Next generation sequencing analysis of platinum refractory advanced germ cell tumor sensitive to Sunitinib (Sutent®) a VEGFR2/PDGFRβ/c-kit/ FLT3/RET/CSF1R inhibitor in a phase II trial

This was a phase II study of sunitinib in refractory male germ cell tumors with the 12 week progression free survival as the primary endpoint. The major inclusion criteria for the phase II study were progressive metastatic GCTs of gonadal or extragonadal origin in males after failure of front-line therapy and at least one salvage regimen. In addition the patients must have evaluable or measurable disease by clinical or radiological studies. Alternatively, in the absence of radiologically evaluable or measurable disease, two sequentially rising marker values each one week apart attributed by treating physician to germ cell tumor was permitted; either beta HCG above 50 mIU/ml and/or AFP above 20 ng/ml qualified as eligible. A major exclusion was prior VEGFR/PDGFR inhibitor therapy.

Once registered, treatment was started no later than 7 days. Patients were treated with sunitinib malate 50 mg orally daily for 4 weeks every 6 weeks. A cycle was defined as a planned 6-week treatment interval. It was preferred that patients complete at least 2 cycles of therapy unless there is evidence of rapid disease progression. Response evaluations were performed every 6 weeks according to RECIST criteria [9],[10]. The University of Texas MD Anderson cancer IRB approved this clinical trial and all patients were followed according to the protocol. The trial was an investigator initiated single institutional trial supported by Pfizer inc. (New York, NY) which provided Sunitinib (Sutent®) to the patients enrolled on the trial was closed early due to slow accrual.

Next generation targeted exomic sequencing was performed for genomic profling.200-500ug of genomic DNA from each sample was sheared by sonication using the Covaris E220 instrument (Covaris). Library preparation utilized the KAPA kit following the “with beads” manufacturer protocol using KAPA HiFi polymerase (6 cycles). The capture included all exons from 202 cancer-related genes. Biotin labeled DNA probes were designed using Roche Nimblegen for capturing target regions (over 5,000 exons in 202 genes) and followed manufacture’s protocol for the capture process. Probes (50–105 bp long) were tiled at a minimum coverage of 2× and balanced across the target regions to ensure uniformity. Captured libraries were sequenced on a HiSeq 2000 (Illumina Inc., San Diego, CA, USA) on a version 3 TruSeq paired end flowcell according to manufacturer’s instructions The resulting BCL files containing the sequence data were converted into “.fastq.gz” files and individual libraries within the samples were demultiplexed using CASAVA 1.8.2 with no mismatches.

Deep sequencing data was aligned to to hg19 using BWA [11]. Duplicate reads were removed via Picard [12]. Single nucleotide variant (SNVs), small indels and and copy number alterations were called using an in-house pipeline or a previously published algorithm, respectively [13]. This supported identifying genome aberrations by next-generation sequencing (NGS) based assays to identify genomic alterations in 200 cancer related genes.

The validation of the copy number alterations, was done using the OncoScan, a MIParray V3. technology [14].

A 52-year-old Caucasian male had been diagnosed at age 29 with a right sided testicular GCT. He underwent a radical, right orchiectomy through an inguinal incision. The primary tumor was classified as a clinical stage I, nonseminomatous GCT. AFP level was above 400 prior to orchiectomy and subsequently normalized appropriately following surgery. He did not receive any adjunctive chemotherapy. For 15 years, serial CT scans of abdomen and pelvis along with serum tumor markers found no evidence of tumor recurrence. However, in February 2009, as a part of work up for abdominal pain, a CT scan of the abdomen and pelvis revealed new and multiple enlarged retroperitoneal lymph nodes (intra-aortocaval and para-aortic). A subsequent CT of the chest showed bilaterally, several small, scattered pulmonary nodules; the largest being a 6 mm × 4 mm, noncalcified nodule in the right, lower lobe. An MRI of the brain found no evidence of intracranial metastases. A scrotal ultrasound revealed a homogenous echo-texture throughout the left testicle without evidence of abnormal masses or scars. Serum alfa-fetoprotein (AFP) and β-hCG levels were elevated at 34.8 ng/ml (normal ranges: 0.0- 5.0) and 22 mIU/ML (0.0- 1.0), respectively.

The patient received standard 3 cycles of bleomycin, etoposide, and cisplatin(BEP) and 1 cycle of etoposide and cisplatin (EP). Serum AFP level a month after his last cycle remained elevated at 56 ng/ml (0.0-5.0). CT scan of the chest, abdomen and pelvis, post-chemotherapy in June 2009, continued to show stable small posterior right lower lobe mass, and a mass in the right retroperitoneum. Because the patient had persistent back pain that did not resolve with the resolution of the retroperitoneal adenopathy, an MRI of the spine was performed in July 2009. This MRI revealed a 2.5 cm enhancing mass in the right retroperitoneum at the level of L2. This mass measured 2.7 × 2.2 cm and comparison to the CT scan done in June 2009 showed an increase in size from 2 × 1.6 cm in just a month. The patient was referred to our institution for further treatment.

The patient was presented at our multi-disciplinary tumor board. A decision to proceed to retroperitoneal lymph node dissection was jointly made. In August 2009 a complete, non-nerve sparing, bilateral retroperitoneal lymph node dissection extending from the renal vessels superiorly to the crossing of the ureters over the common iliac arteries inferiorly; onto the anterior spinous ligaments posteriorly and laterally to the ureters was completed without complications. Pathologic review of the resected tissue demonstrated residual viable malignant GCT within 1 out 9 precaval lymph nodes and 1 out of 8 retrocaval lymph nodes. The histologic classification of the GCT was 80% embryonal carcinoma and 20% yolk sac tumor. Following recovery from surgery, the patient received 2 cycles of adjuvant chemotherapy consisting of paclitaxel, ifosfamide, and cisplatin (TIP). This treatment was complicated by acute renal failure and ifosfamide induced CNS toxicity. Adjuvant chemotherapy was therefore altered for the third cycle to actinomycin-D, cyclophosphamide, and etoposide (ACE). A CT abdomen and pelvis showed no evidence for recurrent disease. He was followed closely with serial CT scans along with serum tumor markers. In 2010 a new pulmonary nodule (Figure 1A) and rising serum tumor markers were detected. He was then enrolled on the clinical study with sunitinib for chemotherapy-refractory germ cell tumors in June 2010. He received sunitinib 50 mg orally for 4 weeks on and 2 weeks off-cycle. By 09/20010 a decline in serum tumor markers was observed (Figures 2 and 3; The arrow points to date of sunitinib initiation). His most significant response was in serum beta-hCG with a more mild and fluctuating AFP response. He developed hypothyroidism while on treatment with sunitinib, a documented side effect [15] and began levothyroxine therapy with an appropriate response. He otherwise suffered no other treatment related G3 or 4 toxicity related to the sunitinib. Subsequent CT scans show a continued response to the treatment and has not revealed any new disease sites (Figure 1B). He continued to be followed up with clinical and biochemical response with a very good performance status. Unfortunately, in November 2011, he experienced progressive weakness of his legs accompanied by saddle anesthesia, and urinary incontinence. MRI spines showed progressive disease involving the cauda equina and biopsy was consistent with metastatic GCT - immunostain results were positive for cytokeratin, CD30, SALL4, and focally positive for Oct3/4. He was taken off the study at that time, 17 months after starting sunitinib. Because of worsening performance status he elected to receive supportive care and was referred to hospice care.

This patient was enrolled on the unusual responder program at our institution, a program that applies appropriate next generation sequencing and other genomic and proteomic technologies to tumor and germline samples from patients with exceptional responses to chemotherapies and targeted regimens in an attempt to identify potential genetic explanations for the outlier response [16]. Next generation sequencing of unusual responder patient with testicular cancer indicated tumor harboring multiple copy number aberrations. Several genetic alterations were identified in this patient’s tumor. Of these, some of the variants identified represented potential candidates for further exploration to understand this patient’s response to sunitinib. The only alterations were found to have reported relevant information in tumors/cells treated with sunitinib were: RET amplification, PTEN loss, EGFR and KRAS amplification (Table 2). We carried out an extensive literature search in National Library of Medicine’s MEDLINE database on the five alterations and tabulated the levels of evidence/significance of genes relevant to sunitinib based therapy (Table 3). As the next step, to validate these genes an oncoscan MIP array were employed to validate the copy number analysis. The RET amplification, EGFR and KRAS amplification were validated by the second method. The PTEN loss was not validated by PTEN IHC.