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BACKGROUND: Deregulation of cellular proliferation represents a defining hallmark of cancer. The mechanisms driving aberrant cell cycle progression across different breast cancer subtypes is highly diverse and likely contributes to therapeutic resistance in a variety of contexts. The significance of cell cycle heterogeneity is most clear in the context of metastatic HR+/HER2- breast cancer, wherein acquired and intrinsic resistance to CDK4/6 inhibition represents a significant clinical challenge. However, aberrant cell cycle regulatory networks likely further confound the use of targeted therapies in the context of TNBC. METHODS AND RESULTS: We employed unbiased analyses of large number of breast cancer models to decipher the genetic requirements for different CDK and Cyclins which drive proliferation and the significance to response to CDK4/6 inhibitors. Breast cancer cells harbor heterogeneity in the requirement for a given G1/S regulatory CDK or Cyclin. These dependencies are conditioned by the expression of the respective CDK or Cyclin, as well as other gene expression or genetic features present within the tumor that could represent putative biomarkers. In general, conventional dependency for CDK4 and Cyclin D1 is strongest in HR+/HER2- breast cancer models, whereas TNBC exhibits dependency on a variety of different CDK or Cyclin genes. Resistance to CDK4/6 inhibitors in HR+/HER2- breast cancer models, while occurring through a number of different primary mechanisms, functionally deregulates the phosphorylation of RB. However, genetically enforcing RB activation is sufficient to block proliferation, underscoring the opportunity to ameliorate resistance to CDK inhibitory strategies. CRISPR screens in HR+/HER2- and TNBC models are identifying new mediators of sensitivity and resistance to CDK4/6 inhibitors. Inherently, elevated expression of Cyclin E and p16INK4A are associated with resistance to multiple therapeutic approaches, including CDK4/6 inhibitors, but denote a requirement for CDK2. The cell cycle conditions disparately dependent on CDK4/D1 vs. CDK2/E can be visualized by multispectral imaging and used to direct CDK inhibitor based strategies. CONCLUSIONS: Together these studies illustrate the complex nature of breast cancer cell cycles which can confound therapy. However, understanding of this heterogeneity can create new opportunities for precision approaches targeting CDK/Cyclin-dependence therapeutically. Citation Format: Erik S. Knudsen, Vishnu M. Kumarasamy, Jianxin Wang, Agnieszka Witkiewicz. Deciphering the heterogeneity of cell cycle in breast cancer and relevance to clinical application of CDK inhibitors [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-10-01.

Background: Breast cancer with low HER2 expression (HER2-low) is of high clinical relevance because of new therapeutic options with antibody-drug conjugates. We have recently shown in a large cohort from neoadjuvant clinical trials that HER2-low breast cancer has different molecular characteristics as well as different clinical outcomes compared to HER2-zero. Considering the positive correlation between HER2-low expression and hormone receptor positivity observed consistently in many investigations, we have extended our analysis to HR+ tumors from the post-neoadjuvant PenelopeB trial. In PenelopeB, patients with HR+ breast cancer and residual disease after neoadjuvant chemotherapy (NACT) were randomized to post-neoadjuvant palbociclib versus placebo in addition to endocrine therapy. We evaluated the molecular phenotype and clinical outcomes of HER2-low compared to HER2-zero patients. Methods: A total of 1250 patients were randomized, HER2 status was available for 1151 tumors from pretherapeutic core biopsy, determined mainly by local pathology, and from 1213 tumors from the post-NACT sample, determined as part of central pathology. For 1119 patients a paired HER2-status was both available. HER2-zero was defined as IHC0 and HER2-low-positive was defined as IHC1+ or IHC2+/ISH-. Gene expression analysis of 2549 genes using the HTG oncology biomarker panel was performed in 620 pretherapeutic biopsies and 780 post-NACT residual tumor samples, with 539 paired gene expression samples. Breast cancer subtypes were determined using the AIMS approach. Results: In pretherapeutic biopsies, 695 tumors (60%) were HER2-low and 457 (40%) were HER2-zero. A HER2-low status in the biopsy was significantly linked to improved iDFS (HR 0.76 (0.60-0.96; p=0.02). In residual tumors, 632 tumors (60%) were HER2-low and 581 (40%) were HER2-zero, without any prognostic impact of HER2 low status. In addition, a shift of HER2-low-status comparing core biopsy and residual tumor was observed in 415 (37%) of 1119 tumors. 161 (14%) had a shift from HER2-zero to HER2-low and 254 (23%) shifted from HER2-low to HER2-zero. A shift from HER2-zero to HER2-low in the post-NACT samples was significantly linked to reduced iDFS (HR 1.43 [95%CI 1.01-2.01]), p=0.04), compared to HER2-low group, while a shift from HER2-low to HER2-zero was associated with better iDFS compared to HER2-zero group, although not statistically significant (p=0.17). We did not observe a significant correlation of HER2-low status and AIMS molecular subtypes. In particular, the HER2-enriched (HER2E) subtype was assigned to only 4.3% of HER2-zero and 3.1% of HER2-low tumors. Significant iDFS differences were observed for HER2-low-status in combination with AIMS subtypes (lumB/basal/HER2E vs. lumA/normL; overall p-value < 0.0001) for both pretherapeutic biopsies and residual tumor. Patients with post-NACT HER2-low tumors had an improved survival in the subgroups of aggressive AIMS subtypes (lumB/basal/HER2E), but not in the less aggressive AIMs subtypes (lumA/normL), with a positive test for interaction (p=0.02). For the pre-NACT samples a similar, but non-significant trend was observed. We evaluated a total of 620 core biopsies for differences in gene expression comparing HER2-low and HER2-zero tumors. A total of 417 genes were statistically significantly different, but in a hierarchical clustering there was no clear separation of HER2-low and HER2-zero tumors. Conclusions: In the PenelopeB cohort of HR+ tumors, a HER2-low status in pretherapeutic core biopsies is related to improved disease-free survival, especially for those tumors that have a more aggressive intrinsic subtype. A shift of HER2-low status was observed before and after chemotherapy, indicating an adaptation of the pathway activity to therapy-induced stress, which might become relevant for future diagnostic and therapeutic approaches. Citation Format: Carsten Denkert, Miguel Martín, Michael Untch, Hervé R. Bonnefoi, Erik S. Knudsen, Seock-Ah Im, Angela DeMichele, Agnieszka Witkiewicz, Laura Van ’t Veer, Sung-Bae Kim, Harry D. Bear, Nicole McCarthy, Karen Gelmon, Frederik Marmé, José Ángel García-Sáenz, Nicholas Turner, Federico Rojo, Martin Filipits, Lesley-Ann Martin, Peter A. Fasching, Christian Schem, Catherine M. Kelly, Toralf Reimer, Masakazu Toi, Hope Rugo, Michael Gnant, Andreas Makris, Yuan Liu, Karsten Weber, Sivaramakrishna Rachakonda, Sibylle Loibl. HER2-06 Outcome analysis of HER2-zero or HER2-low hormone receptor-positive (HR+) breast cancer patients - characterization of the molecular phenotype in combination with molecular subtyping [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr HER2-06.

Fbln5 and α5β1 integrin expression pattern in mouse and human (S1); Characterization of KIC and KPC mice (S2); Examination of ROS levels in normal pancreas and NAC-treated KIC and RGE KIC tumors (S3); Reduced microvessel density (MVD) in RGE KIC and RGE KPC tumors compared to KIC and KPC tumors (S4); Examination and quantification of MVD in pancreatic tissues from KIC and RGE KIC, WT and RGE mice (S5); Fbln5 expression in Fbln5 WT, KO and RGE MEFs (S6); NADPH oxidase (Nox) is not the source of ROS induction for RGE MEFs when plated on FN (S7).

Description of additional methods and procedures used in the study.

Elevated oxidative stress is an aberration seen in many solid tumors, and exploiting this biochemical difference has the potential to enhance the efficacy of anticancer agents. Homeostasis of reactive oxygen species (ROS) is important for normal cell function, but excessive production of ROS can result in cellular toxicity, and therefore ROS levels must be balanced finely. Here, we highlight the relationship between the extracellular matrix and ROS production by reporting a novel function of the matricellular protein Fibulin-5 (Fbln5). We used genetically engineered mouse models of pancreatic ductal adenocarcinoma (PDAC) and found that mutation of the integrin-binding domain of Fbln5 led to decreased tumor growth, increased survival, and enhanced chemoresponse to standard PDAC therapies. Through mechanistic investigations, we found that improved survival was due to increased levels of oxidative stress in Fbln5-mutant tumors. Furthermore, loss of the Fbln5–integrin interaction augmented fibronectin signaling, driving integrin-induced ROS production in a 5-lipooxygenase–dependent manner. These data indicate that Fbln5 promotes PDAC progression by functioning as a molecular rheostat that modulates cell–ECM interactions to reduce ROS production, and thus tip the balance in favor of tumor cell survival and treatment-refractory disease. Cancer Res; 75(23); 5058–69. ©2015 AACR.

Background: In high-risk hormone-receptor (HR)+/HER2- BC patients germline (g) mutations can be found in approximately 14% in BRCA1/2 and in BRCA1/2 and other BC predisposition genes in 20% (Pohl-Rescigno E, et al. JAMA Oncol 2020). In metastatic BC CDK4/6 inhibitors may have greater activity in patients with a BRCA mutation detected in ctDNA (André F, et al. J Clin Oncol 2020). The PENELOPE-B trial did not to show an improved invasive disease-free survival (iDFS) by adding palbociclib to ET in high-risk HR+/HER2- BC (Loibl S, et al. J Clin Oncol 2021). Methods: Blood samples from 898 of 1250 PENELOPE-B patients were available. 445 patients were sampled following a case-cohort design (220 cases defined as patients with any event during follow-up and 225 randomly selected patients without any event [non-cases]) and analyzed for germline variants in BRCA1/2 and 16 non-BRCA1/2 cancer predisposition genes (ATM, BARD1, BRIP1, CDH1, CHEK2, FANCM, MRE11A, NBN, PALB2, PTEN, RAD50, RAD51C, RAD51D, STK11, TP53, XRCC2) by targeted next generation sequencing (NGS). The primary definition of mutational status was the prevalence of a pathogenic mutation (mt) in one or more analyzed BC predisposition genes. Statistical analyses for time-to-event endpoints (iDFS, distant disease-free survival [DDFS], and overall survival [OS]) were based on inverse probability weighting: weighted Cox proportional hazard models and Kaplan-Meier estimates were used. Results: 442 of 445 patients (placebo arm: 104 cases and 105 non-cases; palbociclib arm: 114 cases and 119 non-cases) were successfully analyzed for mutational status. A total of 42 (9.5%) patients (placebo arm: 9.1%; palbociclib arm: 9.9%) carried any mutation. 15 (3.4%) patients had a gBRCA1/2 mt (one of whom carried a gATM mt and one a gCHEK2 mt in addition to gBRCA2 mt) and 29 (6.6%) had mutations in one of the other BC predisposition genes (n=8 CHEK2, n=7 PALB2, n=5 ATM, n=2 RAD50, n=1 for BARD1, FANCM, MRE11A, RAD51C, RAD51D, TP53 and n=1 both RAD51D and BRIP1). The mutational status with respect to all genes analyzed showed no significant correlation to clinical baseline variables. With regard to gBRCA1 and gBRCA2 genes only, the mutational status significantly correlated with age but not with other clinical variables: all 15 (100%) gBRCA mt carriers were younger than 50 years compared to 238 (56%) wildtype (wt) patients (p=0.002). The iDFS rate after 3 years was 80.9% in patients with any mutation and 79.5% in patients without. Mutational status (mt vs. wt) based on all genes analyzed was not prognostic (iDFS: hazard ratio 1.015, 95%CI 0.558-1.784; DDFS: 0.970, 95%CI 0.521-1.758; OS: 0.768, 95%CI 0.274-1.615). Neither the mutated patients had a benefit from palbociclib treatment (palbociclib vs placebo; iDFS: hazard ratio 0.766, 95%CI 0.263-3.022; DDFS: 0.897, 95%CI 0.275-3.489; OS: 0.666, 95%CI 0.063-5.671) nor the wt patients (iDFS: hazard ratio 0.918, 95%CI 0.650-1.303; DDFS: 0.966, 95%CI 0.679-1.393; OS: 0.901, 95%CI 0.573-1.433); interaction tests for treatment arm/mutational status for all time-to-event endpoints were not statistically significant. Analysis in the subgroups of patients by gBRCA1/2 showed similar results but had less statistical power. Conclusions: In this case-cohort analysis of 442 patients enrolled in the PENELOPE-B trial, the detection of BC predisposition genes was lower than expected with 10%. This is probably due to the low rate of gBRCA1/2 carriers (3.4%), which could be influenced by the selection criteria of the trial. Patients with gBRCA1/2 or other BC disposition genes had a comparable outcome to non-carriers in the PENELOPE-B trial. Citation Format: Sibylle Loibl, Jan Hauke, Karen Gelmon, Frederik Marmé, Corinna Ernst, Miguel Martin, Michael Untch, Hervé Bonnefoi, Erik Knudsen, Seock-Ah Im, Angela DeMichele, Laura Van’t Veer, Sung-Bae Kim, Harry Bear, Nicole McCarthy, Nicholas Turner, Agnieszka Witkiewicz, Federico Rojo, Peter A Fasching, José A García-Sáenz, Catherine M Kelly, Toralf Reimer, Masakazu Toi, Hope S Rugo, Carsten Denkert, Michael Gnant, Andreas Makris, Yuan Liu, Olga Valota, Bärbel Felder, Karsten Weber, Valentina Nekljudova, Eric Hahnen. Germline BRCA1/2 and other predisposition genes in high-risk early-stage HR+/HER2- breast cancer (BC) patients treated with endocrine therapy (ET) with or without palbociclib: A secondary analysis from the PENELOPE-B study [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-13-36.

Background: Molecular plasticity of breast cancer is crucial for the development of therapy-resistant disease. In this investigation, we studied changes in molecular signatures between pretherapeutic (pre-Tx) and post-therapeutic (post-NACT) tumor samples from patients included in the PENELOPE-B (NCT01864746) trial. The phase III PENELOPE-B study did not show a significant benefit from palbociclib in women with centrally confirmed HR+, HER2- primary breast cancer without a pathological complete response after taxane-containing neoadjuvant chemotherapy (NACT) and at high-risk of relapse (CPS-EG score ≥3 or 2 and ypN+) (Loibl et al. JCO 2021). However, first translational investigations showed that a small number of patients with a luminal-B tumor subtype, based on absolute intrinsic molecular subtyping (AIMS, Paquet & Hallet, JNCI 2014) subtyping after NACT, had a numerical benefit from post-NACT palbociclib. We have therefore extended the analysis and included a cohort of paired pre-Tx and post-NACT samples. Methods: We investigated gene expression in pre-Tx (n=259) tumor tissue samples using the HTG EdgeSeq Oncology Biomarker Panel including 2549 genes (HTG Molecular Diagnostics Inc.); for the same patients the same panel on post-NACT residual tumor samples were available. The paired samples were selected based on a case-cohort approach. Based on 91 genes of this panel, the AIMS subtype was calculated. In addition, we performed exploratory biomarker analyses to identify genes and gene signatures with prognostic and predictive relevance. After completion of NACT, PENELOPE-B patients were randomized to palbociclib versus placebo in addition to standard endocrine therapy. Results: The prevalence of AIMS subtypes, in particular LumA vs LumB, changed in pre-Tx and post-NACT tumors. In the pre-Tx samples, 115 (44%) and 123 (47%) of tumors had LumA and LumB subtypes, respectively, as expected from a high-risk cohort. However, in the post-NACT samples, LumA tumors were predominant (n=183, 71%) over LumB (n=30; 12%). 78 (30%) and 6 (2%) tumors switched their subtype from LumB to LumA and LumA to LumB, respectively. For further analyses, we compared the groups of low proliferating (LumA and NormL) and high proliferating subtypes (LumB, BasalL and HER2E). In bivariable Cox regression analysis, the grouped pre-Tx and post-NACT AIMS subtypes were independent prognostic factors for iDFS: HR=1.85 (1.16-2.98, p=0.011) for pre-Tx LumB/BasalL/HER2E vs LumA/NormL and HR=2.18 (1.24-3.84, p=0.007) for post-NACT. Similar results were found when adjusted for prognostic clinical factors and for DDFS and OS endpoints although the pre-Tx subtype did not reach significance. These and further Cox models investigating interaction effects show that patients with tumors developing from high (pre-Tx) to low proliferation (post-NACT) had a higher iDFS risk compared to stable low proliferating tumors but a lower iDFS risk compared to stable high proliferating tumors. Neither in the pre-Tx LumB/BasalL/HER2E nor in the pre-Tx LumA/NormL subgroup a benefit from palbociclib was observed. Based on the results of the AIMS subtyping, we extended the exploratory analysis to identify genes that might be involved in the prognostic and predictive effects as well as genes driving the subtype switch. The analysis is ongoing and the relevant genes will be presented at the conference. Conclusions: Our findings show that the switch from high-risk molecular subtypes (in particular LumB) to low-risk subtypes (in particular LumA) is common in neoadjuvant therapy of luminal tumors. The adaptation of luminal high-risk tumors to therapy-induced stress is crucial for the clinical outcome and the results suggest that molecular defined tumor subtypes might not be as stable as originally thought. Citation Format: Carsten Denkert, Frederik Marmé, Miguel Martin, Michael Untch, Hervé Bonnefoi, Sung-Bae Kim, Harry Bear, Agnieszka Witkiewicz, Seock-Ah Im, Angela DeMichele, Laura Van’t Veer, Nicole McCarthy, Thorsten Stiewe, Karen A. Gelmon, José A. García-Sáenz, Catherine M. Kelly, Toralf Reimer, Erik Knudsen, Nicholas Turner, Federico Rojo, Peter A. Fasching, Julia Teply-Szymanski, Yuan Liu, Masakazu Toi, Hope S. Rugo, Michael Gnant, Andreas Makris, Bärbel Felder, Karsten Weber, Sibylle Loibl. Molecular plasticity of luminal breast cancer and response to CDK 4/6 inhibition - The biomarker program of the PENELOPE-B trial investigating post-neoadjuvant palbociclib [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD2-04.

Background: The Penelope-B trial did not show improvement in invasive disease-free survival (iDFS) with the addition of palbociclib to endocrine therapy (ET) in patients with high-risk early breast cancer (BC) after neoadjuvant chemotherapy (NACT). Biomarkers may be able to identify subgroups of patients deriving benefit from Palbociclib and guide future studies. Estrogen-receptor (ER), progesterone-receptor (PgR) and Ki-67 might be helpful in identifying patients benefiting from palbociclib. Concordantly, tumors with elevated expression of Cyclin D1 and phosphorylated retinoblastoma protein (phospho-RB) may harbor more dependency on CDK4/6 and thus higher sensitivity to palbociclib. Methods: The percentage of positive ER and PgR cells and Ki-67 assessed in surgical specimens after NACT were combined to obtain the immunohistochemical score 3 (IHC3, Cuzick et al JCO 2011, low vs high based on the median IHC3 value). Cyclin D1 and phospho-RB Ser 807/811 immunoreactive (phospho-RB) scores were analyzed in residual tumors after NACT (range 0-12 each). Proportional hazard regression model was used to assess the predictive and prognostic value of IHC3 and treatment on iDFS. Subgroup analysis was performed according to BC intrinsic subtypes (luminal-A/normal-like, luminal-B/HER2-enriched/basal) and HER2-status (HER2 0, HER2 low). Cox/Fine-Gray regression was used to define the predictive and prognostic value of CyclinD1 (≤1, >1), phospho-RB (≤2, >2) as dichotomized and continuous variables on iDFS, distant DFS (DDFS), locoregional invasive recurrence-free interval (LRRFI) and overall survival (OS). Multivariate analyses (MVA) were adjusted for age (≤50 vs >50), Ki-67 (≤15 vs >15), region (non-Asian vs Asian), ypN (ypN0-1 vs ypN2-3), risk status (CPS-EG=2 ypN+ vs ≥3), cT (cT1-2 vs cT3-4), ypT (ypT0-2 vs ypT3-4), and grade (G1-2 vs G3). The MVA for IHC3 includes all the covariates above except Ki-67. p< 0.05 was defined as statistically significant. Results: Data for ER, PgR, Ki-67, HER2, Cyclin D1 and phospho-RB were available for 1250 patients. Overall, 98.9% of the patients had ER+ tumors, 75.0% PgR+, 52.2% had HER2 low, 25.5% Ki-67>15, 50% had IHC3 score higher than median, 93.9% had Cyclin D1 >1, 57.8% had phospho-RB >2. Patients with IHC3 score high had a worse iDFS compared to patients with IHC3 score low (MVA HR 2.28 95%CI (1.78-2.91), p< 0.0001). Patients with luminal-A/normal-like tumors and IHC3 low had an improved iDFS with the addition of palbociclib to ET (MVA HR 0.35 95%CI (0.14-0.90), test for interaction p=0.01). No difference was observed according to HER2 status. Cyclin D1>1 has no predictive value but is prognostic for better iDFS (MVA HR 0.62 95%CI (0.41-0.94), p=0.023), LRRFI (MVA HR 0.30 95%CI (0.15-0.63), p=0.001) and OS (MVA HR 0.50 95%CI (0.28-0.89), p=0.019). Similar results were obtained when Cyclin D1 was analysed as a continuous variable. Phospho-RB had neither predictive nor prognostic value. Phospho-RB highly correlates with Ki-67 (p< 0.001, Spearman correlation 0.248). Conclusions: Patients with high Cyclin D1 expression had a favorable prognosis independent of treatment arm, but patients with luminal-A/normal-like tumors and IHC3 low after NACT had an improved outcome when receiving palbociclib in addition to adjuvant ET. Theses exploratory studies suggest specific signatures/phenotypes could predict benefit from Palbociclib in high-risk early breast cancer. Citation Format: Erik S. Knudsen, Sivaramakrishna Rachakonda, Frederik Marmé, Miguel Martín, Michael Untch, Hervé R. Bonnefoi, Wolfgang D. Schmitt, Sung-Bae Kim, Harry D. Bear, Agnieszka Witkiewicz, Seock-Ah Im, Angela DeMichele, Laura Van’t Veer, Nicole McCarthy, Bruno V. Sinn, Karen Gelmon, José Ángel García-Sáenz, Catherine M. Kelly, Toralf Reimer, Nicholas Turner, Federico Rojo, Martin Filipits, Peter A. Fasching, Christian Schem, Lesley-Ann Martin, Yuan Liu, Masakazu Toi, Hope Rugo, Michael Gnant, Andreas Makris, Jenny Furlanetto, Karsten Weber, Carsten Denkert, Sibylle Loibl. Immunohistochemical markers and determinants of clinical response in the Penelope-B trial [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD17-06.

Background: In spite of widespread use and known mechanism of action, predictive biomarkers for the use of CDK4/6 inhibitors in conjunction with endocrine therapy have yet to emerge. Here a cohort of patients treated with standard-of-care combination regimens was utilized to explore features of disease and determinants of progression-free survival (PFS). Patients and Methods: In this cohort of 235 patients, >90% of patients were treated with Palbociclib in combination with either an aromatase inhibitor (AI) or fulvestrant (FUL). The PFS mirrored that observed in randomized clinical trials. A total of 151 patient tumor tissues were used for targeted gene expression analyses with the HTG-Oncology Biomarker Panel. The association of disease state and gene expression analyses were interrogated for disease evolution and association with PFS. Results: HER2 immunohistochemistry (HER20, Her21+, Her22+) was not associated with PFS in full cohort, or AI and FUL subgroup analyses. The lack of progesterone receptor (PR+, PR-), was associated with shorter PFS in the full patient cohort (p=0.012) and selectively in patients treated with AI (p=0.005), but not FUL. Gene expression-based subtyping indicated that the majority of patients, as expected, had luminal breast cancer; however, the predominant subtypes changed with treatment and disease evolution. Primary tissue from tumor resection was dominated by luminal A subtype, which diminished in the context of metastatic disease, and was rare in post-progression specimens. The luminal B, HER2, and basal subtypes exhibited shorter PFS in CDK4/6 inhibitor combinations (AI, p=0.01; FUL, p=0.03). Existing clinically developed breast cancer signatures (e.g. breast cancer index) had variable associations with PFS; however, high expression of gene signatures associated with cell cycle were broadly associated with short PFS. Concordantly, utilizing unbiased analyses, gene expression programs linked to cell cycle were associated with short PFS, while interferon response processes were associated with longer PFS. Algorithms that incorporated standard pathological and clinical variables with the gene expression data were developed that exhibited potent predictive power. Conclusions: Tumor evolution occurs on treatment with CDK4/6 inhibitors; however, analyses of pretreatment biopsies can inform the duration of PFS. These data support discrete biological processes associated with sensitivity/resistance. Predictive algorithms could be developed to inform features of treatment decision which will require prospective validation which is ongoing. Citation Format: Agnieszka Witkiewicz. Biomarker associated with response to CDK4/6 inhibitors in metastatic hormone receptor positive breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-02-08.

Background: The PENELOPE-B (NCT01864746) and PALLAS (NCT02513394) trials are large prospective, randomized, phase III trials that evaluated adjuvant palbociclib (PAL) + endocrine treatment (ET) vs ET in patients with hormone receptor–positive/human epidermal growth factor receptor 2–negative (HER2–) early breast cancer (EBC). Both studies did not meet the primary endpoint of improving invasive disease-free survival (iDFS). We conducted biomarker analyses to identify patients who might benefit from PAL + ET in EBC. Methods: Resected tumor tissue was collected from consenting patients. Gene expression analyses were conducted using the HTG EdgeSeq Oncology Biomarker Panel including 2549 genes. Based on 91 genes from the HTG panel, the intrinsic molecular subtypes were calculated using Absolute Intrinsic Molecular Subtyping (AIMS). Potential predictive treatment biomarkers were established in PENELOPE-B (n=906 with resected tissue) as the development set using an outcome-oriented approach based on iDFS with a selection procedure that maximized the log-rank statistic to estimate a standard Z score–based optimal cutoff. Independent validation was conducted on PALLAS (n=2085; PENELOPE-B-like with resected tissue and HTG data). Hazard ratios and corresponding 95% CIs were calculated using the Cox proportional hazards model, and iDFS distributions between treatment arms were compared using the log-rank test. Interaction between treatment and biomarker status was assessed. Results: Patient baseline characteristics were well balanced, with no differences in iDFS between the intent-to-treat set and the biomarker set for both trials. Approximately 73% of patients (PENELOPE-B [n=663] and PALLAS [n=1516]) had luminal A subtypes whereas only 7.1 % (PENELOPE-B [n=64]) and 8.3 % (PALLAS [n=172]) had a luminal B subtype. AIMS subtypes showed overall similar prognostic patterns for iDFS between PENELOPE-B and PALLAS. The biomarker-defined subgroup found in PENELOPE-B with optimal cutoff demonstrated a preferential benefit from PAL + ET (n=364 [96 events]; hazard ratio [95% CI], 0.63 [0.42, 0.95]; P=0.025). Independent validation of the PALLAS subgroup using the pre-defined optimal cutoff confirmed a significant benefit from PAL + ET (n=916 [70 events]; 0.55 [0.34–0.90]; P=0.015) while not in the rest of the patients (interaction p=0.0025). Significant treatment effects remained (0.55 [0.34–0.89]; P=0.015) after adjusting for the randomization stratification factors of PALLAS. Conclusions: The composite predictive biomarker defined from PENELOPE-B was independently validated in a prospectively defined retrospective analysis of a subset of patients selected from PALLAS. The composite biomarker identified a subset of EBC patients deriving benefit from the addition of PAL to ET. This patient stratification approach can potentially be applied to future adjuvant clinical trials for treatment of hormone receptor–positive/HER2– EBC. Citation Format: Sibylle Loibl, Carsten Denkert, Yuan Liu, Erik S. Knudsen, Angela DeMichele, Zhe Zhang, Julia Teply-Szymanski, Martin Filipits, Peter A. Fasching, Michael Gnant, Shibing Deng, Marija Balic, Federico Rojo, Mark Watson, Chetan Deshpande, Nicholas Turner, Otto Metzger, Kathy Puyana Theall, Agnieszka Witkiewicz, Olga Valota, W. Fraser Symmans, Erica L. Mayer. Development and Validation of a Composite Biomarker Predictive of Palbociclib + Endocrine Treatment Benefit in Early Breast Cancer: PENELOPE-B and PALLAS Trials [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD17-05.

Background: Adrenergic stress (AS) reduces anti-tumor response by decreasing the frequency and function of CD8+ T- cells in the tumor microenvironment (TME), resulting in an increase in those with an “exhausted” phenotype.1 Additionally, AS increases the quantity and immunosuppressive phenotype of myeloid-derived suppressor cells (MDSC) in the TME.2 The data above suggests that β-2 AR acts akin to a tumorigenic IC which can be abrogated by using P, a well-known and highly cost efficient non-selective β-blocker. Synergistic activity of anti-PD-1 with P has been reported in several murine tumor models, including the B-16 OVA mouse model.3,4 A retrospective study has shown an improvement in overall survival (OS) in patients (pts) with metastatic melanoma (MM) treated concurrently with non-selective β-blocker and immunotherapy.5 This formed the basis for our phase I study of the combination of P (at dose levels; 10 mg, 20 mg BID, and 30mg BID) and pem 200 mg every 3 weeks in pts with MM. Our published phase I results found all 3 dose levels of P to be well tolerated, and an objective response was observed in 7/9 pts.6 A decrease in perceived stress score (PSS) in pts over time was observed. Intra-tumor ratio of (CD4+T cells + CD8+T-cells)/(MDSC+ Treg) >1 in the pre-treatment biopsy was predictive of treatment response. Based on the results of the phase I study, we chose P 30 mg BID as the recommended phase II dose. These results, though preliminary, strongly support our subsequent phase II clinical trial. Methods: In this prospective, single-arm, phase II, multicenter trial, pts with unresectable stage III/IV MM and measurable disease per RECIST v1.1 will be treated with P (30 mg BID) + Pem. Pts with active CNS disease, prior therapy with PD-1/PD-L1 inhibitors, or contraindications to β-blocker are excluded. The primary objective is to evaluate the overall response rate (ORR) by immune-modified RECIST v1.1. The secondary objectives are the assessment of progression free survival and OS. A Simon two-stage design will be employed, requiring a minimum of 29 pts (17 in stage 1 and 12 in stage 2) to achieve approximately 80% power to detect a 20% increase (0.35 to 0.55) in the ORR. As an exploratory analysis, we will further report a) Baseline and on-treatment PSS and b) Chronotropic effect of P after 5-minute treadmill walk as a biomarker of response; c) Post therapy changes in the TME, with a 12 week on therapy optional biopsy d) Peripheral blood changes in T cell and MDSC subsets, and cytokines/chemokines. To date, 10 pts have been accrued on the study (NCT0384836). Citation Format: Benjamin Switzer, Manu R. Pandey, Alexandra Valentine, Agnieszka Witkiewicz, Erik Knudsen, Kristopher Attwood, Joseph Tario, Pauline Funchain, Joseph J. Drabick, Hemn Mohammadpour, Marc S. Ernstoff, Igor Puzanov, Elizabeth A. Repasky, Shipra Gandhi. β-2 adrenergic receptor (AR): Another immune checkpoint (IC)" A phase II clinical trial of propranolol (P) with pembrolizumab (Pem) in patients with unresectable stage III and stage IV melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT568.

Background: Up to 75% of ovarian cancers will become platinum resistant and survival following the development of resistance is typically less than one year. New therapies with higher curative rates are needed to improve prognosis. There has been increasing interest in inhibitors of sodium glucose cotransporter-2 (SGLT2) in suppressing tumor growth. Use of SGLT2 inhibitors show promise not only due to their ability to inhibit glucose uptake, but also from the subsequent improvements in other metabolic disorders that may affect cancer prognosis. Purpose: We evaluated the associations of tumor SGLT2 protein expression with survival following diagnosis of incident epithelial ovarian cancer (EOC). Methods: Tumor samples were obtained from 492 patients undergoing debulking surgery for primary EOC at Roswell Park Comprehensive Cancer Center between 1995 and 2002. Only incident cases and patients who did not receive neo-adjuvant chemotherapy were included in analyses (n=396). Tissue samples were compiled into tissue microarray blocks that were sectioned, stained, imaged, and analyzed for SGLT2 expression. Data on patient, clinical, and tumor characteristics, along with available information on components of metabolic disorders, were linked to each patient. Multivariate Cox proportional hazards models were performed to determine hazards ratios (HR) and 95% confidence intervals (CI) for EOC-specific and all-cause mortality. Results: In our analysis of 396 EOC patients seen at Roswell Park, we observed significant associations between high SGLT2 expression and decreased risk of both EOC-specific (HR: 0.68; 95% CI: 0.49-0.94) and all-cause mortality (HR: 0.71; 95% CI: 0.53-0.95). In particular, among those with advanced-stage tumors, there was a significant association between high SGLT2 expression and lower risk of EOC-specific (HR: 0.61; 95% CI: 0.42-0.91) and all-cause mortality (HR: 0.64; 95% CI: 0.45-0.90). A lower risk of all-cause mortality was observed among those with high-grade tumors (HR: 0.69; 95% CI: 0.48-0.98). Trends of increasing SGLT2 associated with decreased risk of mortality varied by histological subtype. The inverse association between high SGLT2 and EOC-death was stronger among those with a history of hyperlipidemia (HR: 0.46; 95% CI: 0.19-0.99) and obesity (HR: 0.43; 95% CI: 0.23-0.82) compared to other metabolic syndrome components. These associations were less robust for all-cause mortality. Conclusion: Contrary to our initial hypothesis, higher SGLT2 expression among EOC patients may be associated with more favorable prognosis. This association may be stronger for high grade and advanced stage tumors as well as non-serous subtypes. Identifying other factors associated with levels of expression could aid in the development of potential screening interventions and more personalized treatment. Citation Format: Jennifer M. Mongiovi, Jo L. Freudenheim, Jeff C. Miecznikowski, Susan E. McCann, Ashley E. Stenzel, Wiam Bshara, Agnieszka Witkiewicz, Leighton Stein, Lisa D. Kellenberger, Ahmad Al Sulimani, Kirsten B. Moysich. Associations of sodium glucose cotransporter-2 (SGLT2), metabolic disorders, and survival following incident epithelial ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6402.

Inactivating mutations in TP53/P16 are found in most types of non-small cell lung cancer while RB1/P16 losspredominates in small cell lung cancer (SCLC). NRF2 activating mutations are also found frequently in lungcancer, especially in lung squamous cell carcinoma. However, how concurrent mutations in these genes maycontribute to lung cancer development is not fully understood. To address this problem, we compared lung tumordevelopment in a genetically-engineered mouse model (GEMM) with dual Trp53/p16 deficiency, +/- expressionof one of the most common mutations in NRF2 found in human tumors, Nrf2E79Q/+. Surprisingly, both groups,Nrf2E79Q/+ and Nrf2+/+, developed combined-SCLC (C-SCLC): a mixture of SCLC and large cell neuroendocrine(NE) carcinoma along with pure-SCLC (P-SCLC). The appearance of C-SCLC implicates Trp53/p16 double lossin the development of this type of lung cancer. Both groups developed C-SCLC at the same penetrance andincidence; all tumors labeled positive for NE-markers by immunohistochemistry (IHC) including ASCL1, SYP andINSM1. However, C-SCLCs developed by Nrf2E79Q/+ mice did not show NRF2 labeling by IHC, despiterecombination of the Nrf2E79Q/+ allele. In contrast, the Nrf2E79Q/+ mice showed significantly higher incidence of P-SCLC compared to Nrf2+/+ mice. All P-SCLC from Nrf2E79Q/+ mice were NRF2-positive by IHC, while the fewtumors developed by Nrf2+/+ mice were negative. All P-SCLC lesions labeled positive for NE-markers includingASCL1, CGRP & SYP. Both C-SCLCs & P-SCLCs were positive for NKX1.2 (lung cancer-marker) and negativefor P63 (squamous cell-marker). Interestingly, phospho-RB1 was positive for both C-SCLCs & P-SCLCs,suggesting the loss of p16 in our GEMM inactivates Rb1 through loss of inhibition of the cyclin dependent kinases4/6 (CDK4/6). This is the first study showing that the concurrent inactivation of Trp53/p16 in mice drivesdevelopment of C-SCLC. Our study also implicated activation of NRF2 signaling in the progression of SCLC.Our next studies will dissect the mechanisms by which NRF2 activation contributes to the development of SCLCdevelopment. Citation Format: Samera Hamad, Stephanie A. Montgomery, Jeremy M. Simon, Brittany M. Bowman, Kyle B. Spainhower, Ryan M. Murphy, Agnieszka Witkiewicz, Suzanne E. Fenton, Scott H. Randell, Neil Hayes, Erik Knudsen, Trudy G. Oliver, Ben Major, Bernard E. Weissman. The Nrf2E79Q activating mutation accelerates growth of pure-small cell lung cancer but not combined small cell lung cancer Tp53floxed/floxed/Cdkn2afloxed/floxed mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 921.

Efficient anaerobic digestion (AD) of sludge is crucial for the overall energy balance, minimization of carbon footprint, and the economy for most wastewater-treatment plants (WWTPs). In recent yea ...

Background The spectrum of clinical findings associated with PTEN tumor suppressor gene germline mutations, referred to as PTEN hamartoma-tumor syndrome (PHTS), includes Cowden and Bannayan-Riley-Ruvalcaba syndromes. Although the skin is the ectodermal structure most often affected by these autosomal dominant genodermatoses, abnormalities of neural tissues are frequently observed. Observations We describe a 5-year-old boy with macrocephaly, prominent corneal nerves, and progressive development of multiple painful, dome-shaped, translucent pink to skin-colored papules on the vermilion portion of the upper lip, fingers, palms, and shins. Histologic evaluation demonstrated dermal proliferation of well-demarcated nerve bundles associated with abundant mucin and surrounded by a distinct perineural sheath, findings diagnostic of a nonencapsulated neuroma. Genetic analysis revealed a novel heterozygous germline nonsense mutation in PTEN , predicted to result in a truncated PTEN protein. To our knowledge, this represents the first report of multiple neuromas as the sole mucocutaneous manifestation of PHTS. Conclusions This article highlights neuromas as a cutaneous sign of PHTS, drawing attention to manifestations of PHTS in neural tissues of the skin, eye, gastrointestinal tract, and brain. Along with multiple endocrine neoplasia type 2B, PHTS should be considered in the differential diagnosis of multiple mucocutaneous neuromas, particularly those involving extrafacial sites.

A 4-year-old girl presented with a 3-year history of demarcated, salmon-pink, hyperkeratotic plaques, which were symmetrically distributed on the elbows, knees, ankles, and dorsal aspects of the hands and feet. A diffuse, orange-pink palmoplantar keratoderma was also evident. Clinical and histologic findings were consistent with a diagnosis of pityriasis rubra pilaris (PRP), type IV (circumscribed juvenile). Type IV PRP develops in prepubertal children, is typically localized to the distal aspects of the extremities, and has an unpredictable course. Although ultraviolet (UV) radiation can potentially exacerbate PRP, our patient has improved with broad-band UVB phototherapy.

A 3-year-old girl presented with a 6-month history of multiple, light-pink, flat-topped papules over the dorsal aspects of the metacarpophalangeal and interphalangeal joints of the hands and feet. Nailfold telangiectases, ragged cuticles, and a heliotrope color of the upper eyelids were also evident, but there was no clinical evidence of muscle weakness and levels of muscle enzymes were normal. A biopsy specimen from one of the papules showed a vacuolar interface dermatitis consistent with a diagnosis of dermatomyositis. This report draws attention to juvenile amyopathic dermatomyositis, which is an uncommon subtype of dermatomyositis with an excellent prognosis.

We compared 4 breast cylindromas with 50 dermal cylindromas and 8 adenoid cystic breast carcinomas. Except for a modest increase in the number of eccrine ducts and reactive Langerhans cells in dermal cylindromas, breast and dermal cylindromas showed identical histologic and immunohistochemical features. Both were characterized by epithelial islands containing central basaloid cells and peripheral myoepithelial cells surrounded by a thickened, continuous, periodic acid–Schiff–positive basement membrane that was immunoreactive for collagen IV. Clusters of sebaceous cells and a few eccrine ducts are described in breast cylindromas. Cytokeratin 7 labeled predominantly the central basaloid cells, and smooth muscle actin stained peripheral myoepithelial cells in breast and dermal cylindromas. Eccrine ducts were highlighted by epithelial membrane antigen and carcinoembryonic antigen. S-100 protein and CD1a showed a variable number of dendritic Langerhans cells. Cylindromas of the breast and skin did not express cytokeratin 20, gross cystic disease fluid protein 15, or estrogen or progesterone receptor. Breast cylindroma might be confused with the solid variant of adenoid cystic carcinoma, especially in needle core biopsy specimens, because they share nodular and trabecular patterns, basaloid cells, myoepithelial cells, eccrine ducts, and hyaline globules of basement membrane material. However, adenoid cystic carcinoma displays an infiltrative growth pattern, cytologic atypia, and mitotic figures and lacks the continuous, thickened basement membrane.

A 4-year-old girl presented with a 3-year history of demarcated, salmon-pink, hyperkeratotic plaques, which were symmetrically distributed on the elbows, knees, ankles, and dorsal aspects of the hands and feet. A diffuse, orange-pink palmoplantar keratoderma was also evident. Clinical and histologic findings were consistent with a diagnosis of pityriasis rubra pilaris (PRP), type IV (circumscribed juvenile). Type IV PRP develops in prepubertal children, is typically localized to the distal aspects of the extremities, and has an unpredictable course. Although ultraviolet (UV) radiation can potentially exacerbate PRP, our patient has improved with broad-band UVB phototherapy.