IHC Status Research Articles

HISTOMORPHOLOGIC SPECTRUM OF HODGKIN AND NON-HODGKIN LYMPHOMAS ACCORDING TO WHO CLASSIFICATION 2016 IN A TERTIARY CARE HOSPITAL IN MAHARASHTRA

Introduction: Lymphoma is a group of lymphoproliferative malignancies that can originate in B lymphocytes, T lymphocytes, or natural killer (NK) lymphocytes. They account for 3% of all cancer types. The WHO's 2008 World Health Organization (WHO) classification of hematopoietic and lymphoid tumors was refined in 2016. This study screened surgically excised or biopsied specimens for lymphoma in lymphadenopathy. This research aimed to classify lymphomas in India based on histopathological and immunohistochemical findings. Methods: The study included 71 cases suspected/probable/proven to have lymphomas and H and E-suggestive lymphoid malignancies were recruited. Clinical details, histopathological reports, and lymphoma panel IHC status were analyzed. Age, sex, biopsy site, B-symptom history, and clinical findings were recorded. Paraffin blocks were prepared from biopsy specimens in buffered formalin using standard automated methods. All cases' slides were examined to confirm lymphoma. Tissue blocks were selected for IHC staining. All the slides were evaluated and classified as per the WHO 2016 lymphoma classification. Results: The mean age of the patients was 44.58 years, and 54 patients were males, while 17 were females. 61 of 71 cases were detected to be of NHL and 10 cases were of HL. The majority of NHL cases were formed by B cell neoplasms, constituting 72.1%, whereas T cell neoplasms constituted 24.6%. 2 of 61 cases showed CD56 positivity apart from T-cell markers and were categorized as T/NK cell NHL. As per WHO classification, different types of NHL and HL were classified. Among NHL, follicular lymphoma (FL) was predominant with 15 cases, while in CHL, mixed cellularity CHL prevalence was high. Ki67 expression in 15 cases of FL showed 1 case of grade 1, 3 cases of grade 2 and 2 cases of grade 3 follicular lymphoma with a Ki67 index of more than 40%. Conclusion: Histomorphology and immunohistochemistry helps diagnose lymphoma. NHL was more common among nodal and extranodal lymphomas. The NHL and HL were dominated by men. Follicular lymphoma (FL) was the most common subtype of B-cell NHL. Most HL patients had mixed-cellularity CHL.

Clinical Validation of MCL35 in Mantle Cell Lymphoma Patients ≥65 Years Receiving Bendamustine-Rituximab

Clinical Validation of MCL35 in Mantle Cell Lymphoma Patients ≥65 Years Receiving Bendamustine-Rituximab

EGFR High Copy Number Together With High EGFR Protein Expression Predicts Improved Outcome for Cetuximab-based Therapy in Squamous Cell Lung Cancer: Analysis From SWOG S0819, a Phase III Trial of Chemotherapy With or Without Cetuximab in Advanced NSCLC

BackgroundThe phase III S0819 trial investigated addition of cetuximab to first-line chemotherapy (CT) in NSCLC. Subgroup analyses suggested an OS benefit among patients with EGFR copy number gain in squamous cell carcinomas (SCC), (HR = 0.58 [0.39-0.86], P = .0071). A more detailed model based on EGFR FISH, EGFR IHC and KRAS mutation status was evaluated to yield a more precise predictive paradigm of cetuximab-based therapy in advanced NSCLC. MethodsFISH was performed using the Colorado Scoring Criteria; H-Score was used to quantify EGFR IHC expression (cut-off ≥ 200). A Cox model was used to assess treatment effects for OS and PFS within biomarker and clinical subgroups. KRAS mutation was analyzed using Therascreen. The false discovery rate controlled for multiple comparisons. S0819 ClinicalTrials.gov Identifier: NCT00946712. ResultsOf 1,313 eligible patients, assay results were obtained for FISH on 976 patients (41% positive), for IHC on 945 patients (31% positive), and KRAS mutation status on 627 patients (26% positive). In SCC patients, OS was significantly improved with addition of cetuximab when both EGFR FISH and EGFR IHC were positive (N = 58), (OS HR: 0.32 [95% CI 0.18-0.59]; P = .0002, q = 0.08), median 12.6 versus 4.6 months. The results were independent of KRAS mutation status. In Non-SCC, no predictive value of EGFR IHC, EGFR FISH status and/or KRAS status was seen. ConclusionsIn NSCLC SCC, a combination index of EGFR FISH plus EGFR IHC results was associated with improved OS when cetuximab was added to CT, representing a potential predictive molecular paradigm for patients suitable for EGFR-antibody therapy.

271P First-in-human, phase I dose escalation and expansion study of anti-HER2 ADC MRG002 in patients with HER2 positive solid tumors

MRG002 is an ADC composed of a humanized anti-HER2 mAb conjugated to MMAE via a vclinker. MRG002 is presently being investigated as monotherapy in an ongoing phase I study for safety, tolerability, PK, and preliminary antitumor activity in patients (pts) with HER2+ advanced or metastatic solid tumors. In phase Ia dose escalation utilizing a “3+3” design, a total of 25 pts with BC (19), SGC (3), GC (2), and CRC (1) were enrolled to identify MTD/RP2D. The median age was 53 (30, 66) years. The median number of prior therapies was 5 (1, 11). The starting dose of MRG002 was 0.3 mg/kg, followed by 0.6, 1.2, 1.8, 2.2, 2.6, and 3.0 mg/kg, all pts with BC and GC had HER2+ per CAP guidelines. For the other cancers, pts had an IHC status of 2+ or 3+, regardless of HER2 FISH results. Each pt received MRG002 Q3W for a maximum of 8 cycles. Commonly observed TRAEs were neutrophil count decrease (56%), AST increase (56%), WBC decrease (56%), lactate dehydrogenase increase (44%), alopecia (44%), CPK increase (40%), ALT increase (36%), triglyceride increase (32%), cholesterol increase (32%), GGT increase (32%), appetite decrease (32%), and hypoaesthesia (32%). Majority of AEs were mild to moderate in severity. The most commonly reported TRAEs ≥ Grade 3 were neutrophil count decrease and triglycerides increase in 3 pts (12%), respectively. The treatment-related SAEs were reported in 6 pts (24%) including GGT increase, AST/ALT increase, neutrophil count decrease, LEVF decrease, fatigue, peripheral neuropathy. There was no death due to AEs. Among 21 pts who had at least one tumor assessment, 9 PR (43%), 8 SD (38%), and 4 PD (19%). The investigator assessed ORR was 43% and the DCR was 81%. Of these 21 pts, 16 pts were HER2+ breast cancer, 8 PR, 5 SD, and ORR was 50% (8/16), DCR was 81%(13/16). There were two DLTs at 3.0 mg/kg including LVEF decrease and CPK elevation. Accordingly, the RP2D determined at 2.6 mg/kg which is currently being evaluated in the phase Ib dose expansion. The dose escalation of MRG002 showed manageable safety profile and encouraging antitumor activity in pts with HER2+ solid tumors including BC, GC and other cancer types, which will be further investigated in phase II studies.

PMS2 is the most frequently mutated Lynch syndrome (LS) gene in women with endometrial cancer (EC): what is the role that low penetrance LS genes play in EC?

Objectives: To determine the frequency and spectrum of LS pathogenic variants in EC patients using upfront germline testing for cancer susceptibility. Secondary objectives included determining the sensitivity and specificity of current LS screening. Methods: Women diagnosed with EC were prospectively enrolled at 9 institutions. Clinical germline testing (panel testing for common cancer susceptibility genes) was performed for 840 unselected EC patients between 10/1/2017 and 10/1/2020. Mismatch repair (MMR) IHC, MLH1 methylation status and relevant clinicopathologic data were abstracted from patients’ records. Detailed family history data were obtained for all subjects. Results: Germline testing revealed 26 of 840 EC patients (3.1%) had a pathogenic/likely pathogenic MMR gene mutation. Mutations in PMS2 were most frequent (10/26, 38%), followed by MSH6 (8/26, 31%), MSH2 (6/26, 23%), and MLH1 (2/26, 8%). Mean BMI for PMS2 carriers was greater than other mutation carriers (31.3 vs 27.8). Age at diagnosis was also higher for PMS2 carriers (59 vs 52). IHC combined with MLH1 methylation correctly predicted LS for all MLH1 and MSH2 mutation carriers. For MSH6 and PMS2, IHC and germline mutation status concordance was lower. Only 4/9 (44%) PMS2 carriers had isolated PMS2 loss. IHC was not completed for one PMS2 carrier (insufficient tumor). IHC correctly predicted MSH6 mutations in 7/8 (88%) carriers. Family history did not predict LS for PMS2 carriers: using PREMM5 (LS prediction model), all had low ( Conclusions: This is the largest cohort of prospectively enrolled EC patients to undergo germline genetic testing in an unselected manner, irrespective of patient age, family history or tumor results. It shows that a majority of LS mutations in EC are in the low-penetrance genes, MSH6 and PMS2. PMS2 mutations were the most common, 10/840 (1.2%). Universal screening with IHC would have missed three PMS2 carriers and one MSH6 carrier. Accurate identification of patients with MMR defects is critical not only for identification of LS, but potentially in consideration of treatment options. In the past, ascertainment of LS relied largely on a strong family cancer history, which is most often seen with MLH1 and MSH2 mutations. For all PMS2 carriers in our study, family history was not predictive of LS and the proband's EC diagnosis was the sentinel event leading to a LS diagnosis in the family. Our study may highlight an evolving LS phenotype in which gene/environment interactions are changing. The observation that EC patients with PMS2 mutations have higher BMIs leads us to hypothesize the existence of an obesity-mediated pathway important in PMS2-associated EC. It may be that in the general population, PMS2 mutation carriers face higher risks for EC than colon cancer.

Abstract 483: TIGIT/PVR/NECTIN immune checkpoint axis expression across breast cancer subtypes

Abstract Background: Targeting the PD1:PD-L1 pathway revolutionized treatment options for patients with metastatic triple negative breast cancer (TNBC). Other immune checkpoint inhibitors are being investigated in clinical settings, such as anti-TIGIT antibodies. The goal of this analysis was to evaluate the expression of the TIGIT/PVR/NECTIN molecule family in breast cancer subtypes. Methods: We used the publicly available METABRIC dataset (Curtis C, Nature 2012; Pereira B, Nature Comm 2016) to evaluate microarray RNA expression levels for TIGIT, CD226, PVR, PVRIG, NECTIN2, NECTIN3, NECTIN4, PDCD1 and CD274 from primary breast tumors annotated with ER, PR and HER2 IHC status (Hormone receptor positive [HR+]: n=1369; HER2 amplified [HER2+]: n=236; TNBC: n=299). Biomarker association with overall survival (OS) was evaluated using Cox proportional hazard analysis adjusted for clinical covariates. Results: Expression of the TIGIT/PVR/NECTIN members revealed three expression patterns across breast cancer subtypes: 1) highest in TNBC, followed by HER2+ and then by HR+ BC (PVR); 2) similar levels in TNBC and HER2+, and higher than HR+ BC (TIGIT, CD226, PVRIG, NECTIN4); 3) highest in HR+, followed by HER2+ and then by TNBC (NECTIN2). NECTIN3 showed the lowest expression differences across subtypes. Correlation of these genes with each other varied: 1) Receptors TIGIT, CD226 and PVRIG were moderately correlated with each other and with PDCD1 (Pearson correlation index [r]: 0.45 - 0.78) and strongly correlated with the T cell markers CD3E, CD8A, and GZMB (r: 0.53 - 0.90); 2) PVR family ligands PVR, NECTIN2, NECTIN3 and NECTIN4 were not correlated with each other, nor to the receptors or biomarkers for T-cell, macrophage or stromal biology (r: -0.36 - 0.30). CD274, the PD1 ligand, was weakly associated with TIGIT, CD226, PVRIG, PDCD1 and with immune cell markers (0.25 - 0.38). This pattern was similar across breast cancer subtypes. No OS prognostic value was observed for the TIGIT/PVR family members in HR+ or HER2+ BC. Only CD226 was associated with an improved OS in TNBC (HR: 0.69 [0.48-0.99]). As expected, PDCD1, CD274, and T-cell biomarkers were linked to improved OS prognosis (HR: 0.61-0.69), observed only in TNBC. Conclusions: Overall, RNA expression levels of TIGIT/PVR/NECTIN family members are higher in HER2+ and TNBC compared to HR+ in the large breast cancer METABRIC dataset. While TIGIT, CD226 and PVRIG are clearly associated with T cells, the cell subsets expressing the PVR/NECTIN ligands remain to be defined in breast cancer. Our analysis suggests that TNBC and HER2+ are the breast cancer subtypes more likely to benefit from anti-TIGIT therapeutic targeting. Citation Format: Kelly J. DuPree, Luciana Molinero. TIGIT/PVR/NECTIN immune checkpoint axis expression across breast cancer subtypes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 483.

Radiation Treatment Breaks: Are They Detrimental to Outcomes for Oropharyngeal Carcinoma Treated with Definitive Chemoradiation?

Varying practices exist to account for radiation treatment breaks (rTBs) resulting from institutional holidays during treatment of oropharyngeal squamous cell carcinoma (OPSCC) cancer patients undergoing concurrent chemoradiation (CRT). Studies in radiation biology suggest that local therapy delays may result in accelerated repopulation of residual tumor cells, however the clinical effects of this model in the setting of concurrent systemic therapy are unclear. We, therefore, evaluated the effect of rTBs in patients who received CRT on disease-free survival (DFS) in a consecutive cohort of patients with OPSCC. Patients with recurrent OPSCC, M1 disease, less than 6 months follow-up, or those treated with surgery or RT alone were excluded. Data collected included age, sex, KPS, smoking status, T stage, N stage, p16 IHC or HPV ISH status, RT dose and fractions, systemic therapy, total treatment duration, number of total and cumulative rTBs, overall survival/local/regional/distant failure (calculated from start of RT). rTBs were defined as any breaks taken during RT. Cumulative rTBs were defined as largest number of consecutive rTBs, not including weekends. The Kaplan-Meier method was used to estimate time-to-event outcomes on univariate analysis (UVA) and the Cox proportional hazards model was used to determine the effects of covariates on multivariate analysis (MVA). 462 OPSCC patients meeting inclusion criteria treated between 1/1/2010 and 12/31/2015. Median follow up was 5 years (6 months -10 yrs). Median total treatment duration was 48 days (41-64 days). 457 (99%) patients received 70 Gy of RT, 1 patient received a 400 cGy boost, and 4 patients received 66-68 Gy. 54 (12%) patients received induction chemotherapy. 369 (80%) patients received a cisplatin-based regimen and 93 (20%) received a non-cisplatin regimen. 345 (75%) were HPV or p16 positive, 45 (10%) were negative, and 72 (15%) were unknown. Table 1 describes the rTBs experienced by our cohort and results of our analysis. On UVA, only cumulative rTBs of 3 days were found to be significant (p = 0.04). On MVA, however, treatment duration, total rTBs, and cumulative rTBs were not significantly associated with DFS. We did not find a significant decrease in DFS for patients who had rTBs of 3 days in the setting of CRT for OPSCC. However, extended consecutive breaks (>3 days) may be associated with poorer outcomes, warranting further investigation.Abstract 3986; TableResults of UVA and MVA for DFSn (%)UVAMVATotal Breaks (days) 0140 (30) 1166 (36)p = 0.21p = 0.63HR 1.68 [0.21-13.65] 279 (17)p = 0.72p = 0.71HR 1.48 [0.19-11.44] 329 (6)p = 0.95p = 0.96HR 0.93 [0.10-9.32] 417 (4)p = 0.41p = 0.51HR 0.42 [0.03-5.70] 5+*31 (7)p = 0.10p = 0.81HR 1.34 [0.12-15.24]Cumulative Breaks (days) 0147 (32) 1256 (55)p = 0.34p = 0.96HR 0.92 [0.12-7.03] 225 (5)p = 0.53p = 0.71HR 1.55 [0.16-14.59] 314 (3)p = 0.04p = 1.00HR 1.00 [0.10-10.03] 410 (2)p = 0.52p = 0.83HR 0.73 [0.04-13.08] 510 (2)p = 0.38p = 65HR 1.83 [0.14-24.79]*Range 5-14 days. Open table in a new tab

New potential therapeutic-sequence strategies of TDM1 in the revolutionary era of metastatic HER2-positive breast cancer treatment: A monocentric retrospective experience.

e13014 Background: EMILIA study showed benefits in terms of both progression free survival (PFS) and overall survival (OS) administering Ado-trastuzumab emtansine (T-DM1) vs lapatinib-capecitabine in HER2+ mBC patients (pts) treated at least with a previous trastuzumab-taxane-based therapy. However a paucity of data is available on TDM1 efficacy after dual anti HER2 blockade with pertuzumab and trastuzumab (PT). Instead TH3RESA trial revealed the positive impact on OS of T-DM1 also in heavily pre-treated (at least 2 lines of T, lapatinib and taxanes regimens) pts. We conducted a retrospective analysis reporting innovative findings on the sequence of anti-HER2 treatments and how it could be potentially optimized. Methods: Between June 15, 2014 and January 31, 2020 we identified HER2+ (IHC: 3+ or 2+/FISH amplified) mBC pts treated with T-DM1, either as second-line after progression on dual blockade PT (Cohort PT) or after ≤ 3 anti her2-combined regimens (trastuzumab o lapatinib plus chemotherapy) (Cohort T). 74 pts received T-DM1: Cohort T n = 34 (all females, median age = 52,7), Cohort PT n = 40 (39 females, 1 male, median age = 52,2). Within Cohort T: 64,7 % of pts (n = 22) received T-DM1 after 1 previous anti her2-combined regimen, 20,6 % (n = 7) and 14,7 % (n = 5) in third and fourth lines, respectively. Instead, the whole Cohort PT received second line T-DM1 after pertuzumab/trastuzumab. Results: Median progression- free survival was 10,2 in Cohort T and 3,7 months in Cohort PT. As regards the best response rate, 8,8 % (n = 3/34) and 52,5 % (n = 21/40) of pts reported a progressive disease (PD) into the Cohorts T and PT, respectively: stable disease (SD) 38,2 % (n = 13/34) versus 15 % (n = 6/40); partial response (PR) 23,5 % (n = 8/34) vs 15 % (n = 6/40); complete response (CR) 23,5 % (n = 8/34) vs 12,5 % (n = 5/40). Almost 2/3 (n = 21/34) of Cohort T and half (n = 20/40) of Cohort PT pts had a 3+ IHC status. Conclusions: Our data suggest a lower efficacy of T-DM1 after progression on dual HER2-blockade PT. After all, EMILIA trial did not robustly evaluate the role of T-DM1 in this specific subset of pts. Obviously, larger prospective studies are necessary in order to optimize the best sequence strategy in the treatment of metastatic HER2+ BC, in light of the increasingly innovative anti-HER2 agents.

PD-L1 positive esophagogastric (EG) cancer is associated with distinct bacteria.

4568 Background: Pembrolizumab is approved for chemotherapy-refractory PD-L1 CPS >1 mEG cancer. In clinical trials, pts with MSI-H, EBV+ and PD-L1 CPS >10 EG cancers derive the greatest benefit with immune checkpoint blockade (ICB). Pre-clinical data suggest that the gut microbiome modulates response to ICB; however, the EG cancer microbiome has not been characterized in EG cancer with respect to PD-L1 and MSI-H status. Therefore, we evaluated the EG tumor microbiome in the context of PD-L1 expression in order to define biologically unique EG tumor phenotypes for future therapeutic development. Methods: Clinical and pathologic characteristics, including age, stage at diagnosis, tumor PD-L1 CPS, HER2 IHC, EBV ISH, genomic analysis, treatment history and survival status were reviewed. CPS was stratified a priori using cutoffs of >1/>10/>20 due to biologic differences. MSK-IMPACT, a capture-based next-generation sequencing platform that detects mutations, copy-number alterations, and select fusions was used to detect non-human bacterial reads identified in the NCBI NT database. Bacterial species found in >2 pts were analyzed and stratified by highest PD-L1 CPS score for each individual patient (Vanderbilt, AMP 2018) and Bonferroni correction was used for odds ratio (OR) confidence intervals where each unique species was considered an independent hypothesis. Results: Molecular data from 311 pts was clinically annotated. PD-L1 results (Table) correlated with bacterial species identified on tumor sequencing. PD-L1 CPS >1 was associated with Selenomonas sputigena (OR: 8.2, 95% CI:1.2-53.6), and PD-L1 CPS >20 was associated with presence of Bifidobacterium dentium (OR: 7.4, 95% CI:1.1-48.5) and Prevotella denticola (OR: 4.2, 95% CI: 1.1-16.6) after multiple comparison correction for the 166 bacterial species identified in the cohort. No differences were seen between PD-L1 < 10 vs >10. Four patients were also found to have EBV+ tumors using this approach, including the 1/54 patients identified by EBER ISH. Conclusions: PD-L1 > 20 EG cancer represents a biologically unique subset, enriched for Bifidobacterium dentium and Prevotella denticola. Correlation between PD-L1 expression, microbial and immune environment, and survival on ICB is underway. [Table: see text]

First-in-human phase I study of anti-HER2 ADC MRG002 in patients with relapsed/refractory solid tumors.

TPS1101 Background: MRG002 is an antibody drug conjugate (ADC) composed of a humanized anti-HER2 IgG1 monoclonal antibody conjugated to a microtubule disrupting agent monomethyl auristatin E (MMAE). MRG002 is presently being investigated in an ongoing phase I study for safety, tolerability, pharmacokinetics (PK), and preliminary antitumor activity in patients (pts) with solid tumors. Methods: MRG002 is evaluated as monotherapy for the treatment of pts with confirmed HER2 positive locally advanced or metastatic cancers, including breast cancer (BC), gastric cancer (GC), salivary gland cancer (SGC) and others. The primary objective is to determine the maximum tolerated dose (MTD) and a recommended phase II dose (RP2D). Secondary objectives include evaluation of PK, tumor response, and immunogenicity. In the dose escalation phase with “3+3” design, approximately 24 pts will be enrolled to identify MTD. The starting dose of MRG002 is 0.3 mg/kg, followed by 0.6, 1.2, 1.8, 2.2, 2.6, and 3.0 mg/kg. In the dose expansion phase, about 50 pts with HER2 positive advanced cancers will be enrolled to further evaluate the safety, antitumor activity, and PK at an appropriate confirmed dose. In this phase I study, each pt receives single agent MRG002 once every 3 weeks (Q3W) for a maximum of 8 treatment cycles. Pts with BC and GC should have HER2 positive advanced solid tumors per College of American Pathologists (CAP) guidelines. For other cancers, pts must have IHC status of 2+ or 3+, regardless of FISH results. Pts should have either failed or are ineligible for standard treatments. All pts must have at least 1 measurable lesion per RECIST 1.1. ECOG should be 0-1, and bone marrow, hepatic, renal, cardiac functions should be adequate. Observations include adverse events (AEs), dose-limiting toxicity (DLT), and antitumor activity, which is assessed every two treatment cycles. Further clinical trial details can be found on chinadrugtrials.org.cn (CTR20181778). Enrollment is ongoing since November 2018. Clinical trial information: CTR20181778 .

Biological analysis of HER2 equivocal (2+) cases in primary breast cancer.

e12555 Background: Overexpression and/or amplification of HER2 is observed in 15–20% of all invasive breast cancer cases. Breast cancer patients with HER2 overexpression can benefit from anti-HER2 therapy. HER2 IHC results are generally divided into four values (range, 0 to 3+). The cases with a HER2 (2+) value should be assessed using FISH. The aim of this retrospective study was to evaluate the biological variables and prognosis of HER2 (2+) cases according to the FISH status. Methods: Primary breast cancer patients (n = 5419) were enrolled in this study from January 2002 to September 2019. The factors investigated included nodal status, tumor size, nuclear grade, ER/PgR and HER2 status, p53 overexpression, and the Ki-67 index values. Positive for HER2 is categorized as having a HER2 value of 3 + or FISH amplified in equivocal cases. Results: The HER2 3+ rate in non-invasive carcinoma was higher than the rate in the invasive carcinoma cases (16.4% vs 12.9%; p < 0.0001). The distribution of HER2 IHC status in invasive carcinoma cases was 3+ (12.9%), 2+ (5.8%) and negative (84.3%). The FISH positive rate of the HER2 2+ cases was 57.0 % (158 cases). HER2 positivity significantly correlated with ER/PgR negative, p53 overexpression, higher Ki-67 value and nuclear grade. The FISH positivity of the HER2 2+ cases significantly correlated with PgR negative, p53 overexpression and higher grade. There were significant differences in biological variables between IHC 3+ and 2+/FISH positive, and between IHC 2+/FISH negative and IHC negative. Patients with HER2 3+ had significantly worse disease-free survival (DFS) rates than the cases before the approval of trastuzumab in Japan. However, there was no difference after trastuzumab was approved. Moreover, there was no difference in DFS according to the FISH status in HER2 2+ cases. Conclusions: HER2 positivity indicated a higher degree of malignancy. HER2 2+ had different independent biological characteristics from HER2 negative (0 & 1+) or positive (3+) cases. Therefore, the difference in biology of HER2 2+ cases may influence the prognosis and should be reconsidered in breast cancer cases.

Updated analysis of the inducible T-cell co-stimulatory receptor (ICOS) agonist, GSK3359609 (GSK609), combination with pembrolizumab (PE) in patients (pts) with anti-PD-1/L1 treatment-naïve head and neck squamous cell carcinoma (HNSCC).

6517 Background: INDUCE-1 (NCT02723955) is a first-in-human study investigating GSK609, an IgG4 ICOS agonist non-T-cell depleting antibody, as monotherapy and combination therapy with anti-cancer agents that includes PE. A range of GSK609 dose levels (≥0.1–1 mg/kg) having biological and clinical activity were identified and evaluated in the expansion phase with GSK609 0.3 mg/kg selected as the dose for further investigation. Results from the HNSCC expansion cohorts (ECs) showed GSK609 has single agent activity in pts with relapsed/refractory disease, and early clinical activity in combination with PE in pts with anti-PD-1/L1 treatment-naïve disease (Rischin, et al. Annals of Oncol 2019;30[Supplement_5]:v454–5). Updated results from the GSK609/PE HNSCC EC are presented. Methods: Eligible pts for the HNSCC EC had anti-PD-1/L1 treatment-naïve disease, ≤5 prior lines of therapy, measurable disease, and no active autoimmune disease. Pts received GSK609 0.3 mg/kg + PE 200 mg every 3 weeks (wks) until disease progression or unacceptable toxicity, up to 2 years (yrs)/35 cycles. Disease assessments were performed every 9 wks through wk 54 then every 12 wks thereafter. Pts were followed for survival and subsequent anti-cancer therapy. Results: As of 11 October 2019, 34 pts were enrolled and evaluable for efficacy analyses. The median age of this population was 61.5 yrs (range: 37–77); 85% were male; 53% received ≥1 prior line of therapy in the metastatic setting. ORR was 26% (95% CI: 12.9, 44.4; n = 9 with 4 complete and 5 partial responses); disease control rate was 68% (95% CI: 49.5, 82.6; n = 23). Among pts with PD-L1 IHC status by 22C3 pharmDx assay (n = 24; 71%), the majority of pts with a response or stable disease (SD) had PD-L1 CPS status < 20 (11 of 15 pts including 1 SD pt with CPS < 1). Median PFS was 5.6 months (95% CI: 3.9, 6,2). Median OS was not reached at time of analysis (95% CI: 8.2, NR); 6-month OS rate was 84% (95% CI: 66, 93). Treatment-related adverse events were reported in 66% of pts; the majority of events were Grades 1 or 2 with < 10% of pts experiencing ≥ Grade 3 events. Conclusions: This updated analysis with a more mature dataset shows promising clinical activity that supports further randomized investigation of GSK609 in combination with PE with an OS endpoint in HNSCC. Clinical trial information: NCT02723955 .

From This Month's Histopathology

From This Month's Histopathology

Abstract P4-10-20: Protein quantitation assays for AKT and PTEN to better understand sensitivity and resistance of breast cancer patients to treatment with AKT inhibitor capivasertib

Abstract Context: The PI3K/AKT/PTEN pathway is frequently aberrantly activated in breast cancer (BC) and involved in resistance to hormonal therapy. Multiple drugs targeting this pathway are approved or in development, including the pan-AKT inhibitor capivasertib (AZD5363). Early phase studies of capivasertib as monotherapy for solid cancers (e.g. NCT01226316, NCT01625286) have used genetic testing to select patients considered most likely to respond based on AKT1, PIK3CA or PTEN mutations. However, not all selected patients responded. Randomised phase 2 studies on Capivasertib as combination treatment showed the importance of context; a more pronounced response in combination with paclitaxel was seen in TNBC patients whose tumors harboured an alteration on AKT1, PIK3CA or PTEN (Schmid et al. ASCO 2018), whereas the same combination in ER+ HER2- advanced/metastatic BC patients did not show a clinical benefit in the overall population nor in the PIK3CA-mutation subgroup (Turner et al. Ann Oncol 2019). The combination with fulvestrant demonstrated an effect in unselected ER+ HER2- advanced BC patients resistant to aromatase inhibitors irrespective of PIK3CA mutations or PTEN IHC status (Jones et al. ASCO 2019). DNA-based assays also have the limitation that potential responders may be missed, e.g., tumors with epigenetic pathway activation but no detectable mutations. Techniques such as IHC and Western Blot (WB) lack the sensitivity, reproducibility and standardization required to precisely quantify these proteins. We hypothesize that precise mass spectrometry (MS)-based methods may facilitate a better understanding of sensitivity and resistance to inhibitors of the PI3K/AKT pathway in future studies. Methods: Protein extracted from tumor tissue lysate is subjected to proteolytic digestion to generate proteotypic peptides used for protein quantitation. Synthetic stable isotope-labeled peptides are added to the digest as an internal standard, after which the target peptides are immuno-enriched with antibodies coupled to magnetic beads. MS analysis is performed by either eluting peptides for measurement with liquid chromatography multiple reaction monitoring tandem MS (LC-MRM-MS/MS) or by spotting beads onto a plate for matrix-assisted laser desorption/ionization MS (MALDI-MS). Calibration curves enable accurate quantitation. A new method for PTEN was applied to 13 breast cancer PDXs. Results were compared to IHC and WB. In a retrospective study, we are applying these methods to test samples from HR+ metastatic breast or gynecological cancer patients (n=25 with PIK3CA-mutations, capivasertib monotherapy; n=26 with PTEN alterations, capivasertib + fulvestrant; n=9 with no PIK3CA, AKT1 or PTEN mutations, not receiving capivasertib). AKT and PTEN quantitation data will be analyzed for a relationship to treatment response as determined by RECIST guidelines (NCT01226316). Results: We validated MS methods to precisely and reproducibly determine AKT and PTEN concentrations in tumor samples. AKT concentrations varied significantly between mutation-positive tumor samples (AKT1: 0.06-0.45, AKT2: 0.05-0.20 fmol/µg total protein). Significant phosphorylation of AKT1-Ser473 or AKT2-Ser474 was observed in 2 out of 17 tested samples. At least 1 mutation-negative sample had a high AKT1 concentration of 0.42 fmol/ug total protein. PTEN concentrations from PDXs were consistent across biological replicates and agreed with IHC and WB. Furthermore, we were able to detect and quantify PTEN from 3 samples that were negative by IHC and/or WB. Conclusion: AKT and PTEN quantitative proteomics assays have the potential to enable more accurate assessment of PI3K/AKT/PTEN pathway activation in tumor tissues from patients treated with inhibitors of this key pathway. Citation Format: Sahar Ibrahim, Constance A Sobsey, Robert Popp, René P. Zahedi, Gerald Batist, Christoph H. Borchers. Protein quantitation assays for AKT and PTEN to better understand sensitivity and resistance of breast cancer patients to treatment with AKT inhibitor capivasertib [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-10-20.

Abstract OT1-07-02: A phase 3, multicenter, randomized, open-label trial of [fam-] trastuzumab deruxtecan (T-DXd; DS-8201a) vs investigator’s choice in HER2-low breast cancer (DESTINY-Breast04)

Abstract Background: Human epidermal growth factor receptor 2 (HER2)-targeted therapies have greatly improved survival in subjects with HER2-positive breast cancer (BC). However, there are many more subjects with BC with HER2-low expression (immunohistochemistry [IHC] 1+ or 2+/in situ hybridization negative), for whom no HER2-targeted therapies are approved. [Fam-] trastuzumab deruxtecan (T-DXd; formerly DS-8201a) is an antibody-drug conjugate with a humanized HER2 antibody, peptide-based cleavable linker, and topoisomerase I inhibitor payload. It has a drug-to-antibody ratio of 7 to 8, and the membrane permeability of the cleaved payload induces a cytotoxic bystander effect. In an ongoing, phase 1 study, T-DXd demonstrated an objective response rate (ORR) of 44.2% (19/43) with a manageable safety profile in heavily pretreated, advanced HER2-low BC (Modi et al, SABCS 2018). Results from the pivotal, phase 2 study of subjects with HER2-positive BC previously treated with [ado-] trastuzumab emtansine (DESTINY-Breast01) confirmed the activity observed in the phase 1 trial and will be presented at the meeting (Krop, et al). Here, we describe the phase 3 trial evaluating T-DXd in subjects with HER2-low BC. Study Description: DESTINY-Breast04 is a randomized, phase 3, 2-arm, open-label, multicenter trial comparing the efficacy and safety of T-DXd with investigator’s choice of treatment in HER2-low (IHC1+ or IHC2+/ISH−), unresectable and/or metastatic BC. The trial started in December 2018 and is recruiting subjects from 223 sites in North America, Europe, and Asia. Approximately 540 subjects (480 hormone receptor [HR]+ and 60 HR-) will be randomized (2:1) to T-DXd (5.4 mg/kg intravenously every 3 weeks) or investigator’s choice (capecitabine, eribulin, gemcitabine, paclitaxel, or nab-paclitaxel). Randomization will be stratified by HER2 IHC status, number of prior treatments, and prior CDK4/6 inhibitor therapy/HR status. HER2 IHC will be assessed and confirmed by central testing based on archival samples. The primary efficacy endpoint is progression-free survival (PFS) per RECIST v1.1, determined by blinded independent central review. Secondary efficacy endpoints include investigator-assessed PFS, overall survival, ORR, and duration of response. Safety endpoints include treatment-emergent adverse events (AEs), serious AEs, and AEs of special interest (including interstitial lung disease/pneumonitis, cardiotoxicity, and infusion-related reactions). For further information about this trial, visit ClinicalTrials.gov (NCT03734029). Citation Format: Shanu Modi, Shoichiro Ohtani, Caleb Lee, Yibin Wang, Kapil Saxena, David A. Cameron. A phase 3, multicenter, randomized, open-label trial of [fam-] trastuzumab deruxtecan (T-DXd; DS-8201a) vs investigator’s choice in HER2-low breast cancer (DESTINY-Breast04) [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr OT1-07-02.

Differences in MSI phenotype between cancer types using a new PCR-based pan-cancer biomarker panel.

e13145 Background: A multiplexed biomarker panel was developed for detection of MSI that is more sensitive than currently available systems. Preliminary data showed an increased MSI sensitivity for colon polyps, endometrial (EC) and skin cancers. This study expands that finding to 14 different types of cancer. Methods: Selection of the best microsatellite markers was done using a cohort of 160 patients ≤55 years with ≥1 colon polyp and 100 EC patients ≤ 50 years. To validate the new panel, a cohort of 100 Lynch syndrome CRC, 100 sporadic MSI-High CRC, 100 sporadic MSI stable CRC and 219 extra-colonic cancers from the Colon Cancer Family Registry were tested for MSI. Samples were screened using a new 8-marker panel and Promega’s MSI Analysis System. Mismatch repair (MMR) gene mutation status and expression were determined. To compare the relative sensitivity of the 8-marker panel across all cancer types we developed a quantitative “MSI-score” for each cancer type. Results: Considerable differences in MSI phenotypes were observed between cancer types and a new approach to scoring MSI in extra-colonic cancers is proposed. Relative intensity of the MSI phenotype was characterized by MSI-Scores ranging from high of 66 (small intestine) to 6 (brain). The mutations in MSI samples were significantly more common and allele size shifts larger with the new biomarkers, making MSI classification more accurate and robost. MSI, IHC and MMR gene mutation status were highly correlated. Conclusions: The MSI sensitivity of the new biomarker panel for most cancer types was significantly higher than currently available PCR-based MSI systems. Development of the new pan-cancer PCR-based MSI system allows laboratories to leverage established equipment and known analysis techniques to keep turnaround time and costs, quick and affordable compared with emerging NGS-based technologies.

A phase III, multicenter, randomized, open label trial of [fam-] trastuzumab deruxtecan (DS-8201a) versus investigator’s choice in HER2-low breast cancer.

TPS1102 Background: Human epidermal growth factor receptor 2 (HER2)-targeted therapies have greatly improved survival in HER2+ breast cancer (BC). However, there are many more BC patients with HER2-low expression (immunohistochemistry [IHC] 1+ or 2+/ in situ hybridization-negative), for whom no HER2-targeted therapies are approved. [Fam-] trastuzumab deruxtecan (T-DXd; formerly DS-8201a) is an antibody-drug conjugate with a humanized HER2 antibody, peptide-based cleavable linker, and a topoisomerase I inhibitor payload. It has a drug-to-antibody ratio of ~8 and the membrane permeability of the cleaved payload enables a cytotoxic bystander effect. In an ongoing phase 1 trial, T-DXd demonstrated an objective response rate (ORR) of 44.2% (19/43) with a manageable safety profile in heavily pretreated, advanced HER2-low BC (Oct 2018 data cutoff; Modi et al, SABCS 2018). Methods: DESTINY-Breast04 is a randomized, phase 3, 2-arm, open-label, multicenter study comparing efficacy and safety of T-DXd vs physician’s choice in HER2-low, unresectable and/or metastatic BC. Approximately 540 subjects will be randomized 2:1 to T-DXd (5.4 mg/kg intravenous every 3 weeks) or physician’s choice (capecitabine, eribulin, gemcitabine, paclitaxel, or nab-paclitaxel). Randomization is stratified by HER2 IHC status, number of prior treatments, and prior CDK4/6 inhibitor therapy/hormone receptor (HR) status. HER2 IHC will be assessed and confirmed by central testing based on archival samples. The primary efficacy endpoint is progression-free survival (PFS) per mRECIST v1.1 determined by blinded independent central review. Secondary efficacy endpoints are investigator-assessed PFS, overall survival, confirmed ORR, and duration of response. Safety endpoints include serious adverse events (AEs), treatment-emergent AEs, and AEs of special interest (interstitial lung disease/pneumonitis, cardiotoxicity, and infusion-related reactions). Primary PFS analysis will occur when 318 events are observed in HR+ subjects; providing 90% power to detect a hazard ratio of 0.68 with 1-sided α of 0.025. Enrollment began in Dec 2018. Clinical trial information: NCT03734029.

Colorectal Carcinomas Containing Hypermethylated MLH1 Promoter and Wild-Type BRAF/KRAS Are Enriched for Targetable Kinase Fusions.

Kinase fusions are rare and poorly characterized in colorectal carcinoma, yet they present unique opportunities for targeted therapy. In this study, we characterized kinase fusions from patients with advanced colorectal carcinoma who had MSK-IMPACT testing of their tumors between January 2014 and June 2018. Patients were analyzed for the presence of fusions, microsatellite instability (MSI), and RAS/BRAF mutations. Mismatch repair (MMR), IHC, and promoter hypermethylation status of MLH1 (MLH1ph) in microsatellite instability-high (MSI-H) colorectal carcinoma with fusions were investigated. Fusion transcripts were confirmed using a targeted RNA-seq panel assay. Of 2,314 colorectal carcinomas with MSK-IMPACT testing, 21 harbored kinase fusions. Overall 57% (12/21) of colorectal carcinoma fusions were MSI-H/MMR-D. Loss of MLH1 and MLH1ph was confirmed in all 12 and all 10 cases with available material, respectively. Fusions were present in 5% of MSI-H/MMR-D colorectal carcinoma compared with 0.4% of MSS/MMR-P colorectal carcinoma (P < 0.001) and 15% of MSI-H/MMR-D colorectal carcinoma with wild-type RAS/BRAF. Of 24 total MLH1-deficient colorectal carcinomas with MLH1ph and wild-type RAS/BRAF, 10 (42%) harbored kinase fusions. Kinase fusions in MSI-H colorectal carcinoma were associated with sporadic MLH1ph rather than with Lynch syndrome, and these patients may be eligible for kinase inhibitors, particularly following resistance or toxicity in response to immunotherapy. These findings identify a molecular subset of colorectal carcinoma with kinase fusions that may be responsive to kinase inhibitors.Significance: A high frequency of targetable kinase fusions in BRAF/RAS wild-type, MSI-H colorectal carcinoma offers a rationale for routine screening to identify patients with colorectal carcinoma with kinase fusions that may be responsive to kinase inhibitors.See related commentary by Valeri, p. 1041.

Abstract P4-02-11: Computer vision detects morphological correlates of HER2 positive breast cancer in H&amp;E stained histological images

Abstract Background/Objectives: The determination of HER2-positivity by IHC or FISH is critical for identifying patients most likely to benefit from anti-HER2 therapy. However, these methods do not always provide an accurate indication of HER2 overactivity, which can occur without gene amplification or overexpression of the HER2 protein. Our study objective was to determine if a deep learning convolutional neural network (CNN) could be trained, using IHC HER2 staining, to learn a morphological signature for HER2 positivity in H&amp;E stained slides. Methods: For training, we used H&amp;E images (whole slides scanned at 40x) from 10 HER+ patients (IHC 3+) and 15 HER2− patients (IHC 0 or 1+) along with their adjacent HER2 IHC images. We first annotated non-cancer regions in H&amp;E images. We then identified tumor regions in HER2 IHC that were positive (intense/complete circumferential stain) or negative (no or weak stain). We ignored any regions with equivocal IHC response as well as whole slides of IHC 2+ patients. For rigorous testing, slides from a separate set of 7 HER2+ and 19 HER2− patients were used. Digitized slides and expert consensus IHC HER2 status for each patient were provided as part of an international HER2 IHC scoring competition organized by the University of Warwick. The computer vision pipeline comprised four-stages. First, we color-normalized the H&amp;E images to reduce unwanted color variation between slides. Second, a pre-trained neural network (NN1) marked all nuclear centroids to make it easier for subsequent stages to focus on nuclear morphology and inter-nuclear spatial arrangements. Third, a neural network (NN2) trained on sub-images of size 100x100 centered at nuclear centroids classified nuclei as non-cancer vs. cancer. Fourth, a final neural network (NN3) sub-classified cancer nuclei into HER2+ or HER−. In H&amp;E images of held-out test patients, the percent of cancer nuclei scored as HER2+ was analyzed. Results: NN2 had a classification accuracy exceeding 97% on a validation set of 25,000 nuclei set aside from training patients, while NN3 had a validation accuracy of 88% on 7,500 test nuclei. On the set aside test patients, among the seven HER2+ patients an average of 49.8% cancer nuclei were scored HER2+, while among the 19 HER2− test patients the corresponding proportion was only 24.7% (p &amp;lt; 0.01). The AUC of binary classification of test patients into HER2+/− based on percent of HER2+ nuclei was 0.815. Upon closer inspection, the H&amp;E morphology of some of the misclassified HER− patients showed visual similarity to the correctly classified HER2− patients and vice versa. Conclusions: Even when morphological patterns associated with cancer subtypes are too subtle for humans to reliably detect, H&amp;E stained slides analyzed by CNNs may be able to geographically map a HER2 signature. It is unclear if misclassification with respect to IHC status is a reflection of morphological confusion or discordance between genomic subtype and IHC response. By training multiple neural networks to detect morphological signatures corresponding to different molecular subtypes of breast cancer, we may be able to detect and study intra-tumor heterogeneity in a cost effective and/or complementary way compared to multi-region sequencing. Citation Format: Dhage S, Anand D, Kumar N, Gann PH, Sethi A. Computer vision detects morphological correlates of HER2 positive breast cancer in H&amp;E stained histological images [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-02-11.

OA02.07 Updated Results of Phase 1 Study of DS-8201a in HER2-Expressing or –Mutated Advanced Non-Small-Cell Lung Cancer

DS-8201a is a HER2-targeting antibody-drug conjugate with a novel peptide-based cleavable linker, a topoisomerase I inhibitor payload, and a high drug-to-antibody ratio (7 to 8). In preclinical studies, DS-8201a showed broad antitumor activity, in a wide range of tumors. The ongoing phase 1 trial has a dose-escalation (part 1) and -expansion (part 2) and includes subjects with advanced breast cancer, gastric cancer, and other HER2-expressing/-mutated solid tumors. Here, we present updated results for subjects with HER2-expressing or -mutated non-small cell lung cancer (NSCLC). Subjects with HER2-expressing (defined as IHC ≥1+ or amplified) or –mutated (detected by NGS or other platforms) NSCLC were eligible to enroll. HER2 expression and mutation were assessed using archival tissue. Adverse events (AEs), objective response rate (ORR), disease control rate (DCR: CR + PR + SD), and duration of response (DOR) were assessed. [Results will be updated for presentation at meeting] As of Apr 18, 2018, 12 subjects with HER2-expressing and/or -mutated NSCLC received ≥1 dose of DS-8201a at 6.4 mg/kg. Median age was 58.5 y with median of 3 prior regimens. At data cutoff, 8 of 12 (66.7%) subjects remain on treatment. HER2 IHC status was available for 7 subjects. Median duration of treatment was 3.66 months (range 0.69, 14.19). Eight of 10 (80.0%) subjects with ≥1 post-baseline scan (ps) experienced tumor shrinkage (100.0% of them at 1st ps at 6 weeks). Overall, confirmed ORR and DCR in the evaluable subjects was 5 of 8 (62.5%) and 6 of 8 (75.0%), respectively. Among subjects with HER2 IHC 2+ or IHC 3+ expression, 2 of 5 (40.0%) had a PR. Overall, median DOR was 11.5 months (range 0.03+, 11.53). Three of 12 (25.0%) subjects experienced a grade ≥3 AE. Common AEs included decreased appetite 66.7% (0.0% grade ≥3), nausea 58.3% (0.0% grade ≥3), alopecia 41.7% (0.0% grade ≥3), and fatigue 41.7% (0.0% grade ≥3). One fatal case of interstitial lung disease was reported in this subgroup. DS-8201a demonstrated promising antitumor activity in heavily pretreated NSCLC subjects.