An occult urothelial carcinoma with wide multiorgan metastases and its genetic alteration profiling: Case report and literature review.

RATIONALE: Urothelial carcinoma, also named transitional cell carcinoma, is the most frequent occurring malignancy in the urinary system. It mainly invades the surrounding tissues and metastasizes to distant organs in later stages. PATIENT CONCERNS: Here, we presented an unusual case of occult urothelial carcinoma primarily manifested as a multiorgan metastatic cancer in a 59-year-old man. The patient complained of pain on the left thigh root for a month. The imaging and histopathological examination revealed multiple malignancies in lung, bone, and liver. DIAGNOSES: The histological evaluation and the immunohistochemistry (IHC) profile of liver, lung, and bone were consistent with the diagnosis of metastases from the original urothelial cancer, while imaging examination was not able to detect a primary lesion in the urinary system.
INTERVENTIONS: Based on the mutation of STK11 M51Ifs*106 detected by next generation sequencing (NGS), we started targeted therapy with everolimus. OUTCOMES: The patient deteriorated after 3 months of treatment and passed away. LESSONS: In this initial report of occult urothelial carcinoma, we obtained information on genetic variations of tumor tissue which could provide important information for subsequent studies on this kind of disease.

Introduction

In the diagnosis and treatment of cancers, it is essential to identify the source of tumor cells because this determines treatment options. Occult cancer is quite rare and defined as a clinically unknown malignancy in which the primary lesion is difficult to identify. The apparent metastatic tumor is diagnosed by clinicians and pathologists according to histological examination. For some patients, the micro primary carcinoma can be detected when a patient is being evaluated for autopsy.[ To date, most occult cancers have been reported in breast cancer and a few in thyroid and gynecologic cancers.[ Also, a few occult somatic malignant tumors may also present acute cerebral infarction as the first clinical manifestation.[ The occurrence of occult cancer is so rare that it only accounts for about 0.3% to 1.0% of all newly diagnosed breast cancer cases.[

Urothelial carcinoma, which is also known as transitional cell carcinoma, is a malignant tumor that arises from the transitional epithelium. It commonly occurs in the urinary tract. Metastasis of urinary tract cancer to distant organs is rare and usually occurs in advanced stages after diagnosis. To date, the initial clinical manifestation of urothelial carcinoma as metastasis has not been reported. Here, we present an unusual case of occult urothelial cancer which manifested in multiorgan metastasis including lung, liver, and bone at an early stage.

Case presentation

The patient was a 59-year-old man who complained of continuous acupuncture-like pain (VAS score of about 8) for a month which started on April 10, 2017 in the left upper thigh. There was no visible lesion on the local skin and the pain did not radiate or affect movement, and therefore, no treatment was given. A month later, the pain gradually worsened and began to radiate toward the sacrococcygeal region and the right buttocks, and increased when the patient was walking. The patient had a history of smoking 20 cigarettes per day and drinking 1 to 1.5 mL of alcohol a week for about 20 years. No positive found in family history. The patient went to a local hospital for medical care on May 22, 2017. Computed tomography (CT) examination showed space-occupying lesions on the tip of the upper lobe of the right lung, a few small lymph nodes in the mediastinum, multiple locations of bone destruction on the body and pedicle of L4 lumbar vertebra, and space-occupying lesions with slightly low density on the left lateral lobe of the liver (Fig. 1). The abnormal images of the lung, mediastinum, liver, and bone were all considered highly probable malignant lesions (either primary or metastatic) by radiologists. Further examination was recommended to make a clear diagnosis.

Figure 1

CT examination in local hospital on May 22, 2017. A. Lung window in CT scan of a space-occupying focus on the tip of the upper lobe of the right lung. B. Mediastinal window in CT scan of a space-occupying focus on the tip of the upper lobe of the right lung. C. CT scan of the fourth lumbar spine destruction. D. CT scan of a space-occupying lesion on the left lobe of the liver with a size of about 3.0 × 2.4 cm. CT = computed tomography.

The patient went to our hospital for further medical care on May 27, 2017. Tumor markers were detected as follows: total prostatic specific antigen (T-PSA) 0.55 ng/mL (normal: 0–4), carcinoembryonic antigen (CEA) >100.00 ng/mL (normal: ≤5 ng/mL), carbohydrate antigen 125 (CA-125) 46.8 U/mL (normal: ≤ 35 U/mL), CA-153 157.8 U/mL (normal: ≤31.3 U/mL), and squamous cell carcinoma associated antigen (SCC) 4.64 ng/mL (normal: 0–1.5 ng/mL). CT results showed a right lung upper lobe mass shadow with size of about 3.1 × 2.5 cm, 4 to 6 small lymph nodes in the mediastinum and multiple low-dense nodules in the thyroid gland. CT reconstruction bone window showed bone destruction of the right fourth rib, L4 vertebral body, and bilateral iliac with soft tissue mass formation. Slightly low-density nodules (3.0 × 2.5 cm) in the left lateral lobe of the liver were slightly enhanced on contrast, and multiple low-density nodules were found in other regions of the liver parenchyma (Fig. 2). No abnormalities were found in gallbladder, bile duct, spleen, pancreas, kidneys, adrenal glands, bladder, or rectum. Whole body bone emission CT (ECT) imaging on June 1, 2017 showed multiple abnormal concentrations of radioactivity on both sides of the ilium. The skeletal radioactive distribution in the skull, sternum, clavicle, scapula, spine, ribs, limbs, and other parts were uniform and symmetrical. The active metabolism of bilateral iliac bone was considered to be possible metastases (Fig. 3). Pelvic magnetic resonance imaging (MRI) showed bone destruction with soft tissue mass formation on bilateral iliac and upper femur segments, and showed iso signal intensity in a T1-weighted image and heterogeneous slightly high signal intensity in a T2-weighted image (Fig. 4). Combined with medical history, it was considered to be possible bone metastases.

Figure 2

CT examination in our hospital on May 27, 2017. The upper lobe mass of the right lung, about 3.1 × 2.5 cm in size, is characterized by burrs and lobulations (A. lung window; B. mediastinum window; C. mediastinum window and contrast-enhanced). Both sides of iliac bone destruction and surrounding soft tissue swelling with a size of about 4.5 × 4.8 cm (D, right) and 3.2 × 2.0 cm (E, left). A slightly low-density nodule on the left lateral lobe of liver about 3.0 × 2.5 cm in size, and slightly enhanced by the contrast (F).

Figure 3

Whole-body bone scan by emission CT (ECT) in our hospital on June 1, 2017. Multiple spots and flaky radioactive concentrating foci could be seen on both sides of the iliac bone.

Figure 4

Magnetic resonance imaging of pelvis in our hospital on June 12, 2017. The fat suppression images indicated bilateral iliac bone destruction with soft tissue mass formation, with a size of about 5.8 × 6.7 cm (A. T2WI; B. DWI; C. T1WI). Coronal position images showed uneven contrast-enhancement of bilateral iliac lesions (D. pre-contrast; E. post-contrast). The fat suppression images indicated left acetabular lesion (F. T2WI; G. T1W1; H. T2WI contrast-enhanced; I. T1WI contrast-enhanced). DWI = diffusion-weighted imaging; T1WI = T1-weighted imaging; T2WI = T2-weighted imaging.

Although all previous examinations suggested the possibility of malignancy, they were still insufficient for diagnosis. For confirmation, biopsies were done with the consent of the patient. In the days following, the left iliac, liver, and pulmonary were punctured under the guidance of B-mode ultrasonography. Representative pictures of pathological examination of each specimen are showed in Fig. 5. The pathological evaluation of bone, liver, and lung suggested a high-grade metastatic urothelial carcinoma. Based on results of bone IHC, (Creatine Kinase) (CK)5/6+ scattered, CK7+, GATA binding protein 3 (GATA3)+, P40+ few, CK20-, Villin-, thyroid transcription factor-1 (TTF-1)-, CDX2-, Glypican3- and Alpha-1 Antitrypsin (AAT)+ few), liver IHC (CK7+, CK20-, Villin-, TTF-1-, paired-box-8 (PAX-8)-, GATA-3-, CDX-2+, Ki-67+ about 60%), and lung IHC (CK5/6-, P40-, CK20-, GATA-3+ partial, CK7+ strong and widespread, TTF-1-, NapsinA-, Ki-67+ 40%), it did not support adenocarcinoma or squamous cell carcinoma from lung, liver, intestines, or stomach. In conclusion, combining the images of the hematoxylin and eosin (H&E) staining and IHC staining, all tumor tissues most likely came from urothelium tissue rather than lung or digestive system tissue. On June 22, 2017 after a multidisciplinary consultation, the patient was diagnosed with occult urothelial carcinoma with bone, liver, and lung metastases, without primary urinary tract lesions.

Figure 5

Pathological examination of biopsy specimens. Biopsy results for the left iliac bone. (A) H&E staining: the cells were arranged in sheets and variable in size, 200×; IHC results: (B) Gata-3+ 200×; (C) Villin- 100×; (D) CDX2- 50×. Biopsy results for the liver. (E) H&E staining: the cells were arranged in sheets and variable in size, 200×; IHC results: (F) Glypican3- 100×. Biopsy results for the lung. (G) H&E staining, 100×; IHC results: (H) GATA-3 + partly 200×; (I) TTF-1 - 200×. CDX2 = homeobox protein CDX-2, GATA-3 = GATA3 GATA binding protein 3, H&E = hematoxylin and eosin; IHC = immunohistochemistry; TTF-1 = thyroid transcription factor 1.

After some pretreatment preparation, the patient began to receive radiotherapy on July 4, 2017. Target delineation: CT imaging of lumbar and pelvic tumors were gross tumor volume (GTV)1 and GTV2, respectively. The vertebral body and the appendage of GTV1 was clinical target volume (CTV)1. CTV2 included GTV2 expanded 1 cm and the imaging of the area of muscle edema. CTV1 externally expanded 5 mm was prophylactic CTV (pCTV)1. CTV2 expanded 1 cm on top and bottom, and 5 mm in other directions was PCTV2. The dose of irradiation was pCTV1: 33.8 Gy/13f, pCTV2: 39 Gy/13f. One week later, the patient expressed relief of pelvic bone pain, but had worse pain in the right rib. CT results suggested progressive disease (PD). Since the tumor of the ribs and cervical vertebrae were close to the spinal cord and liver tumors had increased in size, the radiotherapy regimen changed. Target delineation: the CT imaging of right fourth rib metastases and the surrounding soft tissues was GTV3; CT imaging of the metastatic tumor of the cervical spine was GTV4. GTV3 and GVT4 expanded 5 and 3 cm were the pCTV3 and pCTV4, respectively. The CT imaging of the liver left lobe tumor was GTV5. GTV5 expanded 1 cm upwards and underneath and 0.7 cm on the other direction were the pCTV5. The dose of irradiation was pCTV3-5: 50 Gy/25f. pCTV3-5 radiotherapy was performed 14 times, but the patient's pain was not controlled well. CT re-examination on July 26, 2017 revealed the soft tissue masses around the 4th ribs were larger than when examined on May 2, 2017. Low density nodules in the left lateral lobe of the liver were enlarged to 5.2 × 3.3 cm (5IMA11) in size, and a new nodule with the diameter of 0.8 cm (5IMA9) was found in the left lateral lobe of the liver. The other images had little change compared with the previous CT (Fig. 6). The Response Evaluation Criteria in Solid Tumors (RECIST) assessment was PD and the patient was discharged from the hospital under his own insistence on July 31, 2017.

Figure 6

CT re-examination in our hospital on July 26, 2017. Compared with CT images on May 27, 2017, the size change of upper lobe mass of the right lung remained about the same (3.2 × 2.8 cm vs 3.1 × 2.5 cm. [A] Lung window; [B] mediastinum window). The bone destruction in the fourth sides of the right side, and the surrounding soft tissue mass increased (C and D). (E) The nodule on the left lateral lobe of liver was significantly larger than before (5.2 × 3.3 cm vs 3.0 × 2.5 cm). CT = computed tomography.

To seek the possibility of targeted therapy, the liver biopsy specimen of the patient was sent to Origimed on June 24, 2017. Of the 450 tumor related genes that were identified, 7 clinically significant genetic variants, including MET amplification, STK11 M51Ifs∗106 exon1, KRAS G12C exon2, NCOR1 S1952∗ exon37, RBM10 E832∗ exon22, SETD2 Q2030∗ exon13 and SUFU c.1022+1G>T, were detected. Based on the highest variation abundance of STK11 M51Ifs∗106 exon1, the patient was given 10 mg qd. po. everolimus on July 29, 2017 and had a dose adjustment to 250 mg bid. po. on September 3, 2017. During the whole course of treatment, the pain was not alleviated and his general condition continued to deteriorate until the patient finally died on October 17, 2017. This study was approved by the ethics committee of Affiliated Tumor Hospital of Guangxi Medical University (Ethical approval number: LW2018019). Informed consent for publication of the case has been obtained in writing from the deceased patient's next of kin.

Discussion

Urothelial carcinoma is the most common malignancy in the urinary system. It mostly invades the tissues and organs around the primary focus, and rarely metastasizes to distant organs. Bone (about 35%) is the most common distant metastatic organ in advanced urothelial carcinoma and the vertebrae is the most common site (about 40%) in all cases of bone metastasis.[ In our case, the first complaint of the patient was bone-ache, and further examination found that the urothelial carcinoma had occurred widely in bone metastases, which conformed with the general characteristics of urothelial carcinoma. However, it was quite rare to find metastatic lesions of bone, liver, and lung, rather than lesions of the urinary system.

Occult carcinoma cases have been described in breast, thyroid, and genitourinary system cancers (Table 1). Occult breast cancer accounts for 0.3% to 1% of all breast cancers, and the first manifestation is axillary nodal metastasis.[ Thus, it would be expected that occult primary breast cancer treatment would focus on local control of axillary disease, and the vast majority of patients underwent axillary lymph node dissection. Occult thyroid carcinoma includes papillary, follicular, and medullary carcinoma, with papillary carcinoma being the most common subtype.[ It was reported that occult thyroid carcinoma is popular (with proportion of 36%) in autopsy specimens.[ Occult genitourinary system tumors mainly include ovarian, cervical, renal, and prostate cancer but are rarely reported. The incidence of sporadic multifocal renal cell carcinoma (both manifest and occult) ranges from 5.3% to 25% where occult multifocality accounts for the majority.[ Prevalence of occult prostate cancer is >30% in men over 50 years old and >60% to 70% in men over 80 years old.[ Clinical data suggest that >30% of men without prostate cancer history have occult prostate cancer.[ The major treatment for occult genitourinary system tumor is resection.[ As far as we know, this case is the first report of occult urothelial carcinoma. However, the biological mechanism of the unusual phenomenon is not clear yet. Occult carcinoma is different from that of the carcinoma of unknown primary (CUP). The former can identify the origin of the primary tissue or organ by pathological examination, whereas the CUP cannot. However, judging from this case, poor prognosis is a common challenge.[ Proper identification of the primary tumor origin is essential for determining a correct therapeutic strategy.

Table 1

Occult cancer data from previous reports.

In addition, we acquired genetic alteration information of the cancer cells by next generation sequencing (NGS) analysis of the patient's liver cancer specimens. Seven genetic variants with clinical significance, including MET amplification, STK11 M51Ifs∗106 exon1, KRAS G12C exon2, NCOR1 S1952∗ exon37, RBM10 E832∗ exon22, SETD2 Q2030∗ exon13, and SUFU c.1022+1G>T were detected in 450 cancer related genes.

Tyrosine kinase c-MET, encoded by the METa proto-oncogene, is a receptor of hepatocyte growth factor (HGF).[ C-MET is mainly expressed in epithelial cells, and its ligand HGF is expressed and released by surrounding mesenchymal cells.[ In a normal physiological state, the combination of HGF and c-MET can activate a variety of downstream signaling pathways, including RAS/RAF/MAPK, PI3K/Akt/mTOR, SRC/FAK, and JUN, which can lead to the cell cycle process, proliferation, movement and migration, survival and cell transformation.[MET gene mutations or amplification have been found in many human cancers, most commonly seen in lung and breast cancer. It can activate c-MET signaling and promote uncontrolled cell proliferation and tumor metastasis.[MET gene amplification is often found in metastatic tumors, suggesting that it mainly plays a role in the process of tumor metastasis.[MET gene variation (amplification or mutation), combined with overexpression of the protein, occurs in about 2% to 10% of bladder cancer cases and is associated with bladder cancer metastasis and poor survival.[ In this report, the extensive multiple organ metastasis (including liver, bone, and lung) of occult urothelial carcinoma might be related to MET amplification. Abnormal activation of MET can be inhibited by specific targeted inhibitors, such as cabozantinib and crizotinib. Cabozantinib can inhibit the proliferation of gastric cancer cell lines with MET amplification, but it is not effective for cell lines without MET amplification.[ A lung adenocarcinoma patient carrying MET gene amplification and 14 exon skipping mutation achieved complete remission after being treated with cabozantinib.[

The protein encoded by the STK11 gene belongs to serine/threonine kinase family and functions as a tumor suppressor.[ In this report, the STK11 M51Ifs∗106 mutation can be described as the 51st codon of methionine changed into isoleucine and the 106th codon becoming a stop codon in the new open reading frame. This mutation caused premature termination codons (PTCs) and produced new transcripts that can be degraded by an mRNA surveillance pathway termed nonsense-mediated mRNA decay (NMD). Although there are no anti-tumor drugs targeting the STK11 gene, the inhibition of mTOR can be used as a treatment for cancer patients with STK11 dysfunction. In the STK11–/– mouse breast cancer tumor model, mTOR inhibitor AZD8055 can inhibit tumor growth successfully.[ A patient with metastatic atypical pituitary adenomas with STK11 F298L variation was treated with radiotherapy and everolimus, and MRI and positron emission tomography and computed tomography showed stable disease (SD) over 6 months.[ Another study involving a Peutz-Jeghers syndrome associated advanced pancreatic cancer patient, achieved partial response (PR) with the treatment of everolimus.[ In this case, based on the mutation of STK11 M51Ifs∗106, the patient was treated with everolimus, but it was not very effective and the patient's condition continued to deteriorate. We considered that this might due to the severity of illness. In addition, the patient carried a variety of tumor related gene mutations. Under these circumstances, choosing a treatment strategy with a combination (such as imimols + cabozantinib) or crizotinib alone still needs further study.

KRAS belongs to the RAS family and is one of the most mutated oncogenes in human tumors. KRAS mutations mainly occur in the 12th, 13th and 61st codons, which may affect RAS mediated guanosine triphosphate (GTP) hydrolysis and lead to the continuous activation of RAS, promoting the malignant transformation of cells.[ G12C is one of the most common mutations in KRAS located in the 2nd exon. The glycine or proline of the 12′ position can maintain the alpha helix structure in the GTP binding domain, which is crucial to inhibit the function of KRAS in promoting cell carcinogenesis.[ Compared with the mutation of position 13, the mutation of the 12th position has a stronger ability to resist cell death and promote transformation of cancer cells.[ Although there is no anti-tumor drug targeting the KRAS gene, MEK inhibitors and PI3K pathway inhibitors have a certain therapeutic effect in treating cancer patients with KRAS mutations.[

Both enhanced and blocked functions of the NCOR1 gene have been reported in tumors. The expression level of NCOR1 protein in gastrointestinal stromal tumor is lower than that in normal gastric tissue, and it is significantly correlated with patient survival.[ However, in breast cancer, patients with a high expression of NCOR1 have a better prognosis than those with low expression.[ In this case, the patient with occult urothelial carcinoma had a NCOR1 S1952∗ mutation which resulted in protein truncation. The truncated NCOR1 lost the functional domain of the C1D interaction zone, RARA interaction region, and ID1 and ID2 region, meaning that this mutation may affect the normal function of NCOR1. It was speculated that the expression or function of NCOR1 may be inhibited during the development of urothelial carcinoma. Presently, there is not an anti-cancer drug targeting the NCOR1 gene.

RBM10 encodes a RNA-binding protein and regulates the alternative splicing of NUMB which is the antagonizing modulator of the Notch signaling pathway, and displays negative effects on the proliferation of cancer cells. The deletion or mutation of NUMB frequently occurs in lung cancer.[RBM10 mutation often coexists with known drive gene mutations.[ In this case, the mutation of RBM10 E832∗, as a first reported novel mutation, produced a truncated protein, which lost the G-patch domain (858–904 amino acid residues). The mutation of RBM10 E810∗, which has been reported in lung adenocarcinoma, also produced a truncated protein with G-patch domain deletion.[ It was suggested that RBM10 E832∗ might be a harmful mutation affecting the normal function of RBM10 protein. To date, an anti-cancer drug targeting the RBM10 gene has not been found.

The mutations or translocations of the histone methyltransferase SETD2 are found in leukemia and solid tumors.[ There are many variants of SETD2 mutated at different sites in the same tumor and this will have a selective advantage in the process of renal cell carcinoma.[ In this case, the SETD2 Q2030∗ mutation caused the glutamine of position 2030 mutated to stop codon. The C-terminal 2030–2564 amino acid residue of the truncated SETD2 protein contains the region interacting with POLR2A (the 2457–2564 amino acid residue).[ It was suggested that the variation of SETD2 Q2030∗ might affect the normal function of SETD2. There is no anti-cancer drug targeting the SETD2 gene at present.

As a tumor suppressor, loss of function of SUFU can increase the activity of Hedgehog (Hh) signaling. Abnormal activation of the Hh pathway is associated with tumor formation in various tissues.[SUFU c.1022+1G>T mutation makes the G base in the splicing donor site into a T, which might cause abnormal mRNA splicing. This mutation has been reported as either a germline mutation or a somatic variation in Gorlin syndrome 136 and medulloblastoma.[ Real-time reverse transcription polymerase chain reaction confirms that SUFU c.1022+1G>T mutation causes SUFU exon 8 skipping, and forms a stop codon in the 9th exon. This incomplete protein loses binding ability to GLI or GLI2, and results in the accumulation of GLI in the nucleus and, therefore, cannot inhibit GLI mediated transcriptional activation.[ There is no anti-cancer drug targeting the SUFU gene at present.

Based on gene test results, the patient was treated with everolimus, an inhibitor that indirectly targeted STK11, but the effect was poor. The patient's condition continued to deteriorate until he passed away. This generates further questions as to whether or not it is necessary to use a combination of targeted drugs for cancer patients with multiple genetic variants.

Conclusion

This patient was diagnosed with occult urothelial carcinoma by pathological evaluation and networking between the clinician and pathologists. In addition, the 7 gene alterations of liver cancer tissue were detected by NGS analysis. From this case report, we can increase our knowledge of occult urothelial carcinoma and provide insight into combining multiple targeted drugs for the treatment of cancer patients with multiple driven genes.

Author contributions

Conceptualization: Bixun Li.

Data curation: Kunpeng Bu, Yang Lu, Juan Zhao.

Investigation: Kunpeng Bu, Yang Lu, Juan Zhao.

Supervision: Bixun Li.

Writing – original draft: Kunpeng Bu.

Writing – review & editing: Zeyan Shi.