A 24-year-old woman was referred from an outside hospital where she had presented a few days earlier with increasing abdominal discomfort, bloating, anorexia and weight loss. She was found to have a 45 cm complex cystic mass occupying most of her abdomen () and a hemoglobin level of 7 g/dl. The patient was transferred for definitive treatment. No liver or lung metastases were observed. She underwent arteriography with successful transarterial alcohol embolization of a left anterolateral artery that was presumed to represent the left renal artery. During the procedure, 1.6 l of a dark, rusty fluid with a small amount of fatty-appearing debris was drained, and biopsies were performed. The biopsy material was necrotic, and no definitive pathological diagnosis could be established. Her hemoglobin level stabilized after the procedure. Contrast-enhanced brain MRI showed no evidence of brain metastasis. 4 weeks later, the patient was electively readmitted for surgical resection. The mass was adherent to the mesentery, spleen and pancreas, which necessitated partial colectomy, splenectomy and partial pancreatectomy.
Figure 1 CT and 18FDG-PET/CT imaging throughout treatment. CT performed a | at presentation, b | after surgery and c | after treatment with mTORC1 inhibitors. Arrows show the largest paraspinal mass. 18FDG-PET/CT performed d | before 2DG therapy, e | after treatment (more ...)
Pathological studies revealed two tumor masses attached to each other measuring 45 cm and 13 cm at their largest diameter. Microscopic analyses showed a high-grade adenocarcinoma with fibrovascular papillae lined by stratified, large, pleomorphic cells with eosinophilic cytoplasm and large, prominent nucleoli (). The tumor invaded into the pancreas and involved multiple lymph nodes, including pericolonic nodes. Immunohistochemical analyses showed the tumor cells to be strongly positive for vimentin, focally positive for CD10, and negative for cytokeratins 7 and 20. Overall, the diagnosis was most consistent with a type-2 papillary renal cell carcinoma (pRCC-2).
Figure 2 Representative photomicrographs of tumor sections, showing fibrovascular papillae lined by stratified, large, pleomorphic cells with eosinophilic cytoplasm and large, prominent nucleoli. Hematoxylin and eosin staining, a | original magnification ×200, (more ...)
The patient recovered well from the surgery, and received meningococcal, pneumococcal and Haemophilus influenzae
type b vaccines prior to discharge. Approximately 1 month after surgery, CT of the chest, abdomen and pelvis showed several discrete enhancing nodules in the abdomen and paravertebral area that had increased in size and measured up to 3 cm in diameter (). Temsirolimus, an inhibitor of mammalian target of rapamycin complex 1 (mTORC1; also known as mTOR) that has shown unrestricted activity against RCC,1
was started. Temsirolimus was given at the standard dose of 25 mg intravenously once a week; however, the patient had difficulty attending weekly infusions, and, after two doses, the treatment was switched to daily, oral everolimus 10 mg. Everolimus, like temsirolimus, is approved for renal cancer, and although studied in a different context,2
both drugs are sirolimus analogs and are likely to act in the same manner. 3 months later, CT showed a modest reduction in the size of the metastases (). The largest paraspinal mass was treated with additional stereotactic radiation therapy.
The patient had no family history of cancer and both her parents were alive and well, but, given her young age and advanced presentation with an uncommon tumor type, consideration was given to the possibility that a de novo
germline predisposing mutation had arisen. In particular, given the tumor histology, there was a suspicion of hereditary leiomyomatosis and renal cell cancer (HLRCC). HLRCC is a syndrome with an autosomal dominant pattern of inheritance caused by germline loss-of-function mutations in the gene encoding fumarate hydratase (FH).3,4 FH
functions as a classic two-hit tumor suppressor gene,5
and the resulting tumors typically exhibit loss of heterozygosity (LOH).6
HLRCC is characterized by cutaneous and uterine leiomyomas and highly aggressive renal cell carcinomas (typically type-2 papillary tumors).7
although no cutaneous leiomyomas were observed on skin examination, she was noted to have uterine fibroids. The patient was initially uncertain about genetic testing, but she subsequently agreed. Sequencing analyses of DNA from peripheral blood mononuclear cells showed a heterozygous germline FH
mutation (c.1021G>A) (). The mutation resulted in a nonconservative substitution of an evolutionarily conserved residue (Asp341Asn). This variant was not known to represent a polymorphism, and was not found among previously reported mutations.8
Figure 3 FH gene sequencing and enzymatic activity analyses. a | DNA sequence chromatograms from PBMCs and tumor cells. b | FH enzymatic activity in the pRCC-2 vs a panel of five ccRCCs. Data are means ± SE. *P <0.01. Abbreviations: ccRCC, clear-cell (more ...)
Importantly, sequencing studies of tumor DNA indicated LOH with nearly undetectable amounts of the wild-type FH
allele (). In addition, enzymatic assays showed, in comparison to a panel of clear-cell RCC (ccRCC) tumors, very low levels of FH activity (). FH functions as a tetramer,9
and studies of the previously reported crystal structure and reconstitution experiments suggested that the patient’s mutation interfered with oligomerization and that mutant FH did not form stable tetramers (Box 1
Box 1 | Characterization of the FH mutation
To evaluate the effects of FHAsp341Asn, we first examined the crystal structure of the FH holoenzyme (Protein Data Bank ID 3E04). We found that Asp341, an acidic, negatively charged amino acid, was involved in an intramolecular interaction with Lys337, a basic, positively charged residue, and that this interaction was buried deep within the intermolecular interface (). A substitution of Asp341 for Asn, an uncharged amino acid, would leave Lys337 unpaired, resulting in an energetically unfavorable net positive charge in the hydrophobic intermolecular interface, which would be expected to destabilize the tetramer. To determine experimentally whether FHAsp341Asn would form stable tetrameric complexes, the mutation was engineered by site-directed mutagenesis and introduced into FH-deficient UOK262 cells (). In contrast to UOK262 cells in which wild-type FH was introduced, tetramers did not form in FHAsp341Asn-reconstituted cells (). These data suggest that FHAsp341Asn does not form homotetramers, and this observation may explain the lack of FH activity in the tumor.
Abbreviation: FH, fumarate hydratase.
Figure 4 Characterization of the mutant FHAsp341Asn protein. a | Quaternary structure of the human wild-type FH tetramer, color-coded to show each subunit, with a depiction of the intramolecular interaction between the negatively charged carboxylic acid group (more ...) FH
-deficient cells express high levels of glucose transporters,10-12
leading us to hypothesize that 2-deoxy-2-(18
FDG)-PET (which is not recommended for routine RCC evaluation) might be a suitable imaging modality to use in this patient. 18
FDG-PET/CT showed diffuse 18
FDG uptake throughout the abdomen and pelvis, indicative of peritoneal carcinomatosis (). Importantly, the size of tumor implants was such that they would have escaped recognition by CT alone. In addition, a dilated loop of small bowel suggestive of a partial obstruction was noted. Owing to the presence of progressive disease, everolimus treatment was stopped.
There are no established therapies for metastatic FH
-deficient pRCC-2. While pRCC-2 also occurs sporadically, FH
has not been found to be mutated in sporadic tumors;13,14
indeed, whether sporadic and familial pRCC-2 are related entities remains to be determined. Germline FH
mutations are uncommon, and there is no established treatment for metastatic pRCC-2 in HLRCC patients.15
even for sporadic pRCC-2, which may represent a different clinical entity, the role of other RCC therapies, including small-molecule tyrosine kinase inhibitors like sunitinib (which have been largely evaluated against tumors with a clear-cell component) is questionable. Whereas a retrospective study suggested that sunitinib may be active against non-clear-cell RCC,16
a small prospective study in patients with papillary RCC showed very disappointing results.17
In addition, these agents can cause intestinal perforation: although this is a rare occurrence, the risk may be increased in the context of peritoneal carcinomatosis and an impending partial small bowel obstruction. On this basis, and following a discussion with the patient about the situation, we decided to explore alternative treatment options.
We determined that the glycolytic inhibitor 2-deoxy-d
-glucose (2DG) might be beneficial for the patient, on the basis that FH
deficiency disrupts the tricarboxylic acid (TCA) cycle, resulting in profoundly impaired mitochondrial ATP production and a reliance on glycolysis for energy production.12,18,19
There is currently great interest in the development of glycolytic inhibitors, such as 2DG, as cancer treatments.20
2DG was particularly attractive in the current case because the tumor had shown high avidity for 18
FDG, which, like 2DG, is a glucose analog with a substitution of the hydroxyl group at position 2, suggesting that 2DG would similarly accumulate in tumor cells. 2DG is transported into cells by glucose transporters, and, once inside cells, is phosphorylated by hexokinase to 2DG-6-phosphate. However, in contrast to glucose-6-phosphate, the lack of a hydroxyl group at position 2 precludes further metabolism. Thus, 2DG-6-phosphate acts as a competitive inhibitor of glucose-6-phosphate isomerase, which catalyzes the next step in glycolysis, as well as of glucose-6-phosphate dehydrogenase, which is involved in the pentose phosphate pathway.
Two phase I clinical trials have evaluated 2DG in solid tumors.21,22
The studies differed in the maximum tolerated dose, which was reached in one study because of the observation of prolongation of the corrected QT interval (QTc),21
but not in the other.22
However, both studies concurred that 2DG was well tolerated.
As there were no clinical trials of 2DG open, and following approval by the institutional review board, a single patient emergent investigational new drug request was filed with the FDA, and the drug was requested from the investigational new drug holder, threshold Pharmaceuticals (Redwood City, CA).
Owing to the patient’s heterozygous FH
state, and an expected reduction in FH activity, there was a concern that the patient would be at increased risk of 2DG-related toxicities. Furthermore, there have been reports of mutations in patients that not only result in loss-of-function, but that are also dominant-negative and interfere with the function of the wild-type protein encoded by the remaining wild-type allele.23
While our studies suggested that Asp341Asn interfered with tetramerization, suggesting that it may not act in a dominant-negative fashion, further studies would be required to exclude this possibility. As a precaution, 2DG was started at one-eighth of the target dose of 63 mg/kg. 2DG was administered orally once daily after an overnight fast under direct medical supervision. The patient tolerated the first dose without any problems. Given the advanced nature of her disease and concerns about an impending small bowel obstruction, the 2DG dose was rapidly escalated and, within a period of 8 days, the target dose was reached. The patient’s QTc remained within the normal range, and except for grade 1 hypokalemia and hypomagnesemia, no other toxicities were apparent. Unfortunately, however, 1 week after the target dose was reached, the patient developed abdominal pain, nausea and vomiting, and was found to have a complete proximal small bowel obstruction. Exploratory laparotomy revealed diffuse tumor infiltration, but no transition point could be identified. 2DG treatment was stopped, a jejunostomy tube was placed, and the patient was started on total parenteral nutrition (TPN).
2DG did not seem to be effective under the current dosing regimen, and we explored alternative strategies with the use of in vitro
experiments (Box 2
, ). The results prompted us to attempt more-frequent 2DG administration. Once approval for this regimen had been obtained from the FDA, and while the patient was hospitalized and on telemetry, 2DG (63 mg/kg) was given every 8 h for the first 2 days, followed by every 6 h on subsequent days. To minimize 2DG competition by circulating glucose, carbohydrates were eliminated from the TPN infusion and calories were provided in the form of fat and amino acids. On this regimen, the patient developed symptoms of hypoglycemia, including palpitations, clamminess and transient blurred vision. Circulating glucose levels were within the normal range and ketones were present in the urine. There were no electrolyte abnormalities, arrhythmias or QTc prolongation, and echocardiography showed normal cardiac function. After 4 days on 6-hourly dosing and an interval >24 h off 2DG (to avoid competition with 18
FDG-PET/CT was performed to evaluate the antitumor activity of the treatment. Compared to a scan performed 3 weeks earlier (), tumor 18
FDG uptake was increased ().
Box 2 | Sustained 2DG exposure required to abrogate tumor cell proliferation in vitro
Pharmacokinetic studies with a daily 2DG dose similar to that used in our patient had shown a peak blood concentration of 10–12 mg/dl and an elimination half-life of 5–6 h.21
Based on these data, we estimated that 2DG levels would be ~10% of fasting circulating glucose levels for ~4 h. We set out to determine the effects of such an exposure on FH
-deficient tumor cells in culture. UOK262 cells, which do not tolerate low glucose levels, could be cultured on near-physiological glucose concentrations (150 mg/dl). To simulate daily dosing, the medium was supplemented with 10% 2DG for 4 h a day (pulse treatment). While this 2DG regimen decreased cell proliferation (), tumor cells continued to expand even with 2DG concentrations as high as 20% (data not shown). By contrast, sustained, uninterrupted treatment with 10% 2DG suppressed cell proliferation (). These data suggested that greater antitumor activity may be achieved in the patient with more-frequent 2DG dosing.
Abbreviations: FH, fumarate hydratase; 2DG, 2-deoxy-d-glucose.
Figure 5 The effect of 2DG on the proliferation of UOK262 cells. Proliferation curves of UOK262 cells exposed to 0% or 10% 2DG for a | 4 h per day (pulse treatment) or b | continuously (with daily medium exchanges). Despite pulse 2DG treatment, cell numbers continued (more ...)
While the increase in 18FDG uptake may have reflected a compensatory increase in glucose uptake as a result of glycolysis blockade, it could also have indicated tumor progression, and, in the absence of any evidence of antitumor activity, and despite the short treatment period, further 2DG treatment was stopped. Several weeks later, following a course of sunitinib therapy, the patient developed septic shock, which ultimately led to her death.