Genomic case report of a low grade bladder tumor metastasis to lung

Bladder cancer is the fourth most common cancer in men, with an estimated 79,030 new cases and 16,870 deaths expected in the United States in 2017 [1]. Unlike many other cancers, bladder cancer’s mortality rates and treatment options have changed very little in the last 30 years [1, 2]. Twenty-five percent of bladder cancers are muscle-invasive and life-threatening at diagnosis [3]. Non–muscle-invasive bladder cancers can recur [4], but they rarely metastasize to distant organs [4].

We present a genomic case report of a man diagnosed with low-grade bladder cancer and a separate, concurrent high-grade cancer of the upper urinary tract in 2009. The tumors were surgically removed. In 2016, the patient had a lung metastasis that, on pathology, resembled the low-grade bladder tumor that had been removed in 2009. Low-grade bladder tumors rarely metastasize to distant organs, so we used exome sequencing on the patient’s three tumor biopsy specimens (from 2009 and 2016) to investigate the relationship between the metastasis and the initial low-grade and high-grade tumors, to study tumor progression, and to identify therapeutic vulnerabilities. We also carried out primary culture of the patient’s lung cancer cells to study drug response.

In July of 2009, a 56-year-old man with a 40 pack-year smoking history presented with a low-grade papillary urothelial (transitional cell) carcinoma at the right ureteral orifice (primary bladder tumor). He also had a high-grade urothelial carcinoma of the renal pelvis with focal squamous differentiation and extensive renal parenchymal involvement. His right ureter was filled with tumor but did not show intramuscular invasion. Venous and lymphatic invasion of this tumor was absent. The patient first underwent transurethral resection of the bladder tumor (TURBT) of right ureteral orifice for the bladder carcinoma and underwent an ureteroscopic resection of the right ureter. The final pathology report characterized the bladder tumor as low-grade, non-invasive transitional cell carcinoma, and the ureteral resection demonstrated low-grade transitional cell carcinoma. In August 2009 a month later, the patient’s upper urinary tract tumor was removed by hand-assisted laproscopic neproureterectomy. The final pathology on this tumor was pT3 pN0 with negative margins. The patient then underwent an intense course of 6 rounds of bacillus Calmette-Guérin (BCG) treatment in an adjuvant setting, followed by maintenance BCG treatment for 3 years.

In 2016 the patient returned with a lung tumor that, on pathologic evaluation, resembled the low grade right ureteral orifice bladder tumor (transitional cell) from July 2009. The lung tumor was surgically removed. Because low-grade bladder tumors rarely metastasize to distant organs, we consented the patient for an Institutional Review Board–approved research study to investigate the origin of the lung metastasis and also to identify genetic changes that could represent a therapeutic target for any future recurrence or metastasis.

Hematoxylin and eosin (H&E) review of the formalin-fixed paraffin-embedded

(FFPE) tumor biopsy specimens showed more than 70% tumor tissue within all the samples sent for exome sequencing (Fig. 1a). The identified variants are listed in Additional file 1: Table S1, Additional file 2: Table S2, Additional file 3: Table S3, Additional file 4: Table S4. We focused on missense and nonsense somatic mutations present in the three tumor samples. Multiple variants were shared by these three tumors, and others were unique to each individual tumor, as shown by the Venn diagram and heat map (Fig. 1b, c).

Phylogenetic analysis indicates that the lung metastasis and primary bladder tumor are most closely related, and that the upper urinary tract tumor may have developed first (the distances between normal tissue and upper urinary tract tumor and normal tissue and primary bladder tumor are very similar, 168 gain/loss of single nucleotide variants (SNVs) versus 171 (Fig. 1d, Additional file 5: Table S5). No mutations in a known oncogene or tumor suppressor gene are shared by all three tumors; however, the primary bladder tumor and the lung metastasis share known oncogenic mutations frequently found in bladder tumors, such as mutations in the KMT2D and RXRA genes. The mutational signatures, histomorphology, and distinct anatomic sites indicate that the upper urinary tract tumor and the primary bladder tumor likely are unrelated. Conversely, based on mutational profile and our model of tumor evolution, the primary bladder tumor and the lung metastasis may be related.

With evidence that the lung metastasis may be derived from the primary bladder tumor, we analyzed the somatic variants present in the primary bladder tumor and the lung metastasis that might be responsible for tumor initiation and progression. Table 1 lists some of the important variants shared between the primary bladder tumor and the lung metastasis and also variants that are unique to the lung metastasis. The primary and the metastatic tumor have mutations in the KMT2D and RXRA genes. KMT2D encodes the protein histone-lysine N-methyltransferase 2D which is a tumor suppressor [5, 6]. KMT2D is mutated in 28% of bladder tumors [7]. RXRA, which encodes retinoid X receptor alpha (RXR-alpha), is mutated in 10% of bladder tumors [7]. The RXRA S427F mutation present in these patient tumors is a hotspot mutation that predominantly occurs in urothelial tumors [7‐10]. Initial studies show that this particular RXRA mutation regulates lipid metabolism via peroxisome proliferator-activated receptor gamma (PPARG) activation [8]. Among the mutations unique to the lung metastasis, a clinically actionable, activating mutation in mTOR (C1483F) was identified. This particular MTOR mutation is also present in the primary bladder tumor (Table 1), but at a very low frequency (1%). This C1483F mTOR mutation has been shown to activate mTOR downstream signaling via phosphorylation of p70-S6K and 4E-BP1 [11]. Development or selection of a subpopulation of cells with this activating MTOR mutation may be the driving event for lung metastasis within the primary bladder tumor.

We carefully examined the mutations present in the patient tumors based on the base substitutions C > A, C > G, C > T, T > A, T > C, T > G to identify how the patient tumors correlate with known mutational signatures representative of various biological processes. Figure 2a shows the mutational landscape in all the three tumors. More C > T and T > C mutations were found in the three tumors. Next we developed a mutational signature for the patient by combining all the mutations present in these three tumors (Fig. 2b). This mutational signature is characterized by predominantly C > T and T > C mutations (Fig. 2c). This patient’s mutational signature resembles published mutational Signature 1A/B and Signature 5 [12]. Mutational signature 1A/B is related to the relatively elevated rate of spontaneous deamination of 5-methyl-cytosine, which results in C > T transitions and which predominantly occurs at NpCpG trinucleotides [12]. Signature 1A/B exhibits strong positive correlations with age in majority of cancers [12]. Signature 5, characterized by C > T and T > C mutations, is caused by tobacco carcinogens [12]. Our patient had a 40 pack-year smoking history, which suggests that tobacco use played a role in initiation of his tumors.

Using the Drug Gene Interaction Database [13], we identified candidate drugs targeting 12 of the 100 genes with SNVs in the lung tumor (Additional file 6: Table S6). Several FDA-approved anti-cancer therapies were identified, including the RXRA agonist bexarotene and mTOR inhibitors, such as everolimus. We note that this analysis does not consider whether the variant is activating or deleterious, and all candidate therapies need to be evaluated. Primary culture of the lung metastasis was established in the laboratory. Since the lung metastasis has an activating MTOR mutation, we treated these cells with mTOR inhibitor everolimus at two concentrations (10 and 50 nM). The treatment showed a marked inhibition of mTOR activity and downstream signaling via two of its effectors, p70 S6K and 4E-BP1, at both concentrations (Fig. 3a); however, AKT activity increased with everolimus treatment (Fig. 3a). AKT can function both upstream and downstream of mTOR, but an increase in AKT activity could be a mechanism of resistance to the mTOR inhibitor.

Cytotoxicity study showed that at very low concentration (0.1 nM) everolimus reduces viability of these cells by about 60% (Fig. 3b), but even at a high concentration 40% of cells remain viable, indicating a cell population resistant to the drug.

We present a genomic case report of rare distant metastasis of a low grade bladder tumor. Phylogenetic analysis revealed that the primary low grade bladder tumor and the lung tumor are more closely related, and shared several known oncogenic mutations frequently observed in bladder tumors. Both these tumors presented with KMT2D and RXRA gene mutations. KMT2D is a known tumor suppressor that is mutated in a quarter of bladder tumors [5‐7] and also regulates gene transcription [6]. We speculate that the loss of function of this tumor suppressor (KMT2D mutation seen here is likely inactivating) is an important driver for these tumors. Another important variant present in both these tumors is a mutation in the RXRA gene. The S427F RXRA mutation predominantly occurs in bladder tumors [9, 10]. Preliminary studies have shown that this particular RXRA mutation regulates RXRA and PPARG interaction [8]. There is increased activation of PPARG in tumors carrying this particular RXRA mutation, which drives lipid metabolism [7, 8]. We believe that RXRA S427F mutation may play an important role in tumor initiation and progression. Although outside the scope of this study, the role of this RXRA mutation in bladder cancer should be characterized in detail as another potential therapeutic avenue for patients with advanced bladder cancer.

The lung metastasis has an activating mutation in the MTOR gene (C1483F). This particular C1483F mutation was presented with a variant allele frequency (VAF) of 19% (of 172 reads) in the metastatic tumor and has a VAF of 1% (of 160 reads) in the primary bladder tumor. This indicates that a small population of cells in the primary tumor probably developed this particular MTOR mutation and this clonal population likely migrated and was able to seed in the lung. Thus, we think that the activating MTOR mutation could be one of the important drivers that lead to distant metastasis, perhaps thru evolution of a subclone of the low-grade bladder tumor. Treating the lung metastasis with everolimus showed a marked decrease in p70 S6K and 4E-BP1 activity, but a concurrent increase in activated AKT. Cytotoxicity assay showed that 60% of these cells are sensitive to everolimus treatment.t. We speculate that increase in AKT activity with the drug treatment could be a mechanism of resistance to mTOR inhibition and could explain the 40% viable cells post-treatment.

Finally, we tried to understand whether the mutations present in these tumors represent a known oncogenic process. The mutational signature in this patient was dominated by predominantly C > T and T > C mutations—the hallmark of a mutational signature caused by tobacco carcinogens [12]—suggesting that our patient’s tobacco use was perhaps responsible for his tumors.

Here we used genomic analysis to study cancer evolution and present a rare case where a low grade bladder tumor metastasis to the lung. We used phylogenetic analysis to prove that the low grade bladder tumor and the lung metastasis are closely related. We also conclude that this patient may benefit from an mTOR inhibitor in case of disease recurrence since his metastatic disease carries an activating mTOR mutation and cancer cells cultured from his lung tumor showed vulnerability to mTOR inhibition. This case report points to the importance of genomic analysis of patient tumors to understand tumor biology, evolution and for personalized patient care.