Exploiting antitumor immunity to overcome relapse and improve remission duration

Cancer survivors often relapse due to evolving drug-resistant clones and repopulating tumor stem cells. Our preclinical study demonstrated that terminal cancer patient’s lymphocytes can be converted from tolerant bystanders in vivo into effective cytotoxic T-lymphocytes in vitro killing patient’s own tumor cells containing drug-resistant clones and tumor stem cells. We designed a clinical trial combining peginterferon α-2b with imatinib for treatment of stage III/IV gastrointestinal stromal tumor (GIST) with the rational that peginterferon α-2b serves as danger signals to promote antitumor immunity while imatinib’s effective tumor killing undermines tumor-induced tolerance and supply tumor-specific antigens in vivo without leukopenia, thus allowing for proper dendritic cell and cytotoxic T-lymphocyte differentiation toward Th1 response. Interim analysis of eight patients demonstrated significant induction of IFN-γ-producing-CD8+, -CD4+, -NK cell, and IFN-γ-producing-tumor-infiltrating-lymphocytes, signifying significant Th1 response and NK cell activation. After a median follow-up of 3.6 years, complete response (CR) + partial response (PR) = 100%, overall survival = 100%, one patient died of unrelated illness while in remission, six of seven evaluable patients are either in continuing PR/CR (5 patients) or have progression-free survival (PFS, 1 patient) exceeding the upper limit of the 95% confidence level of the genotype-specific-PFS of the phase III imatinib-monotherapy (CALGB150105/SWOGS0033), demonstrating highly promising clinical outcomes. The current trial is closed in preparation for a larger future trial. We conclude that combination of targeted therapy and immunotherapy is safe and induced significant Th1 response and NK cell activation and demonstrated highly promising clinical efficacy in GIST, thus warranting development in other tumor types

Exploiting antitumor immunity to overcome relapse and improve remission duration.

Cancer survivors often relapse due to evolving drug-resistant clones and repopulating tumor stem cells. Our preclinical study demonstrated that terminal cancer patient's lymphocytes can be converted from tolerant bystanders in vivo into effective cytotoxic T-lymphocytes in vitro killing patient's own tumor cells containing drug-resistant clones and tumor stem cells. We designed a clinical trial combining peginterferon α-2b with imatinib for treatment of stage III/IV gastrointestinal stromal tumor (GIST) with the rational that peginterferon α-2b serves as danger signals to promote antitumor immunity while imatinib's effective tumor killing undermines tumor-induced tolerance and supply tumor-specific antigens in vivo without leukopenia, thus allowing for proper dendritic cell and cytotoxic T-lymphocyte differentiation toward Th1 response. Interim analysis of eight patients demonstrated significant induction of IFN-γ-producing-CD8(+), -CD4(+), -NK cell, and IFN-γ-producing-tumor-infiltrating-lymphocytes, signifying significant Th1 response and NK cell activation. After a median follow-up of 3.6 years, complete response (CR) + partial response (PR) = 100%, overall survival = 100%, one patient died of unrelated illness while in remission, six of seven evaluable patients are either in continuing PR/CR (5 patients) or have progression-free survival (PFS, 1 patient) exceeding the upper limit of the 95% confidence level of the genotype-specific-PFS of the phase III imatinib-monotherapy (CALGB150105/SWOGS0033), demonstrating highly promising clinical outcomes. The current trial is closed in preparation for a larger future trial. We conclude that combination of targeted therapy and immunotherapy is safe and induced significant Th1 response and NK cell activation and demonstrated highly promising clinical efficacy in GIST, thus warranting development in other tumor types

KIT activation & up-regulation, concomitant parallel induction of ET3, KIT⁺Melan-A^–- progenitor cells, and melanocyte regeneration in proportion to sun-exposure.

<p>(<b><i>A</i></b>), IHC of KIT and ET3 on serial sections of human skin specimen obtained from a lower extremity-amputation. Sole represents active suppression of melanogenesis (<i>a</i> and <i>d</i>), dorsum of big toe represents intermediate sun-exposure (<i>b</i> and <i>e</i>), and lateral lower leg represents heavy sun-exposure (<i>c</i> and <i>f</i>). (<b><i>B</i></b>), IHC of KIT, Melan-A, and ET3 on serial sections of human skin punch biopsy specimens obtained from sun-protected axilla (<i>g</i>, <i>i</i>, <i>k</i>) and chronic heavy sun-exposed forearm (<i>h</i>, <i>j</i>, <i>l</i>) from the same individual. Lymphocytes serve as internal negative control for KIT, ET3 and Melan-A; mast cells serve as internal positive control for KIT. Together, these images demonstrate that human skin exhibits sun-exposure-dependent up-regulation of KIT (<i>a-c</i>) and concomitant parallel sun-exposure-induced increasing induction of ET3 (<i>d-f</i>). Chronic sun-exposure induces intense dendritic pattern of KIT expression as well as a large increase in the number of KIT-expressing-cells in the basal layer (<i>h</i>) consisting of KIT⁺Melan-A⁺ mature melanocytes (<i>j</i>) and KIT⁺Melan-A^–melanocyte progenitor cells as evidenced by the difference between (<i>h</i>) and (<i>j</i>).</p

Autophosphorylation, internalization, and nuclear localization of activated KIT with tyrosine phosphorylation at 568/570 (pY568/pY570KIT).

<p>(<b><i>A</i></b>), IHC of frozen sections of an aggressive GIST (<i>a-c</i>) and a normal human adult testis as external control (<i>d-f</i>) using pan-KIT antibody (<i>a</i> and <i>d</i>), pY568/pY570KIT antibody (b and <i>e</i>, red arrow indicates nuclear localization), and pY703KIT antibody (<i>c</i> and <i>f</i>) respectively. (<b><i>B</i></b>), <i>In situ</i> IHC to assess kinetics of SCF-induced nuclear translocation of pY568/pY570KIT using WM793 melanoma cells cultured in 4-well chamber tissue culture treated glass slides. Control (<i>g)</i> without SCF stimulation, after addition of SCF to culture media, the nuclear localization of pY568/pY570KIT increases progressively (<i>h-j</i>) in more than 90% of WM793 cells, reaches a plateau about 40–60 minutes (<i>i</i> and <i>j</i>), begins to decrease at 90 minutes (<i>k</i>), and is completely absent in nucleus with relocation back to the cytoplasm at 4 hours, some residual cytoplasmic staining remains visible (<i>l</i>).</p

Microarray analysis comparing a highly aggressive GIST with an indolent GIST: A selective list of target genes whose expression was up- or down-regulated by KIT signaling.

<p>Microarray analysis comparing a highly aggressive GIST with an indolent GIST: A selective list of target genes whose expression was up- or down-regulated by KIT signaling.</p

Immunohistochemical studies on human colon myenteric plexus demonstrate that 48 hours fasting results in activation of SCF-KIT signaling and concomitant parallel induction of endothelin-3.

<p>Human colon specimens post 48 hours fasting demonstrate intense KIT staining in ICCs within the myenteric plexus (<i>a</i>), and intense ET3 staining within the myenteric plexus (<i>b</i>). In sharp contrast, the surrounding longitudinal and circular smooth muscle cells (SM) show negative ET3 staining.</p