Radical nephrectomy and regional lymph node dissection for locally advanced type 2 papillary renal cell carcinoma in an at-risk individual from a family with hereditary leiomyomatosis and renal cell cancer: a case report

Hereditary leiomyomatosis and renal cell cancer (HLRCC, Online Mendelian Inheritance in Man accession number 605839) is a recently identified autosomal dominant tumor susceptibility syndrome that is characterized by a predisposition to develop benign leiomyomas of the skin and the uterus (fibroids and myomas), as well as aggressive renal cell cancer with papillary type 2 (pRCC2) or collecting duct histology [1‐3]. The disease-related gene has been identified as fumarate hydratase (fumarase, FH, Online Mendelian Inheritance in Man accession number 136850) located at 1q43. FH encodes an enzyme that is part of the mitochondrial tricarboxylic acid (TCA) cycle involved in cellular energy metabolism and appears to function as a tumor suppressor since its activity is very low or absent in tumors from individuals with HLRCC. HLRCC-associated kidney cancer has distinctive architectural and morphologic features, is particularly aggressive, and tends to metastasize to regional lymph nodes and distant organs early [4]. Therefore, a high detection rate of mutations in HLRCC families may enable early identification of at-risk individuals and allow early initiation of therapy while their tumors are still small. However, it stills remains controversial how we should treat HLRCC-associated kidney cancer [5]. So far, there have been several case reports regarding HLRCC-associated kidney cancer, however, most of those were reporting the mutation analysis of FH, pathological features, and clinical course. Furthermore, to our knowledge, there have been no case reports of the patients of at-risk of HLRCC-associated with kidney cancer who received active surveillance and were treated successfully, and little is known about the relationship between the clinicopathological features and molecular changes associated with targeting therapy in this disease. In the present study, we successfully treated a patient with locally advanced HLRCC-associated pRCC2 by neoadjuvant administration of axitinib and subsequent radical nephrectomy and extended retroperitoneal lymph node dissection.

FH-deficient RCC is characterized by enhanced aerobic glycolysis and increased anti-oxidant response phenotype [6, 7]. Overactivation of phosphatidylinositol 3‘kinase (PI3K), serine/threonine protein kinase B (Akt), and mammalian target of rapamycin (mTOR) pathway has been reported in RCC. Inhibition of Akt disrupts transcription of glucose transporter protein-1 (GLUT1) and its translocation to the plasma membrane to promote glucose utilization independent of an effect on cell proliferation [8]. Phosphorylation at two sites is required for full activation of Akt, since it is phosphorylated by PI3K-dependent kinase-1 (PDK1) at a threonine residue in the catalytic domain (Thr-308) and by PI3K-dependent kinase-2 (PDK2) at a serine residue (Ser-473) in the carboxy-terminal hydrophobic motif [9]. mTOR has dual rapamycin-sensitive (mTOR-raptor complex: mTORC1) and rapamycin-insensitive (mTOR-rictor complex: mTORC2) functions. mTORC1 is activated by PI3K-Akt and it phosphorylates S6 and eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1), thereby promoting translation and protein synthesis. mTORC2 regulates the actin cytoskeleton and also possesses PDK2 activity that phosphorylates Ser-473 at the carboxy-terminus of Akt, which is essential for activation of Akt [10, 11], and mTORC2-pAkt(Ser-473) signaling affects energy metabolism and cell survival [12]. Activation of Akt may increase cell viability after inhibition of mTORC1 [9]. Hypoxia-inducible factor (HIF)1α expression is dependent on both raptor and rictor, whereas HIF2α expression only depends on rictor, with HIF2α and mTORC2 being more important in RCC [13]. Moreover, phosphorylation of Ser-473 in Akt is considered to be key molecular step in the progression of RCCs and could be a potential target [10, 11, 14]. Furthermore, available reports support HIF-dependent pseudo-hypoxia manner as the mechanism of tumorigenesis in HLRCC [15]. In FH-deficient kidney cancer cells, increased fumarate inactivate prolyl hydroxylases, leading to stabilization of HIF, and increased HIF target genes such as GLUT1, vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and transforming growth factor (TGF)α, which facilitate tumor growth [7, 16].

On the other hand, HIF-independent manner has been recently reported [17]. FH-deficiency leads to succination of Kelch-like ECH-associated protein 1 (Keap1), stabilization of nuclear factor E2-related factor 2 (Nrf2), and induction of stress-response genes including HMOX1, which is important for the survival of FH-deficient cells. The Keap1-Nrf2 pathway is the major regulator of cytoprotective responses to oxidative and electrophilic stress. Although cell signaling pathways triggered by the transcription factor Nrf2 prevent cancer initiation and progression in normal and premalignant tissues, in fully malignant cells Nrf2 activity provides growth advantage by increasing cancer chemoresistance and enhancing tumor cell growth, and high Nrf2 protein level is associated with poor prognosis in cancer [18]. FH loss results in Keap1 inactivation and Nrf2-dependent activation of anti-oxidant pathways [19, 20].

Axitinib is a potent, selective, second-generation inhibitor of VEGF receptor (VEGFR) 1, 2, and 3 that blocks VEGFRs at sub-nanomolar drug concentrations [21], and relative potency of axitinib is 50–450 times greater than that of the first-generation VEGFR inhibitors like sorafenib or sunitinib [22]. In order to investigate the roles of Akt-mTOR pathway and Nrf2 anti-oxidant response element transcription pathway in HLRCC-associated kidney cancer, we examined the expressions of phosphorylated-Akt (Ser-473) (pAkt(Ser-473), phosphorylated-Akt (Thr-308) (pAkt(Thr-308), phosphorylated-S6 ribosomal protein (Ser-235/236) (pS6), and Nrf2 in surgically resected samples. We also investigated FH mutations by sequencing the coding exons and intron flanking regions in both blood and tumor samples by targeted next-generation sequencing analysis. Such information might be useful to understand the signaling pathway in HLRCC-associated kidney cancer from a molecular point of view.

A 48-year-old woman (III-8, a sister of the proband from this HLRCC family) underwent abdominal ultrasonography annually at a local clinic after 2007, and presented with a left renal mass detected by an ultrasonography and was introduced to our hospital in March 2013 (Additional file 2: Figure S1).

She had undergone enucleation myomectomy for uterine leiomyomatosis at the age of 29 years at another hospital, while hysterectomy had been performed for recurrence large uterine leiomyomatosis at the age of 39 years at other hospital. In 2007 (when she was 40), her sister was diagnosed with HLRCC having a novel FH mutation at 241,671,938 bp (C574T) by direct sequencing of the FH gene from leukocyte DNA. Her sister subsequently died of HLRC-associated advanced renal cancer. In 2007, sequencing of DNA extracted from blood cells of this patient confirmed that she also had the same FH mutation as her sister [23]. After 2007, we recommended that 13 members of this family with the FH mutation should receive active surveillance by annual imaging (abdominal plain computed tomography (CT) or ultrasonography) at a convenient clinic (Fig. 1).

Laboratory tests revealed moderate anemia (hemoglobin: 9.3 g/dl) and elevation of serum C-reactive protein (CRP: 3.19, normal < 0.3 mg/dl). Karnofsky performance status (KPS) was 100 %. Plain CT scans obtained at our hospital showed a left renal tumor with a diameter of 7 cm and involvement of multiple regional para-aortic lymph nodes, but no distant metastases (cT3aN1M0) (Fig. 2a). Positron emission tomography (PET) showed fluorine-18-deoxyglucose (FDG) accumulation in the renal tumor and the metastatic lymph node and the maximum standardized uptake value (SUVmax) was 15.3 and 7.5, respectively (Figs. 2b,c).

Her risk classification for renal cancer was intermediate risk according to the Memorial Sloan-Kettering Cancer Center (MSKCC) criteria. However, the prognosis of patients with HLRCC-associated renal cancer, in particular those with extrarenal involvement, is extremely poor. Furthermore, her tumors showed a different imaging pattern from that of typical clear cell RCC (Additional file 2: Figure S1), and the histology of the renal cancers in her relatives was non-clear cell RCC (undifferentiated RCC in her mother, pRCC2 in both her sister and maternal cousin). Thus, the tumor of this patient seemed likely to be non-clear cell carcinoma, but we did not perform needle biopsy to avoid dissemination of cancer cells.

In order to decrease the tumor burden and improve the feasibility of surgery, we selected preoperative treatment with a multi-targeted tyrosine kinase inhibitor (TKI). In comparison to first-generation TKIs targeting the VEGFR, axitinib is a potent second-generation inhibitor of VEGFRs with a higher affinity for tyrosine kinase and achieves stronger inhibition of kinase activity with fewer adverse effects such as thrombocytopenia. Additionally, first-generation inhibitors block other targets, such as PDGF receptors (PDGFR), KIT (cluster of differentiation 117: CD117), b-rapidly accelerated fibrosarcoma (RAF), and Fms-like tyrosine kinase 3 (FLT-3), which are not substantially inhibited by axitinib. These off-target activities might contribute to the adverse effects of the first-generation inhibitors, suggesting that more specific inhibitors of VEGFR such as axitinib might have an enhanced therapeutic window. We recently successfully treated a patient who had a large right RCC showing sarcomatoid differentiation that directly invaded the duodenum and inferior vena cava with regional lymph node involvement. In this patient, radical right nephrectomy, cavotomy with thrombectomy, and pancreatoduodenectomy were successfully performed after administration of axitinib as first-line neoadjuvant therapy without severe toxicity [24].

We selected axitinib as preoperative molecular-targeting therapy to decrease the tumor size before surgery with good tolerability. Administration of axitinib starting at 5 mg/day was scheduled for four to six weeks before radical surgery involving left nephrectomy and extended retroperitoneal lymph node dissection (para-aortic and aorto-caval nodes). After 1 week, the dose of axitinib was increased to 14 mg/day. After four weeks of total dose of axitinib of 329 mg (5 mg/day for continuous 7 days and 14 mg/day for following continuous 21 days), there were no apparent adverse events of > grade 3, excluding headache and hypertension (systolic blood pressure > 200 mmHg). Tumor shrinkage and a decrease of SUVmax were observed (Figs. 2d-f). Subsequently, we successfully carried out radical left nephrectomy and extended retroperitoneal lymph node dissection (para-aortic and aorto-caval nodes). Macroscopically, the tumor was an invasive whitish-yellowish mass with partial necrosis. Pathological examination confirmed pRCC2 with Fuhrman grade 3 differentiation (pT3apN1M0). The pathological effect of axitinib was grade 2 (i.e., two-thirds necrosis of the tumor). The patient has been receiving axitinib at 5 mg/day in the manner of one cycle of one week (5 days on - 2 days off) as adjuvant therapy for 33 months, and remains well with no evidence of recurrence at 33 months after the operation.

In the present study, next-generation sequencing revealed that FH mutation and loss of the second allele were completely identical between blood and tissue samples from this patient and her sister (III-9) who died of advanced HLRCC-associated kidney cancer, indicating that FH-deficient RCC is a unique neoplasm that progresses directly by FH mutation.

In FH-deficient RCC, oxidative phosphorylation is impaired and the cells undergo a shift to aerobic glycolysis, consistent with the Warburg effect [6, 7]. The conversion of glucose metabolism from oxidation to glycolysis, the Warburg effect, is one of the representative strategies for generation of adenosine triphosphate (ATP) in cancer cells [27]. Reprogramming of energy metabolism, the conversion of glucose metabolism from oxidation to glycolysis, the Warburg effect, can now be viewed as one of the “hallmarks of cancer” [28]. RCC is characterized by impaired oxidative phosphorylation and a metabolic shift to aerobic glycolysis, a form of metabolic reprogramming. In particular, HLRCC-associated kidney cancer cells have lost the ability to completely cycle through the TCA cycle, due to the loss of FH enzyme activity, and have effectively lost the ability to perform oxidative phosphorylation indicating that these cancers exist in a state of enforced dependence upon glycolysis and represent a notable example of the Warburg effect [7]. Thus, HLRCC-associated kidney cancer might be a clinical model to study energy metabolism deregulation, as well as developing new targeted therapeutic approaches for TCA cycle enzyme-deficient cancers [29]. In this HLRCC-associated pRCC2 case, surgically resected cancer tissue showed high expression of pAkt (Ser-473) and pAkt (Thr-308), as well as very low expression of pS6, indicating that we should study the roles of mTORC2-Akt signaling in HLRCC-associated kidney cancer from the metabolic point of view in more patients in the forthcoming study.

On the other hand, FH-deficient RCC is also characterized by increased oxidative stress and elevated levels of reactive oxygen, thus effective anti-oxidant response is critical for continued growth [6, 7]. In this study, subsequent immunohistochemical staining for Nrf2 protein in the HLRCC-associated pRCC2 also showed intense positive staining (III-9 and −4). At the same time, normal kidney and clear cell RCC tissues showed negative staining. Our findings were consistent with those by previous study [20], indicating that Nrf2 was indeed activated in these FH-deficient RCC tissues. Thus, FH-deficient RCC appeared to be linked to increased expression of anti-oxidant genes with accompanied by the accumulation of Nrf2. In this HLRCC-associated pRCC2 case treated with neoadjuvant axitinib, some of viable cancer cells showed weak reaction for anti-Nrf2 antibodies compared to the tumors of the proband (III-9) and maternal cousin (III-4) who received no prior treatment in which much of cancer cells showed diffusely strong reaction, indicating that axitinib might suppress the Nrf2 pathway by unknown mechanism. Since we do not have preoperative tumor tissues and stored blood samples, we could not compare the VEGF levels between before and after administrating axitinib in the present study. However, in addition to pAkt, pS6, and Nrf2, we should also analyze repeatedly the VEGF, GLUT1 or HIF using tissue and blood samples in order to correspond to dynamic change of the tumor and the general condition which is continuously changed with time in the future.

From a molecular point of view, insight in the cellular pathways involved in pathogenesis of HLRCC might lead to specific options for early diagnosis and targeted therapies. However, since this is only a single case report about our experience with surgery after axitinib treatment for HLRCC-associated kidney cancer, the results should be interpreted with consideration of such limitations and definite conclusions cannot be obtained. While an investigation of the usefulness of axitinib for preoperative or neoadjuvant therapy in patients with locally advanced RCC is now ongoing, availability of axitinib for adjuvant therapy for RCCs has not yet elucidated. Although there was no detailed information regarding histological type of papillary RCC in a previous study using other kinase inhibitors in papillary RCCs, sunitinib seems to be more effective than sorafenib [30]. Furthermore, a phase II trial of bevacizumab and erlotinib in patients with advanced HLRCC-associated pRCC2 as well as sporadic pRCC2 is currently under way (NCT01130519). Therefore, it would be great to conduct in vitro studies using established two HLRCC kidney cancer lines, UOK262 and UOK268 [29, 31].

It is thought that early diagnosis and provision of definitive therapy is the best way to reduce the tumor burden as rapidly as possible. Unlike other hereditary renal cancers, HLRCC-associated kidney cancer is an aggressive tumor characterized by metastasis to regional and distant lymph nodes [4]. A recent report regarding HLRCC-associated kidney cancer recommended that surveillance should preferably be annual abdominal MRI, and that treatment of renal tumors should be prompt and generally involve wide surgical excision with consideration of retroperitoneal lymph node dissection [5]. Thirteen at-risk individuals from this HLRCC family had received active surveillance by annual imaging (abdominal plain CT or ultrasonography) at a convenient clinic, however, as shown in Fig. 2 and Additional file 2: Figure S1, we may overlook the smaller tumors associated with HLRCC in plain CT and/or ultrasonography because of low contrast of the tumors to the normal kidney. Although enhanced CT may be useful, given the radiation exposure and the adverse effect of contrast medium, magnetic resonance imaging (MRI) seems to be suitable for surveillance as recommended [5]. Subsequently, we would ask their attending physicians at a convenient clinic annual MRI imaging study every twelve months.

Currently, surgical intervention is the only therapy available to patients with HLRCC­associated kidney cancers. The patient underwent radical left nephrectomy and extended retroperitoneal lymph node dissection (para-aortic and aorto-caval nodes) after administration of preoperative axitinib. It is difficult issue how we should follow the patient. The patient undergoes imaging examination of chest CT and abdominal MRI at least every three months and PET scan every six months, and remains well with no evidence of recurrence at 33 months after the operation. The patient is receiving axitinib as adjuvant therapy, and we would like to decrease the dose of axitinib gradually. Careful patient selection and meticulous surgical technique are essential in the treatment of patients with HLRCC-associated kidney cancer, and these points should be further emphasized in the era of targeted therapy. Collection of HLRCC-affected family data dedicated to monitoring of patients will provide information on clinical variability and outcome measures that will allow clinicians to adjust diagnostic criteria and management recommendations. It is our hope that more patients with HLRCC-associated kidney cancer will be able to achieve a better outcome.

The patient and many of individuals in this family signed a consent form that was approved by our institutional Committee on Human Rights in Research for the analysis of germline and somatic DNA. On the other hand, some individuals could not come to our hospital and we could not get approval for DNA analysis, however, all participants in this family give approval for the publication of their clinical and other details in written informed consent. Furthermore, if the participant has died, then consent for publication has been sought from the next of kin of the participant. Thus, we have 'consent to publish' from all the individuals represented in the family tree in Fig. 1. This study was conducted in accordance with the Helsinki Declaration and was approved by the Dokkyo Medical University Hospital ethical review board.

Written informed consent was obtained from the patient and their relatives to sequence their DNA and for the publication of this case report and any accompanying sequence data or images. A copy of the written consent is available for review by the Editor of this journal.