Abstract P6-03-03: Enrichment of janus kinase-2 (JAK2)-amplified tumor cell populations in triple-negative breast cancers (TNBC) during chemotherapy treatment
Authors: Schwarz Luis J , Balko Justin M , Giltnane Jennifer M , Estrada Monica V , Sanders Melinda E , Wang Kai , Lin Nancy U , Miller Vicent A , Stephens Philip , Yelensky Roman , Pinto Joseph A , Gómez Henry L , Landis Melissa D , Chang Jenny C , Arteaga Carlos L 
Published: May 2015

Abstract
Neoadjuvant chemotherapy (NAC) is frequently used in the treatment of triple-negative breast cancer (TNBC). Approximately 30% of TNBC patients achieve pathological complete response with excellent prognosis. However, the remaining 70% are at increased risk of recurrence with no molecularly targeted therapeutic options in the adjuvant setting. We performed targeted next-generation sequencing in residual TNBC tumors after NAC to identify potentially actionable genomic alterations. As previously reported, we found amplifications in JAK2 (JAK2AMP) in 7/68 (10.2%) post-NAC tumors compared with 1/30 (3%) in the pre-NAC cohort; all cases were confirmed by JAK2–fluorescence in situ hybridization (FISH). In contrast, JAK2AMP was detected in 1% or less of primary untreated breast tumors in other independent cohorts.
Patients with JAK2AMP after NAC compared with JAK2NORMAL patients were younger (39.9yo vs 47.6yo), more frequently pre-menopausal (71.4% vs 57.9%), and had little or no anti-tumor response to NAC (Miller & Payne I: 42.9% vs 15.8%). RNA expression of the JAK2-activating ligand interleukin-6 (IL6) was also higher in these patients (p=0.008).
In matched untreated and post-NAC specimens, including 2 patient derived xenograft models generated from a single patient’s pre- and post-NAC specimens, FISH analysis identified a subpopulation of tumor cells with JAK2AMP that was enriched after NAC treatment. Preliminary data from RNA in situ hybridization (RNAScope) showed that tumor cells expressing high levels of JAK2 are distinct from their counterparts, with higher IL6 expression, suggesting a paracrine signaling event.
We also explored the intra-tumor heterogeneity of JAK2AMP in cell lines and patient tumors, performing chemical and genetic loss of function studies in 4 TNBC cell lines: HCC-1143 (JAK2DELETED/LOW), SUM-159PT (JAK2HIGH), HCC-38 (JAK2GAIN, HIGH) and HCC-70 (JAK2AMP, HIGH).
In vitro, IL6 expression after adriamycin and/or docetaxel treatment was higher in the JAK2HIGH cell lines compared with the changes registered in HCC-1143 (around 2, 4 and 100 fold increase from their respective IL-6 basal levels in HCC-38, HCC-70 and SUM159PT respectively, compared with 0.5 fold increase in HCC-1143).Treatment with chemotherapy also abrogated the IL-6 downregulation produced by the JAK1/2 inhibitor ruxolitinib. Ruxolitinib decreased mammosphere formation by 50% in SUM-159PT cells, with a 10% reduction of the CD24low/CD44high stem cell compartment. Interestingly, we observed no change with ruxolitinib treatment in the JAK2GAIN/AMP cell lines. However, in HCC-38, siRNA knockdown of JAK2 reduced the CD24low/CD44high compartment (around 15%) and mammosphere formation (around 80%).
These findings suggest that a JAK2AMP cell population may escape from chemotherapy-induced apoptosis resulting in lack of response to treatment and eventual disease recurrence. Furthermore, chemotherapy may induce a wound-healing response in the tumor, upregulating and/or selecting JAK2AMP cells via a paracrine or autocrine IL-6/JAK1/pSTAT3 signal. In vivo models confirming these observations and further exploring the therapeutic potential of ruxolitinib to treat JAK2AMP TNBCs are underway.
Citation Format: Luis J Schwarz, Justin M Balko, Jennifer M Giltnane, Monica V Estrada, Melinda E Sanders, Kai Wang, Nancy U Lin, Vicent A Miller, Philip Stephens, Roman Yelensky, Joseph A Pinto, Henry L Gómez, Melissa D Landis, Jenny C Chang, Carlos L Arteaga. Enrichment of janus kinase-2 (JAK2)-amplified tumor cell populations in triple-negative breast cancers (TNBC) during chemotherapy treatment [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-03-03.