Helen Kakkassery

Inves­ti­gat­ing the immune sur­veil­lance mech­a­nisms in atyp­i­cal respon­ders of stage IV breast can­cers fol­low­ing stan­dard of care chemo — and tar­get­ed therapies

Abstract

“Atyp­i­cal respon­ders” can encom­pass three sub-cat­e­gories of patients: “excep­tion­al respon­ders” (those with an unusu­al­ly favourable treat­ment response), “rapid pro­gres­sors” (unusu­al­ly poor or no response), and “excep­tion­al/­long-term sur­vivors” (out­lived the ini­tial prog­no­sis). Here, we aim to inves­ti­gate the dri­vers of immune sur­veil­lance mech­a­nisms (local, lym­phoid, and periph­er­al) and the nature of immuno­log­i­cal tumour recog­ni­tion in excep­tion­al sur­vivors of stage 4 metasta­t­ic dis­ease fol­low­ing stan­dard-of-care chemo- and tar­get­ed therapies.

Meth­ods

We con­duct­ed a com­ple­men­tary mul­ti-plat­form immune pro­fil­ing study to define dif­fer­ences in the phe­no­typ­ic immune land­scape includ­ing mass-spec­trom­e­try-based pro­teomics on periph­er­al blood mononu­clear cells (PBM­Cs), mul­ti­plex cytokine pro­fil­ing of patient serum, and high-dimen­sion­al flow cytom­e­try on whole blood sam­ples. By stain­ing 500μl of whole blood using 2 pan­els, 353 immune-cell-relat­ed para­me­ters were obtained for com­par­isons among the fol­low­ing groups: “excep­tion­al sur­vivors,” “excep­tion­al respon­ders,” “rapid pro­gres­sors,” “non-excep­tion­al metasta­t­ic patients,” “ear­ly breast can­cer patients,” and “healthy vol­un­teers”. Patients were matched for age, breast can­cer recep­tor sta­tus, and sites of metas­tases where possible.

Results

Pre­lim­i­nary analy­sis of serum bio­mark­ers and pro­teome of the PBM­Cs of the excep­tion­al sur­vivors showed ele­vat­ed lev­els of NK-cell-relat­ed pro­teins such as KCTD10 and RBBP7. Flow cytom­e­try analy­sis revealed the pres­ence of increased acti­vat­ed CD56dim NK cells, CD56bright NK cells, and cen­tral mem­o­ry (CM) (CD45RA-CD27+) CD8 T cells in both the excep­tion­al sur­vivors and the rapid pro­gres­sors. How­ev­er, expres­sion of CD25 appears to be the main mode of acti­va­tion in rapid pro­gres­sors com­pared with increased NKG2D expres­sion in excep­tion­al sur­vivors. Addi­tion­al­ly, acti­va­tion of the uncon­ven­tion­al gd T cells was evi­dent in both groups with ter­mi­nal­ly dif­fer­en­ti­at­ed effec­tor mem­o­ry cells re-express­ing CD45RA (TEM­RA) Vd1 cell pre­dom­i­nat­ing in the excep­tion­al sur­vivors. In the rapid pro­gres­sors, the increased CD25+ TEM­RA Vd2 cells cou­pled with ele­vat­ed serum IL-17A and IL-1b sug­gest their abil­i­ty to gen­er­ate IL-17. Th2 cytokines, IL‑5 and IL-13 were also enriched in rapid pro­gres­sors and con­comi­tant increase in CD25+ Th2 cells reveals a strong Th2-dri­ven immune sig­na­ture in these patients. Final­ly increased CD86+ atyp­i­cal dou­ble neg­a­tive (DN) B cells, most­ly com­prised of the DN2 sub­set were also sig­nif­i­cant­ly enriched in the rapid pro­gres­sors com­pared to the excep­tion­al sur­vivors where DN1 B cells were prevalent.

Con­clu­sion

The mul­ti-plat­form approach to inves­ti­gat­ing immune respons­es present in atyp­i­cal respon­ders has iden­ti­fied sev­er­al dis­tinct immunophe­no­types, in which the extreme out­liers dif­fer in their poten­tial immune sur­veil­lance mech­a­nisms. Func­tion­al val­i­da­tion of these find­ings through acti­va­tion and cyto­tox­i­c­i­ty assays is cur­rent­ly under­way and future work aims to under­stand the spa­tial biol­o­gy of these cir­cu­lat­ing immune cells with­in the con­text of their pri­ma­ry tumours, metas­tases, and lymph nodes.

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