report the rare and interesting case of a primary lung cancer detected 5 years after cancer of unknown primary (CUP) of a mediastinal lymph node (LN) was resected. A 40-year-old male was diagnosed with adenocarcinoma of unknown primary in a mediastinal lymph node after resection of the mediastinal tumor. Five years after resection of the CUP in mediastinal LN, a small, abnormal nodular shadow in left upper lobe was detected by chest CT. This pulmonary tumor was diagnosed as a lung adenocarcinoma. The pathological and immunohistological findings of the resected pulmonary tumor resembled those of the LN resected 5 years before. We speculated that the pulmonary lesion represented primary lung cancer that enlarged later than the metastatic mediastinal LN. This case illustrates the importance of careful observation and long-term follow-up in patients treated for CUP of a thoracic LN.

GATA-3 and FOXA1 expression is useful to differentiate breast carcinoma from other carcinomas.

GATA-3, a member of the GATA family of zinc-finger DNA binding proteins, and FOXA1, a member of the forkhead transcription factor family, are both associated with estrogen receptor expression. Both GATA-3 and FOXA1 are useful markers for breast carcinoma, but their expression in the different breast cancer subtypes and other neoplasms has not been thoroughly evaluated. We examined the expression of GATA-3 and FOXA1 in estrogen receptor-positive, Her2/neu-positive, and triple-negative breast carcinomas as well as in 10 other common carcinomas, including hepatocellular, colonic, pancreatic, gastric, endometrial (endometrioid), lung, prostatic, renal cell, urothelial, and ovarian serous carcinomas. Primary and metastatic melanomas and mesotheliomas were also evaluated. GATA-3 and FOXA1 staining of estrogen receptor-positive breast carcinomas was seen in 96.6% and 96.2%, respectively. In triple-negative breast carcinomas, GATA-3 and FOXA1 staining was seen in 21.6% and 15.9%, respectively. Among the other tumors, GATA-3 staining was only seen in urothelial carcinoma (70.9%) and FOXA1 staining was only seen in prostatic (87.5%), urothelial (5.1%) carcinomas, and mesotheliomas (40.0%). In conclusion, GATA-3 and FOXA1 are excellent breast carcinoma markers; however, their utility is limited in the triple-negative subtype. The utility of FOXA1 in diagnosing prostatic carcinoma and mesothelioma warrants further investigation.

Cytologic evaluation of cervical lymph node metastases from cancers of unknown primary origin.

Fine-needle aspiration biopsy (FNAB) is often the first diagnostic procedure performed in patients with head and neck (HN) masses. Metastatic squamous cell carcinoma (SCC) to cervical lymph nodes is by far the most common malignancy aspirated in the HN, but in approximately 3-10% of patients, a primary tumor will not be found even after complete clinico-radiological workup. Several HN cancers are associated with oncogenic viruses, including HPV-associated SCC and EBV-associated nasopharyngeal carcinoma (NPC). While the primary tumor is sometimes small or undetectable, patients often present initially with cervical lymph node metastases. HPV-associated SCC and EBV-associated NPC are typically non-keratinizing carcinomas that can mimic several other poorly differentiated HN cancers by FNAB but have a significantly better prognosis. Therefore, the precise classification of the metastatic disease in the FNAB material is very useful since it can facilitate the subsequent location of the primary tumor, and it can provide prognostic and therapeutic information as well. In this review, we discuss the major entities that can present as a metastatic cancer of unknown primary in cervical lymph node other than supraclavicular, including their cytologic features and the role of ancillary studies.

Autophagy and redox status in carcinoma of an unknown primary.

AIM AND BACKGROUND: The purpose of the study was to investigate the role and clinical implications of autophagy and reactive oxygen species-related proteins in carcinoma of an unknown primary (CUP). METHODS AND STUDY DESIGN: Tissue microarray was constructed for a total of 77 CUP cases. Immunohistochemical stains conducted were as follows: autophagy-related beclin-1, LC3A, LC3B, and p62; redox-related catalase, thioredoxin reductase, glutathione S-transferase π, thioredoxin-interacting protein, and manganese superoxide dismutase. Immunohistochemical results were then related to their clinicopathologic parameters. RESULTS: The degree of LC3A expression showed a difference according to histologic subtype. In undifferentiated carcinoma, LC3A had the highest expression and adenocarcinoma had the lowest expression (P = 0.021). According to clinical subtype, there was a significant difference between LC3A and glutathione S-transferase π in expression. LC3A had the highest expression in single-organ types and the lowest in intermediate and carcinomatosis types (P = 0.003). Glutathione S-transferase π showed the highest expression in nodal-type tumors and the lowest in carcinomatosis types (P = 0.010). In univariate analysis, shorter overall survival was related to tumor glutathione S-transferase π negativity (P = 0.030). CONCLUSIONS: Different expression levels of the autophagy and reactive oxygen species-related proteins, LC3A and glutathione S-transferase π, were observed according to histologic and/or clinical subtype of carcinoma of an unknown primary.

[Carcinoma unknown primary-clinical practice].

Carcinomas of unknown primary are cancers that are pathologically diagnosed as carcinomas based on the findings of metastatic lesion biopsy but in which the primary site remains unknown even after thorough and detailed examinations.In the clinical setting, pathological diagnosis and diagnostic imaging are very important for cases of carcinoma of unknown primary. When examinations do not identify the primary tumor, carcinoma cases are treated as carcinoma of unknown primary.In clinical practice, it is very important not to miss cases of favorable prognosis group, which is 20% of unknown cancer. Recently, microarray studies have been performed for cases of carcinoma of unknown primary.

Assigning site of origin in metastatic neuroendocrine neoplasms: a clinically significant application of diagnostic immunohistochemistry.

The neuroendocrine epithelial neoplasms (NENs) include well-differentiated neuroendocrine tumors (WDNETs) and poorly differentiated neuroendocrine carcinomas (PDNECs). Whereas PDNECs are highly lethal, with localized Merkel cell carcinoma somewhat of an exception, WDNETs exhibit a range of "indolent" biologic potentials-from benign to widely metastatic and eventually fatal. Within each of these 2 groups there is substantial morphologic overlap. In the metastatic setting, the site of origin of a WDNET has significant prognostic and therapeutic implications. In the skin, Merkel cell carcinoma must be distinguished from spread of a visceral PDNEC. This review intends to prove the thesis that determining the site of origin of a NEN is clinically vital and that diagnostic immunohistochemistry is well suited to the task. It will begin by reviewing current World Health Organization terminology for the NENs, as well as an embryologic and histologic pattern-based classification. It will present population-based data on the relative frequency and biology of WDNETs arising at various anatomic sites, including the frequency of metastases of unknown primary, and comment on limitations of contemporary imaging techniques, as a means of defining the scope of the problem. It will go on to discuss the therapeutic significance of site of origin. The heart of this review is a synthesis of data compiled from >100 manuscripts on the expression of individual markers in WDNETs and PDNECs, as regards site of origin. These include proteins that are considered "key markers" and others that are either useful "secondary markers," potentially very useful markers that need to be further vetted, or ones that are widely applied despite a lack of efficacy. It will conclude with my approach to the metastatic NEN of unknown origin.

Molecular profiling diagnosis in unknown primary cancer: accuracy and ability to complement standard pathology.

BACKGROUND: Molecular tumor profiling (MTP) is a potentially powerful diagnostic tool for identifying the tissue of origin in patients with cancer of unknown primary (CUP). However, validation of the accuracy and clinical value of MTP has been difficult because the anatomic primary site in most patients is never identified. METHODS: From March 2008 through January 2010, clinicopathologic data from 171 CUP patients who had MTP (CancerTYPE ID; bioTheranostics, Inc, San Diego, CA) performed on archived material were evaluated. The accuracy of MTP diagnoses was evaluated by comparison with (1) latent primary tumor sites found months/years later; (2) initial single diagnoses by immunohistochemistry (IHC); and (3) additional directed IHC and/or clinicopathologic findings evaluated after MTP diagnoses. RESULTS: A single MTP diagnosis was made in 144 of 149 patients with adequate tumor specimens. Eighteen of 24 patients with latent primaries discovered months to years later had correct diagnoses by MTP (75%), and these diagnoses compared favorably with IHC. Single IHC diagnoses matched MTP diagnoses in 40 of 52 patients (77%). IHC predictions of 2 or more possible primaries compared poorly with MTP diagnoses. However, additional targeted IHC and clinical/histologic evaluation supported the MTP diagnosis in 26 of 35 patients (74%). Clinical features were usually consistent with MTP diagnoses (70%). CONCLUSIONS: The diagnostic accuracy of this MTP assay was supported by a high level of agreement with identified latent primaries (75%), single IHC diagnoses (77%), and additional directed IHC and/or clinical/histologic findings (74%) prompted by the MTP diagnoses. MTP complements standard pathologic evaluation in determining the tissue of origin in patients with CUP, particularly when IHC is inconclusive.

p16 expression in carcinoma of unknown primary: diagnostic indicator and prognostic marker.

BACKGROUND: Carcinoma of unknown primary (CUP) of the neck are heterogeneous tumors in their clinical and biological characteristics, and a preoperative prognostic marker is desirable to optimize staging and therapy and to improve outcome and survival. For CUP syndrome, no optimized diagnostic and treatment strategy or biomarker have yet been determined. METHODS: Forty-seven patients presenting with CUP syndrome were analyzed after thorough standard diagnostic staging procedures. All patients were surgically treated with tonsillectomy, neck dissection of the diseased neck, as well as adjuvant chemoradiation. The tissue of lymph node metastases (and, if found, of the primary tumor) was analyzed regarding expression of p16, epidermal growth factor receptor (EGFR), and presence of human papillomavirus (HPV) DNA. RESULTS: In 39% of all cases (20 of 47), the primary cancer was found during diagnostic workup. If HPV DNA was detected in the neck lymph node metastasis, the primary cancer was significantly more frequently found in the oropharynx (p = .002). Patients with a p16-positive tumor had a significantly higher 5-year overall survival (OS; 33% vs 69%; p = .045, disease-free survival [DSF] 77% vs 89%; p = not significant [NS]). Patients with p16-positive neck metastasis and no detectable primary cancer had a better prognosis. Expression of EGFR in this series did not have a significant effect on prognosis. CONCLUSION: In patients presenting with CUP syndrome, p16 immunohistochemistry can serve to locate the primary cancer in the oropharynx. It is a positive prognostic indicator in patients with those heterogeneous cancers.

p16 expression in cutaneous squamous carcinomas with neck metastases: a potential pitfall in identifying unknown primaries of the head and neck.

BACKGROUND: Human papillomavirus (HPV) positivity (+) has been used to identify oropharyngeal squamous carcinomas (SCCs) presenting as unknown primaries in the neck. p16 overexpression correlates with HPV+ in the oropharynx; however, the use of p16 alone as a surrogate marker of oropharyngeal HPV+ tumors has not been validated. METHODS: We immunohistochemically analyzed p16 expression in surgically resected aggressive cutaneous head and neck SCC primaries and their nodal metastases from 24 patients to determine the potential overlap of p16 expression outside of the oropharynx. RESULTS: Five of 24 primary tumors (20.8%) and 3 lymph node metastases (12.5%) in levels II, III, and V, and the periparotid region diffusely expressed p16. HPV (high-risk types by in situ hybridization) was negative. CONCLUSIONS: p16 expression is relatively common in lymph node-positive cutaneous head and neck SCCs; thus, p16 expression as an independent biomarker and mechanism to determine the oropharyngeal source of an unknown primary is not advised.

Is CDX2 immunostaining useful for delineating anorectal from penile/vulvar squamous cancer in the setting of squamous cell carcinoma with clinically unknown primary site presenting with histologically confirmed inguinal lymph node metastasis?

AIMS: Penile, vulvar and anal squamous cell carcinomas (SCCs) share histomorphological overlap and are prone to lymphatic dissemination into inguinal nodes. Anal SCCs might derive from the anorectal zone (ARZ), anal transitional zone, squamous zone or from perianal skin. These anatomically distinct zones differ in terms of their embryological development. We sought to investigate the role of caudal-related homeobox 2 (CDX2), a homeobox gene implicated in the development and anterior/posterior pattern specification from duodenum to rectum including the ARZ, in terms of narrowing the possible sites of origin to be considered in the setting of SCC with unknown primary presenting with histologically confirmed inguinal lymph node metastasis. METHODS: By immunohistochemistry (IHC) employing a panel of antibodies directed against CK5/6, CK7, CK20, p63, p16, CEA and CDX2, we compared 89 penile, 11 vulvar and eight anal SCCs with respect to their staining profiles. Moreover, anal SCCs were subjected to in situ hybridisation (ISH) for high-risk human papillomavirus (HPV) subtypes. RESULTS: By IHC, CDX2 expression was observed in 2/8 anal SCCs (25%) while being absent from all penile and vulvar SCCs examined. High-risk HPV subtypes were detected by ISH in all anal SCCs examined, which were uniformly p16-positive by IHC. CONCLUSIONS: CDX2 might be valuable in terms of narrowing the possible sites of origin to be considered in the setting of SCC with unknown primary presenting with inguinal lymph node metastasis. However, despite its favourable specificity, the diagnostic benefit achieved by this observation is limited by the low sensitivity.

Comparison of histopathology to gene expression profiling for the diagnosis of metastatic cancer.

BACKGROUND: Determining the primary site of metastatic cancer with confidence can be challenging. Pathologists commonly use a battery of immunohistochemical (IHC) stains to determine the primary site. Gene expression profiling (GEP) has found increasing use, particularly in the most difficult cases. In this pilot study, a direct comparison between GEP and IHC-guided methods was performed. METHODS: Ten archived formalin-fixed paraffin embedded metastatic tumor samples for which the primary site had been clinically determined were selected. Five pathologists who were blinded to the diagnosis were asked to determine the primary site using IHC and other stains selected from a panel of 84 stains. Each pathologist was provided patient sex, biopsy site and gross sample description only. Slides were digitized using ScanScope®XT at 0.25 µm/pixel. Each evaluating pathologist was allowed to provide a diagnosis in three stages: initial (after reviewing the H&E image), intermediate (after reviewing images from the first batch of stains) and final diagnosis (after the second batch of stains if requested). GEP was performed using the only FDA-cleared test for this intended use, the Pathwork Tissue of Origin Test. No sample information was provided for GEP testing except for patient sex. Results were reported as the tumor tissue type with the highest similarity score. RESULTS: In this feasibility study, GEP determined the correct primary site in 9 of the 10 cases (90%), compared to the IHC-guided method which determined the correct primary site for 32 of 50 case evaluations (average 64%, range 50% to 80%). The five pathologists directing the IHC-guided method ordered an average of 8.8 stains per case (range 1 to 18). GEP required an average of 3 slides per case (range 1 to 4). CONCLUSIONS: Results of the pilot study suggest that GEP provides correct primary site identification in a higher percentage of metastatic cases than IHC-guided methods, and uses less tissue. A larger comparative effectiveness study using this study design is needed to confirm the results. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1749854104745508.