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Clinical trials on adoptive T-cell therapy (ACT) using expanded tumor-infiltrating lymphocytes (TIL) have shown response rates of over 50% in refractory melanoma. However, little is known how clinical and pathologic features impact TIL outgrowth isolated from metastatic melanoma tumors.
We analyzed the impact of clinical and pathologic features on initial TIL outgrowth in 226 consecutive patients undergoing tumor resection. Successful initial TIL outgrowth was defined as ≥40 million viable lymphocytes harvested from all tumor fragments in a 5-week culture. To normalize for the different size of resected tumors and thus available tumor fragments, we divided the number of expanded TIL by the starting number of tumor fragments (TIL/fragment).
Overall, initial TIL outgrowth was successful in 62% of patients, with patients ≤30 years of age (94%; p=0.01) and female patients (71% vs. 57% for males; p=0.04) having the highest rate of success. Systemic therapy 30 days prior to tumor harvest negatively impacted initial TIL outgrowth compared to patients who never received systemic therapy (47% versus 71%, p=0.02). Biochemotherapy within 0–60 days of tumor harvest negatively impacted the initial TIL outgrowth with a success rate of only 16% (p<0.0001).
Parameters such as age, sex, and the type and timing of prior systemic therapy significantly affect the success rate of the initial TIL outgrowth from tumor fragments for ACT; these parameters may be helpful in selecting patients for melanoma ACT.
Metastatic melanoma is one of the most immunogenic cancers, and adoptive T-cell therapy (ACT) using expanded tumor-infiltrating lymphocytes (TIL) has shown great promise as an effective therapy. Since the introduction of ACT in 1988 (1), changes in the preparation regimens and expansion of T cells have produced clinical response rates as high as 51%–72% (2–6).
One of the major limitations of ACT is the ability to generate TIL from surgically resected tumor fragments with a success rate ranging from 31% to 94% (7, 4, 8–10). A critical first step in generating TIL is the initial outgrowth of lymphocytes from the first 5 days in culture. While there is no definitive cut-off that defines a successful initial outgrowth, we have found that at least 40 × 106 TIL are necessary to move forward with the subsequent large-scale expansion. Given the time- and resource- intensive nature of this therapy, its associated morbidity and cost, and the availability of alternative treatment options for patients with metastatic melanoma, parameters that would predict a successful initial TIL outgrowth could be used as a tool for patient selection and treatment prioritization.
Here, we have determined what patient clinical characteristics, primary tumor characteristics, and types of prior systemic therapy affect the rate of TIL expansion or TIL anti-tumor reactivity.
Patients with Stage IV melanoma, Stage III in-transit disease, or recurrent regional nodal disease were enrolled following informed consent. Some patients underwent multiple tumor resections in order to generate TIL, but in order to most accurately define the success rate of the initial TIL outgrowth, we only included the first tumor harvest. Tissue from metastatic surgical resections was used to expand TIL under an IRB-approved protocol (LAB06-0755) approved by the Institutional review Board of University of Texas MD Anderson Cancer Center.
Tumor fragments were processed and TIL expanded according to previously published methods.(3) Briefly, 3–5 mm2 tumor fragments (range of 4–48 tumor fragments) were placed into 24-well plates in 2 ml of culture medium (CM) containing 6,000 IU/ml of IL-2 (Proleukin™; Prometheus, San Diego, CA), and after 5 weeks the viable TIL were counted. The cells cultured from this initial outgrowth are then used to perform a large-scale cell expansion using a “rapid expansion protocol” (REP) to generate the final TIL infusion product of 10–20 billion TIL. After multiple preclinical studies, we determined that 40 × 106 TIL to be the minimal number necessary for the subsequent REP to generate a final GMP-grade TIL infusion product for an ongoing ACT clinical trial at M.D. Anderson Cancer Center (NCT00338377). The size of resected tumor and number of tumor fragments used to generate TIL varied, and in order to normalize for this difference we divided the total viable TIL by the number of tumor fragments to create a ratio of TIL/tumor fragment (TIL/fragment). Values of TIL/fragment are shown as a power of 106, unless otherwise indicated. Anti-tumor reactivity of TIL against melanoma was determined in samples with successful TIL expansion by measuring IFN-γ (≥400 pg/ml by ELISA) in culture supernatants collected from triplicate 24-h co-cultures of 1 × 105 TIL with 1 × 105 autologous or HLA class I-matched allogeneic melanoma lines in 96-well plates.
Clinical and pathologic data was retrospectively obtained from the patient record, including age, gender, biopsy site, melanoma histological type, primary tumor location, serum lactate dehydrogenase (LDH) at the time of tumor resection, and prior treatment history. LDH was determined before tumor resection using a CLIA-certified assay as part of the routine clinical monitoring of patients in the Department of Laboratory Medicine at MD Anderson Cancer Center. A level of ≥618 IU/L was considered as elevated, while values below this were considered as normal.
We categorized patients based on the last systemic therapy prior to tumor harvest. We categorized prior systemic therapy into five groups: IL-2, other immunotherapy, chemotherapy, biochemotherapy, and targeted therapy. Prior radiation was not included as a prior systemic therapy. “Other immunotherapy” was defined as IFN-alpha, anti-CTLA-4, vaccine, or GM-CSF including if given in the adjuvant setting. Chemotherapy included all cytotoxic agents including isolated limb perfusion with melphalan. Biochemotherapy was defined as the combination of cytotoxic chemotherapy with either IL-2 or IFN-alpha. Targeted agents included mTOR and tyrosine kinase inhibitors when given not in combination with chemotherapy. Finally, we included patients who received adjuvant therapy, including interferon, in the previous treated groups.
In order to assess how the timing of the last systemic therapy affected initial TIL outgrowth and TIL/fragment we categorized patients based on the time from the last systemic therapy to tumor harvest into the following four time periods: 0–30 days, 31–60 days, 61–90 days, and >90 days.
Pathologic information of the primary tumor was available on a subset of patients. We used the pathologist review in determining ulceration, mitotic figures, Breslow depth, presence of TIL, and histology of the primary tumor. A subset of our patients underwent testing for mutational analysis in B-RAF, N-RAS, or C-KIT. Mutations were evaluated by CLIA-certified pyrosequencing of B-RAF (exon 15), N-RAS (codons 12, 13, 61), and C-KIT (exons 11, 13, and 17). Patients were classified as having a B-RAF mutation if they harbored any mutation at amino acid 600 or 601 in the sequence. Patients were classified to have an N-RAS mutation if they harbored a mutation at codon 61 (CAA to CGA). Patients were considered to be WT/WT if they did not harbor a B-RAF or a N-RAS mutation. Patients were classified to have a C-KIT mutation if they harbored an activating mutation in either exon 11, 13 or 17 as previously reported.(11)
The success rate of the initial TIL outgrowth was correlated with clinical and pathologic parameters using the Fisher's exact test. Wilcoxon rank-sum test or Kruskal-Wallis test were used to compare TIL/fragment values between or among clinical and pathologic groups. When correlating LDH to TIL/fragment we used Pearson's correlation coefficient. A p-value less than 0.05 was considered as statistically significant. All tests were performed using SAS 9.2 by SAS Institute Inc., Cary, NC, USA.
In total, 226 patients (76 female and 150 male) with a median age of 51 years (range 14 to 70 years) were enrolled in the study. The overall success rate of the initial TIL outgrowth was 62% (n=139). As shown in Table 1, successful outgrowth was associated with a younger age (49.5 versus 52.7 years; p=0.03) with patients in the youngest subgroup (14–30 years) having the highest success rate (94%). Females were more likely to have a successful TIL outgrowth than males (71% versus 57%; p=0.04). Neither disease stage nor LDH (normal or elevated) at the time of resection impacted the success rate of the initial TIL outgrowth. The median number of tumor fragments used to generate TIL was 12 (mean 13.5; range 4–48), and patients with a successful outgrowth actually had a lower mean number of tumor fragments (12.97 versus 14.38; p=0.03).
Table 1 also shows the mean number of TIL per fragment according to age, gender, stage, and LDH status. The mean number of TIL/fragment was 12.84 (median 5.5 × 106; range 0–123.7 × 106) with none of the indicated parameters affecting TIL/fragment yield. However, when analyzed in a linear fashion elevated LDH significantly inversely correlated with TIL/fragment (p=0.03; data not shown).
In some cases, the TIL cryopreserved after expansion from tumor fragments were thawed and further expanded using the REP. TIL from 22 of the 139 patients for whom initial TIL outgrowth was successful had their TIL further expanded using the REP.11 Among these, all TIL successfully underwent secondary expansion in the REP with an average fold-expansion of 1,665-fold ±677 (range of 359- to 2,660- fold; median of 1,656-fold).
Information on the primary tumor was available for a subset of our patients, and we correlated the features of the primary tumor with the success rate of initial TIL outgrowth and TIL/fragment (Table 2). The pathologic features of the primary, the location, the subtype, and the mutation status did not impact the rate of successful initial TIL outgrowth (Table 2). Patients with brisk TIL in the primary tumor (n=2) had a 100% success rate in generating TIL, and conversely none of the patients with absent TIL in primary (n=3) had successful TIL expansion. Patients with a mucosal melanoma primary (n=7) had the highest percentage (86%) of successful TIL generation; however, the numbers were too small to be statistically significant. Patients who had a visceral tumor resected (n=56) had the highest rate of successful initial TIL outgrowth (73% versus 52%, p=0.05).
The only aspect of the primary tumor that correlated with TIL/fragment was mitotic figures. Information on the mitotic figures of the primary was available for 90 patients with the median number of mitotic figures per mm2 being 5 (Table 2). Patients whose primary tumor had greater than the median number of mitotic figures correlated with less TIL/fragment when compared with patients whose primary tumor had less than the median mitotic figures (18.9 × 106 vs. 8.2 × 106; p=0.02).
Table 3 summarizes our analysis of all 226 TIL expansion attempts based on prior systemic therapy before the tumor harvest. The majority of patients (69%) received systemic therapy prior to TIL harvest, and when grouped as a whole, patients who received prior systemic therapy (Table 3: “Prior systemic therapy”) had a statistically insignificant lower rate of initial TIL outgrowth (71% versus 57%, p=0.06) and a significant decrease in TIL/fragment (11.53 versus 15.82 × 106, p=0.02) when compared to patients who did not receive prior systemic therapy. We then analyzed the impact of the type of systemic therapy anytime before tumor harvest (Table 3: “Last systemic therapy before resection”) and found that biochemotherapy negatively impacted both the success rate of the initial TIL outgrowth and yield of TIL/fragment (p=0.003 in both cases). Targeted therapy seemed to have a negative impact on TIL/fragment but not the success rate of the initial TIL outgrowth. However, the number of samples was too small to make any definitive conclusions. Fig. 1 further illustrates this negative effect of prior biochemotherapy on the yield of TIL per tumor fragment of each patient. We found no difference in the mean or median number of tumor fragments placed in culture between the different pre-treatment groups.
The timing of prior systemic therapy also significantly impacted the success rate of the initial outgrowth and TIL/fragment (Table 3: “Impact of timing of last systemic therapy”). Patients who received systemic therapy within 30 days of tumor harvest had the lowest rate of successful initial TIL outgrowth and the least TIL/fragment when compared to patients who did not receive prior systemic therapy (47% success rate, p=0.02; 7.48 × 106 TIL/fragment, p=0.004). Further analysis of the specific type of systemic therapy within 30 days before tumor harvest (Table 3: “Therapy within 0–30 days”) revealed that biochemotherapy and targeted therapy negatively impacted the initial TIL outgrowth and TIL/fragment. It must be noted however that only a few samples (n=3) were in the “Targeted” category. For this reason, this patient sub-group was omitted from further analysis.
Table 4 individually summarizes the effect of the timing of the last systemic therapy given prior to tumor resection. When compared to patients who received no priory systemic therapy, biochemotherapy given 0–30 days or 31–60 days prior to tumor harvest negatively impacted the success rate of the initial outgrowth (11% and 20% respectively) and TIL/fragment. However, when given beyond 60 days, and especially beyond 90 days, biochemotherapy no longer negatively affected the success rate of the initial outgrowth or TIL/fragment. There were no time points when IL-2, other immunotherapy, and chemotherapy negatively impacted the success rate of the initial TIL outgrowth or TIL/fragment.
We also tested the anti-tumor reactivity of TIL in 128 of the 139 patients successfully generating the minimal 40 × 106 TIL (92% of successful growers). The TIL were tested for reactivity against either autologous (when available) or HLA class I-matched melanoma tumor cell lines using IFN-γ release assays. Overall, 77/128 (60%) of the patients tested for TIL reactivity had reactive TIL (see Materials and Methods for reactivity criteria). Of note, this could be an underestimate of reactivity since assays performed in the absence of an available autologous tumor line could miss unique antigens as well as antigens restricted by alternative MHC alleles. We did not find any correlations between the clinical characteristics, primary tumor characteristics, or previous treatment with TIL reactivity (data not shown).
The success rate of expanding TIL from tumor fragments from a first resection (≥40 × 106 TIL yield after 5 weeks) was 62%. Fourteen patients whose initial TIL outgrowth was unsuccessful underwent a second tumor harvest. Five (36%) of these 14 patients successfully generated TIL on the second harvest and none of these patients had received systemic therapies within 60 days prior to the second harvest. Five of the 9 patients (55%) who did not have a successful initial TIL outgrowth on the second harvest received systemic therapy with 60 days of the second tumor harvest.
The purpose of this analysis was to understand what clinical or pathologic characteristics impact successful TIL generation when anticipating treating a metastatic melanoma patient with ACT. An understanding of how parameters affect successful TIL expansion could be used to better select patients who would generate enough TIL for ACT. Our key findings were that younger and female patients were more likely to successfully generate TIL, and that certain types of prior therapy (biochemotherapy in particular) could negatively impact the ability to expand enough TIL for ACT. We also found however that the timing of type of systemic therapy significantly impacted the success of TIL growth with the negative effects of biochemotherapy waning when given later that 60 days before tumor harvest for TIL expansion.
Our institution was able to successfully generate TIL in 62% of all patients on the first tumor harvest, in line with other published reports ranging from 34%–94% (7–10). All attempts at secondary expansion were successful, with an average of 1,665-fold expansion of the cells after the REP. A number of other recent studies have reported somewhat higher success rates for expanding TIL from resected metastatic melanomas. For example, Goff et al. (8) reported a 94% success rate, Nguyen et al. (9) reported a 72% success rate, and Besser et al. (10) reported a 97% success rate. There are a number of caveats in comparing the TIL expansion success rates across different institutions. First, the cut-off for successful initial TIL outgrowth differs between institutions. We defined a successful initial outgrowth as 40 × 106 TIL while others used 5 × 106 (Goff et al.) and 30 × 106 (Besser et al. and Nguyen et al.). Using different thresholds to define successful initial TIL outgrowth will obviously impact the success rate. Second, the methods to isolate and grow TIL differ between centers; with some studies expanding TIL from single cell suspensions from enzymatic tumor digests (Besser et al.), tumor fragments (Nguyen et al.), or a mixture of both tumor fragments and cell suspensions from tumor enzymatic digests (Goff et al.) (8, 9, 10). Third, the prior therapy before tumor resection, as we have shown here, can affect the outcome, and it is likely that the type and timing for prior therapy before tumor harvest differed among the institutions. Finally, in the study by Goff et al. (8) from the NCI (Bethesda, MD), the reported success rate of initial TIL expansion from the tumor included a significant number of patients that had more than one attempt (two-three) to grow TIL after surgery. These issues point to the need to further optimize and then standardize TIL expansion methodologies across centers in order to more accurately gauge the rate of successful TIL expansion and the effects of patient clinical and pathological characteristics on TIL growth across different centers.
Until this analysis, age and gender had not been identified as a factor in TIL expansion success. The reason why younger patients were more successful at growing TIL (p=0.01) is unclear, and one of the following could play a role: higher telomere lengths in lymphocytes from younger people (12, 13), the higher proportion of naïve T cells at the onset of disease (14), or age-associated decrease in T-cell diversity and decline in immune function with older patients harboring more differentiated effector-memory and effector cells with a shorter lifespan. A younger patient age has also been associated with longer TIL persistence after infusion into patients undergoing ACT (15, 16). The reason why female patients were more successful in generating TIL is also unknown, but it could be related to estrogen's inhibitory effect on T-suppressor cells and stimulatory effect on T-helper cells (17), or the increased incidence of autoimmune disease such as lupus (18) or rheumatoid arthritis (19) in females. Lower androgen levels in females may also play a role in regulating stronger T-cell responses, as androgen ablation has been found to have positive immunoregulatory effects (20). The ability to expand adequate TIL from tumor biopsies could also be a functional biomarker indicating the relative “strength” of a patient's immune system against their disease, or the “directing” of an immune response to a larger array of self antigens in females, and perhaps explain why female melanoma patients overall have longer survival than males (21). It is also possible that a host of genetic factors, such as the extent and polymorphism in immune system-related factors (e.g., HLA subtypes, lymphocyte signaling molecules, innate and adaptive immune system cytokines, and immunosuppressive factors) play a role in modulating the parameters measured here.
Characteristics of the primary tumor are well known to predict outcomes in patients with melanoma (22, 23). Ulceration, high mitotic rate, and greater Breslow thickness are associated with a higher stage and worse survival (22, 23). Although the tumor specimens taken for TIL harvest were not from the primary tumor, we hypothesized that the pathologic factors of the primary tumor that lead to a poorer prognosis might also influence the ability to successfully generate TIL or the number of TIL per tumor fragment later on during the disease course. While none of the factors of the primary tumor were associated with the success rate of growing TIL (getting at least 40 × 106 TIL), high mitotic rate was associated with an expansion of fewer TIL/fragment. The etiology of these findings is unclear, but one hypothesis is that metastatic tumors that originate from a primary with a higher mitotic rate might lead to a higher proliferating, more aggressive metastatic tumor that is better able to create an immunosuppressive environment and thus decrease the ability of T cells to proliferate and/or persist in the tumor microenvironment (24, 25). Tumors expressing higher T-cell inhibitory ligands, such as B7H1 (PD-1 ligand) for example, have been shown be more aggressive and suppress T-cell activation and T-cell function (26, 27). It is also possible that more aggressive, faster dividing tumors attract higher numbers of CD4+ Foxp3+ regulatory T-cells that suppress T cell growth and survival in the tumor microenvironment.
The location of the primary tumor is well known to be associated with different mutations suggesting a different biology of the tumors (28). While the tumor used to harvest TIL came from a metastatic and not the primary site, we examined if the location of the primary tumor influenced either successful TIL generation or the total number of TIL per fragment generated. However, we did not find any statistically significant association between the site of the primary tumor and the success rate of expanding TIL as well as TIL per fragment. Of note, acral and mucosal melanomas are known to have the highest frequency of c-kit mutations (11), and all 3 patients in our study who had a c-kit mutation successfully generated TIL and had the highest mean and median TIL/fragment. However, our numbers of c-kit mutant patients were low and this needs to be confirmed with a larger sample size. Another parameter we examined is whether the location of the metastatic tumor used to grow TIL from was associated with the quality and quantity of TIL expansion. Again, as found by other investigators such as Goff et al., we did not find any affect of the tumor location for TIL growth and these parameters. This is actually “good news” for ACT since conceivably patients with metastases in any location in the body would be eligible for TIL expansion and therapy. One location that may be problematic however is brain metastases. We were successful in only one out of four attempts to expand TIL from brain metastases (Table 2). Most of our tumors have been from lymph node, subcutaneous, and other visceral sites. It is noteworthy that we have successfully expanded TIL in 3/5 small bowel metastases suggesting that this is also a viable option if bacterial or fungal contamination can be avoided (the two cases that did not grow were contaminated).
One of the most evident parameters affecting the success rate of TIL generation and TIL/fragment was the type and timing of systemic therapy prior to tumor resection. Patients, who received either no prior therapy, or no therapy within 60 days of TIL harvest, had the highest rate of TIL generation with 71% and 64% respectively. Patients who received either IL-2 or other immunotherapy as their last systemic therapy prior to tumor harvest at any time point before tumor harvest had similar rates of TIL success and TIL/fragment as patients who had no prior systemic therapy. Interestingly, in our cohort of patients, immunotherapy did not significantly increase the yield of TIL per tumor fragment relative to no prior therapy even when given within 30 days of the tumor harvest. Although this data needs to be followed up by monitoring future patients receiving immunotherapies such as IL-2, anti-CTLA4, vaccines, and other immunopotentiators, these results suggest prior immunotherapy with IL-2, for example, is not necessary to successfully expand TIL to large numbers for ACT; again, “good news” for centers contemplating performing ACT, but who do not perform these types of immunotherapy.
Perhaps one of the most interesting findings of our study was that biochemotherapy was the only form of prior therapy that had deleterious effects on successful generation of TIL and the yield of TIL per tumor fragment, while other forms of chemotherapy without IL-2 and IFN-α (including limb perfusion with Melphalan, and systemic therapy with Abraxane, Dacarbazine, Paclitaxel, Vinblastine, and Cisplatin) had very little impact. Studies have shown that the recovery of T-cells after cytotoxic chemotherapy takes weeks to months (29), and we would have expected that patients who received both biochemotherapy or chemotherapy within 0–60 days to have a decreased rate of successful expansion. Little is known about the effects of chemotherapy and biochemotherapy on TIL and whether any particular chemotherapy selects or spares TIL in comparison to other lymphocytes. In addition, at present it is unclear why biochemotherapy in particular was especially deleterious. It is possible that the immune activation induced by IL-2 and IFN-α, may drive T cells into cell cycle where they become susceptible to the chemotherapy agents of the biochemotherapy regimen (cisplatin, vinblastine, and either dacarbazine or temozolomide). Although biochemotherapy was deleterious to TIL expansion, this effect seemed to disappear when the therapy was given over 60 days prior to tumor harvest for TIL growth. Thus, we would recommend that patients do not receive biochemotherapy within 60 days prior to tumor harvest if this is clinically feasible. Unexpectedly, patients pre-treated with targeted therapies had a significantly lower rate of successful TIL generation and TIL/fragment. However, the number of patients treated with targeted therapy 2 months prior to tumor harvest (n=4) was small making it difficult to draw conclusions about the effect of any one specific targeted agent on TIL growth.
Clinically there remains a question of whether a second attempt at TIL generation will be successful if an initial attempt fails. Fourteen patients whose first tumor resection did not successfully expand TIL underwent a second tumor resection, and 5/14 (36%) were able to generate an adequate number of TIL from the second harvest. All 5 patients who generated TIL from the second harvest did not receive systemic therapy within two months prior to the second harvest, while 5/9 (55%) of the patients who did not generate TIL on the second harvest underwent systemic therapy 60 days prior to their second tumor harvest. This further suggests that systemic therapy within 60 days of tumor resection is a powerful inhibiting factor in the generation of TIL. Although there is likely an intrinsic yet unknown biology of who is capable of growing TIL or not, the absence of systemic treatment 60 days prior to resection is the most powerful predictor of successful TIL generation, and if possible we recommend TIL harvest either occur before receiving systemic therapy or waiting at least 2 months after receiving therapy.
In summary, our results have shown that TIL can be successfully expanded from isolated tumor fragments in culture in 62% of cases in an unselected group of metastatic melanoma patients. The most important conclusion was that systemic therapy can adversely impact TIL expansion, especially when given within 60 days prior to tumor harvest, and therefore, we would recommend surgery or biopsies for TIL expansion be performed 60 days after systemic therapy is given. It appears that younger patients and female patients have the best chances of generating TIL, and patients who receive either biochemotherapy 2 months prior to resection have the worst chances of expanding TIL to adequate numbers for ACT. If validated in a “test set” of patients, a combination of these parameters may be a powerful selection tool to decide which patients will likely have a successful TIL expansion for ACT.
At present, the treatment of metastatic melanoma is limited to only three FDA approved regimens: bolus high-dose IL-2, dacarbazine, and ipilimumab. While still experimental, adoptive cell therapy (ACT) using tumor-infiltrating lymphocytes (TIL) derived from metastatic tumor tissue has produced response rates greater than 50%. However, one of the major limitations in ACT is that TIL cannot always be expanded to adequate numbers from all patients for therapeutic use. Our study addressed this issue by determining how clinical characteristics of the patient, including the type of prior systemic therapy, impacts the rate of successful initial TIL outgrowth. We found that some of these parameters, especially prior systemic therapy other than high-dose IL-2, negatively affected successful initial TIL outgrowth. Our data should help guide clinicians on choosing when to refer a metastatic melanoma patient for a tumor resection for the purpose of TIL expansion for ACT.
We thank all participating patients, nurses, laboratory scientist, and data coordinators for help on this project. This work was funded by NIH/NCI grants (1RO1 CA111999-01A2). We are also grateful for support from the Dr. Miriam and Sheldon Adelson Medical Research Foundation (AMRF). In addition, we thank the American Society of Clinical Oncology who in part funded this work through the ASCO Young Investigator Award.