Discordant Tumors Research Articles

Prognosis of P16 and HPV Discordant Oropharyngeal Cancers: Natural Language Processing to Extract Data from Free-Text Pathology Reports

<h3>Purpose/Objective(s)</h3> P16 immunohistochemistry (IHC) is not as specific for detecting human papillomavirus (HPV) related oropharyngeal cancers (OPC) as in situ hybridization (ISH). However, given its high sensitivity and wide availability, P16 IHC is a standard of care requirement for OPC staging and is also used for clinical trial eligibility. As both P16 and HPV testing are not required for routine clinical management, there are varying reports on the prognosis of P16 and HPV discordant tumors. We aimed to assess the prognosis of P16 and HPV discordant OPC tumors, using natural language processing (NLP) techniques to extract results from free-text OPC pathology reports. <h3>Materials/Methods</h3> We reviewed our institutional OPC radiation database and included a series of patients from 7/1994-6/2020 with digitized pathology reports. Patients were excluded if they did not receive curative intent radiation, did not have both P16 IHC and HPV ISH, or if either test was equivocal. 422 patients were included with a median follow-up of 87.8 months (0.3-241.0). We performed text extraction and classification to ascertain P16 and HPV results from free-text pathology reports. All reports were manually evaluated by two independent reviewers as our gold standard. Four groups were identified: P16-negative/HPV-negative (P16-/HPV-, n=60), P16-/HPV-positive (P16-/HPV+, n=9), P16-positive/HPV- (P16+/HPV-, n=49), and P16+/HPV+ (P16+/HPV+, n=304). The Kaplan-Meier method was used to estimate cancer-specific survival (CSS) and overall survival (OS). <h3>Results</h3> 39% of reports could not be processed by our NLP techniques. The algorithm accurately coded 92.6% of P16 and 94.2% of HPV results. Positive predictive value/precision, sensitivity/recall, and F-score for P16/HPV were: 99.5%/99.0%, 91.7%/94.0%, and 95.5%/96.5%. 13.7% of tumors were discordant (P16-/HPV+ or P16+/HPV-). As expected, P16-/HPV- tumors had the worst CSS and OS, and P16+/HPV+ had excellent prognosis (Table 1). P16-/HPV+ constituted only 2.1% of the cohort but outcomes performed favorably with P16+/HPV+. P16+/HPV- tumors had favorable outcomes in the first two years, but with longer follow-up clustered between P16-/HPV- and P16+/HPV+ tumors. <h3>Conclusion</h3> P16 and HPV results were accurately abstracted from the majority of free-text OPC pathology reports. We provide the first example to our knowledge of employing NLP techniques to extract data from head and neck cancer pathology reports and correlate results with clinical outcomes. P16 and HPV discordant tumors constitute a minority of patients, but their divergent prognoses suggest that performing both tests may be of utility—especially for clinical trial eligibility or treatment de-escalation.

OS02.6.A The TeloDIAG: How telomeric parameters can help in glioma rapid diagnosis and liquid biopsies approaches

Abstract BACKGROUND The integration of molecular markers into the WHO 2016 classification has clarified the complex diagnosis of gliomas. Among these biomarkers, the TERT promoter mutation and the loss of ATRX (ATRX loss) are mutually exclusive alterations associated with re-activation of telomerase or alternative lengthening of telomeres (ALT), respectively. Strangely, 25% of gliomas display neither or both these alterations, a situation referred to as abnormal telomere maintenance mechanism (aTMM). MATERIAL AND METHODS To investigate the TMM actually involved in gliomas, the C-circle (CC) assay was adapted to tumor (FFPE and frozen) samples. RESULTS We constructed a CC-based algorithm able to identify the TMM of 284 gliomas with either TERT or ATRX alteration, with a sensitivity of 100% and a specificity of 97.3%, and succeeded in deciphering the TMM involved in 122 aTMM gliomas. Additionally, the combination of the TMM, the mutational status of the Isocitrate dehydrogenase 1/2 (IDH) gene, and the histological grading was used as base for a new classification: TeloDIAG. Six subtypes are defined in this classification: tOD, tLGA, tGBM_IDHmt, tGBM, and tAIV, corresponding to oligodendroglioma, IDHmt low grade astrocytoma, IDHmt glioblastoma, and IDHwt glioblastoma, respectively, the last class gathers ALT+ IDHwt glioma. The TeloDIAG diagnosis is 99% concordant with the WHO classification for glioma displaying typical molecular characteristics (N=312). It modified the classification of 38% (N=156) discordant tumors, such as IDHwt Astrocytoma, aTMM tumors, or gliomas with unexpected TMM (e.g. TERTwt oligodendroglioma, ATRX loss GBM). Interestingly, 20% (N=69) of TERTwt, ATRXwt, or IDHwt GBM were actually tAIV, which is remarkable as tAIV-glioma patients’ survival tended to be longer (21.2 months) than tGBM patients’ survival (16.5 months). Importantly, CC in blood sampled from IDHmt astrocytoma patients was detected with a sensitivity of 56% and a specificity of 95% (N = 206). CONCLUSION In sum, the TeloDIAG is a new, simple, and efficient tool helping in glioma diagnosis and a promising option for liquid biopsy

Comparative study of the loop-mediated isothermal amplification method and the QIAGEN therascreen PCR kit for the detection of EGFR mutations in non-small cell lung cancer.

BackgroundEpidermal growth factor receptor (EGFR) mutations are important biomarkers in the treatment of patients with advanced or metastatic diseases. The therascreen EGFR Rotor-Gene Q (RGQ) PCR Kit® (Qiagen, Inc.) is an approved diagnostic test for EGFR mutations in non-small cell lung cancer (NSCLC). This study aims to investigate the diagnostic capability of a loop-mediated isothermal amplification (LAMP) assay as an accurate, efficient, and cost-effective alternative to the therascreen assay.MethodsEGFR mutations were investigated by LAMP and therascreen assays using tissue samples that were surgically resected or biopsied from 117 consecutive patients with NSCLC tumors. The EGFR status from the LAMP assay was compared with that of the therascreen assay. Next-generation sequencing (NGS) was performed to confirm EGFR status of tumors that did not match in both assays. To establish an optimal LAMP AUC value, receiver operating characteristics (ROC) curve analysis was performed within tumors with exon 19 deletion or L858R point mutation.ResultsOf the 117 tumors assayed, 45 tumors with EGFR mutations and 68 tumors with EGFR wild type were matched in both assays, four tumors having mismatched EGFR statuses. NGS further confirmed that two of the four discordant tumors had the same EGFR status that was determined by the LAMP assay. The AUC values were 0.973 (95% CI: 0.929–1.00) in exon 19 deletion, and 0.952 (95% CI: 0.885–1.00) in L858R point mutation. In exon 19 deletion, sensitivity, specificity, and accuracy were 89.3%, 98.9%, and 96.6%, respectively, and 94.7%, 95.9%, and 95.7%, respectively, in L858R using AUC value of 0.222.ConclusionsThe LAMP assay compared favorably with the therascreen assay and has potential as an effective, simple, rapid, and low-cost diagnostic alternative. Based on these results, a liquid biopsy LAMP system should be developed for point-of-care testing of oncogenes in the near future.

HER2 heterogeneity in adenocarcinoma of the distal esophagus and stomach.

4567 Background: Patients with HER2 positive adenocarcinoma of the esophagus or stomach are eligible for HER2 targeted therapy, which can improve survival in selected patients. Previous research shows that HER2 gene amplification and HER2 overexpression is frequently heterogeneous within these tumors. Biopsies taken from heterogeneous tumors for predictive testing may therefore result in false-negative outcomes. The objective of this study was to assess HER2 amplification and expression in biopsies and paired resection specimens with adenocarcinoma of the esophagus or stomach, from patients who did not receive neoadjuvant systemic therapy. Methods: Paired biopsies and resection specimens of patients with adenocarcinomas of the esophagus or stomach were retrospectively selected. Immunostaining was performed on all samples using antibody 4B5 (Ventana Medical Systems) and Silver-In-Situ-Hybridization was performed in selected cases. Scoring for HER2 was performed according to the method described by Hofmann et al. (2008). Results: We included 378 cases for analysis. In both biopsies and resection specimens 14% of the cases were HER2 positive. Intratumor heterogeneity in HER2 positive tumors was present in 45% ( n= 24/53) in biopsies and 75% ( n= 39/52) in resection specimens. In HER2 positive resection specimens, 65% ( n= 34/52) of paired biopsies were also positive. In the 18 remaining discordant tumors (resection HER2 positive, biopsy negative), intratumor heterogeneity was present in 16/18 cases. For HER2 negative resection specimens all paired biopsies were also HER2 negative. SISH was performed in 110 tumors. Agreement of HER2 gene amplification between biopsy and resection specimens was observed in 86% (n = 95/110). Five HER2 negative biopsies were positive in the resection specimen. Conclusions: The results of this study indicate that predictive HER2 assessment in adenocarcinoma of the esophagus or stomach can lead to false negative results based on biopsies. As a result, patients with HER2 positive tumors can unintentionally be denied neoadjuvant HER2 targeted therapy. The set of patients investigated in this present study is unique because of the absence of any systemic and/or radiation therapy between the biopsy and the resection of the tumor. Hopefully these results can help in developing methods for improved patient selection for HER2 targeted therapy.

TERT, BRAF, and NRAS Mutational Heterogeneity between Paired Primary and Metastatic Melanoma Tumors

Mutational heterogeneity can contribute to therapeutic resistance in solid cancers. In melanoma, the frequencies of intertumoral and intratumoral heterogeneity are controversial. We examined mutational heterogeneity within individual patients with melanoma using multiplatform analysis of commonly mutated driver and nonpassenger genes. We analyzed paired primary and metastatic tumors from 60 patients and multiple metastatic tumors from 39 patients whose primary tumors were unavailable (n= 271 tumors). We used a combination of multiplex SNaPshot assays, Sanger sequencing, mutation-specific PCR, or droplet digital PCR to determine the presence of BRAFV600, NRASQ61, TERT-124C>T, and TERT-146C>T mutations. Mutations were detected in BRAF (39%), NRAS (21%), and/or TERT (78%). Thirteen patients had TERTmutant discordant tumors; seven of these had a single tumor with both TERT-124C>T and TERT-146C>T mutations present at different allele frequencies. Two patients had both BRAF and NRAS mutations; one had different tumors and the other had a single tumor with both mutations. One patient with a BRAFmutant primary lacked mutant BRAF in at least one of their metastases. Overall, we identified mutational heterogeneity in 18 of 99 patients (18%). These results suggest that some primary melanomas may be composed of subclones with differing mutational profiles. Such heterogeneity may be relevant to treatment responses and survival outcomes.

3:18 PM Abstract No. 201 Percutaneous biopsy of hepatocellular carcinoma in the setting of endovascular locoregional therapy

Percutaneous biopsy for the characterization of intermediate and advanced stage hepatocellular carcinoma (HCC) is projected to increase in the era of precision medicine. Given its controversial role in management, the purpose of this study was to profile the safety and efficacy of acquiring high-volume HCC core biopsies. Between April 2016 and September 2019, 30 patients with embolotherapy-naïve advanced HCC, were enrolled in this IRB-approved prospective trial. Multiple co-axial 18G core-biopsies of a targetable HCC were obtained during transarterial chemoembolization (TACE) or radioembolization (TARE). Cores were sent to pathology, the Center for Personalized Diagnostics (CPD) for solid-tumor next generation sequencing, or prepared for custom sequencing using an HCC panel developed in our lab. From 30 patients, 33 LR4 (1) or LR5 (32) HCCs were biopsied. A median of 7 core biopsies were obtained during the initial treatment. Nine tumors were biopsied a second time (post-TACE), and 2 were biopsied a third time (recurrence). Of 44 biopsy encounters, 1 (2.3%) minor (SIR class A or B) and 0 major biopsy-attributable adverse events were reported during the postprocedural period. Three (9.1%) biopsies revealed discordant pathology of cholangiocarcinoma resulting in a change in management. Four (12%) biopsies revealed poorly differentiated carcinoma, ultimately favored to represent HCC over cholangiocarcinoma. Five (15%) biopsies were negative for tumor, only 1 of which was later confirmed as sampling error. Of 12 cores sent to CPD, 9 (75%) were adequate for analysis. In custom sequenced samples, TERT, CTNNB1, and ALB were the most frequent variant alleles in a markedly heterogeneous pool. No changes in genetic mutations were identified in post-TACE samples when compared to pre-TACE biopsies. Obtaining a high-volume of co-axial percutaneous core biopsies during HCC treatment by TACE or TARE is safe, providing desperately needed tissue for the molecular characterization of intermediate and advanced stage HCC. Biopsy results may change management in imaging discordant tumors.

Oncotype recurrence score (RS) and discordance in patients with secondary invasive breast events (SIBE).

e12016 Background: Oncotype RS is a 21-gene assay used to predict the likelihood of distant recurrence and benefit of chemotherapy in patients with node-negative, tamoxifen treated breast cancer (BC). We developed a database to determine rates of SIBE and identify cases of discordance between Oncotype RS and tumor grade (TG). Our goal was to recognize patients with discordant tumors who had SIBE, with implications for patient management. Methods: We identified 618 patients (645 samples) with early-stage, hormone positive BC treated between 2006-2018, with tumor characteristics available for analysis. Among these patients, there were 24 SIBE (n = 24) from 23 patients (3.7%). When stratified by RS using traditional cutoffs (Paik, 2004), rates of SIBE were 2.8%, 5.0% and 4.5% in low, intermediate and high-risk groups respectively. Discordance was defined as 1 or 2-step difference between RS (low, intermediate, high risk) and TG (well (WD), moderately (MD) and poorly (PD) differentiated). Prior studies demonstrated 7-19% “2-step discordance” between TG and RS (i.e. PD tumors with low-risk RS or WD tumors with high-risk RS). N(%) or median were used to describe patients’ characteristics between groups and compared by Mann-Whitney U test at the confidence level of 5%. Results: Of the 24 SIBE, 42% had low RS ( &lt; 18), 46% had intermediate RS (18-30) 12% had high RS ( &gt; 31). Median RS was 22 and median age at secondary event was 61 (67% post-menopausal). At time of SIBE, 54% were metastatic, 25% locally recurrent and 21% contralateral. Within this dataset, 63% were 1-step and 4% were 2-step discordant. Significantly fewer patients were treated with systemic chemotherapy in the discordant group (p &lt; 0.01), though 94% displayed MD or PD TG. Discordant tumors tended to have lower RS (median 14 vs. 24) and be larger. There were no significant differences in menopausal status or Oncotype ER/PR score. Conclusions: Although the sample size of patients with SIBE is small, our data suggests that patients with discordant tumors of low-risk RS but higher TG may be receiving inadequate treatment (i.e. no chemotherapy). In addition to RS, other factors such as discordance, TG and size should perhaps be considered when determining treatment plans.

Estrogen receptor variants in ER-positive basal-type breast cancers responding to therapy like ER-negative breast cancers

Immunohistochemically ER-positive HER2-negative (ER+HER2−) breast cancers are classified clinically as Luminal-type. We showed previously that molecular subtyping using the 80-gene signature (80-GS) reclassified a subset of ER+HER2− tumors to molecular Basal-type. We report here that molecular reclassification is associated with expression of dominant-negative ER variants and evaluate response to neoadjuvant therapy and outcome in the prospective neoadjuvant NBRST study (NCT01479101). The 80-GS reclassified 91 of 694 (13.1%) immunohistochemically Luminal-type tumors to molecular Basal-type. Importantly, all 91 discordant tumors were classified as high-risk, whereas only 66.9% of ER+/Luminal-type tumors were classified at high-risk for disease recurrence (i.e., Luminal B) (P < 0.001). ER variant mRNA (ER∆3, ER∆7, and ERα-36) analysis performed on 84 ER+/Basal tumors and 48 ER+/Luminal B control tumors revealed that total ER mRNA was significantly lower in ER+/Basal tumors. The relative expression of ER∆7/total ER was significantly higher in ER+/Basal tumors compared to ER+/Luminal B tumors (P < 0.001). ER+/Basal patients had similar pathological complete response (pCR) rates following neoadjuvant chemotherapy as ER−/Basal patients (34.3 vs. 37.6%), and much higher than ER+/Luminal A or B patients (2.3 and 5.8%, respectively). Furthermore, 3-year distant metastasis-free interval (DMFI) for ER+/Basal patients was 65.8%, significantly lower than 96.3 and 88.9% for ER+/Luminal A and B patients, respectively, (log-rank P < 0.001). Significantly lower total ER mRNA and increased relative ER∆7 dominant-negative variant expression provides a rationale why ER+/Basal breast cancers are molecularly ER-negative. Identification of this substantial subset of patients is clinically relevant because of the higher pCR rate to neoadjuvant chemotherapy and correlation with clinical outcome.

Mixed response to immunotherapy in lung cancer.

122 Background: Mixed response (MR) to systemic therapy is common and associated with a poor prognosis. MR often represents a therapeutic dilemma resulting in the discontinuation of a therapy to which a portion of the tumor is still sensitive, due to the emergence of resistance in other metastatic deposits. We hypothesize that discordant responses in patients receiving immunotherapy may be associated with genomic variation within discordant tumors. Methods: We defined MR with the following criteria: 1) At least one tumor has increased in size while another tumor has decreased; 2) One or more tumors have remained stable while another has increased; 3) One or more tumors have remained stable while another has decreased. 4) A new tumor has developed while other tumors have decreased or remained stable. Between 2012 and 2017, we identified 112 patients with a MR. Two patients with metastatic lung cancer, treated with nivolumab underwent paired, simultaneous sampling of discordant tumors. Results: Both patients treated with nivolumab demonstrated genomic heterogeneity between discordant tumors on next-generation sequencing, including one patient with SCLC. Conclusions: MR remains a common and challenging therapeutic quandary. Genomic heterogeneity and differences in TMB of metastatic tumors demonstrating a MR, may be factors that contribute to this phenomenon. Further characterization of genomic discrepancies in MR may provide insights into mechanisms of emerging resistance, as well as, more rational and effective combinatorial therapies.[Table: see text]

Abstract P1-07-16: Retrospective analysis of oncotype DX recurrence score (RS) and discordance in patients with node-negative, ER+ breast cancer with recurrence

Abstract BACKGROUND: Oncotype RS is a 21-gene assay used to predict the likelihood of distant recurrence and benefit of chemotherapy in patients with node-negative, tamoxifen treated breast cancer. We developed a database to determine tumor recurrence rates and identify cases of discordance between Oncotype RS and tumor grade (TG). Our goal was to recognize patients with discordant tumors who had breast cancer recurrence and to understand the implications for patient management. METHODS/RESULTS: We analyzed patient and tumor characteristics from 704 breast cancer patients between 2006-2016. Of these patients, there were thirteen recurrences (n=13), or 1.9% recurrence rate at a median follow-up of 4.2 years. When stratified by RS, recurrence rates were 1%, 2.4% and 4.3% in low, intermediate and high-risk groups respectively. Of the 13 patients who recurred, 31% had a low RS (&amp;lt;18), 54% had an intermediate RS (18-30) and 15% had a high RS (&amp;gt;31). The median RS was 23 and median age at time of recurrence was 55 years (62% postmenopausal). Tumor characteristics at time of recurrence were notable for: 77% metastatic, 23% locally recurrent, 85% PR positive, 69% moderately-differentiated (MD) and 31% poorly-differentiated (PD). No well-differentiated (WD) tumors recurred. We defined Oncotype discordance as either 1-step or 2-step difference between Oncotype risk group (low, intermediate, high) and tumor grade (WD, MD, PD). Prior studies have demonstrated 7-19% “2-step discordance” between TG and RS (i.e. PD tumors with low-risk RS or WD tumors with high-risk RS). Of the 13 recurrences in our database, 46% were at least 1-step discordant, compared with 64.3% in our overall database. Among these recurrences, we compared discordant versus concordant tumors using two-sided T-tests. We found that fewer patients were treated with systemic chemotherapy in the discordant group (p=0.045), which was statistically significant. Among discordant patients, only one received chemotherapy, though all displayed MD or PD tumor grade. Discordant tumors tended to have lower RS (mean 17 vs. 27, p=0.34) and tended to be larger (mean 1.88cm vs. 1.33cm), however this was not statistically significant (p=0.84). Notably, the two largest tumors were both discordant. There were no significant differences in terms of age, Oncotype ER/PR score or mitotic count. CONCLUSION: Although the sample size of recurrent patients is small, our data may suggest that patients with discordant tumors of low-risk Oncotype RS but higher TG may be receiving inadequate treatment (i.e. no chemotherapy). In addition to RS, other factors such as discordance, TG and tumor size should perhaps be considered when determining treatment plans. Characteristics of Breast Cancer Recurrences in Oncotype DX DatabaseSubject #Age (years)Oncotype RSTumor GradeDiscordancePath PR%Tumor Size (cm)Chemotherapy14111MDY600.9N25811MDY1001.7N35713MDY301.0N45515MDY902.7N56122MDN301.5Y64522MDN901.8Y75323MDN00.9N83324MDN951.4Y96324PDY802.9U104129MDN801.1Y116530PDY102.1Y124933PDN01.6Y136135PDN51.0YMD=moderately-differentiated, PD=poorly-differentiated, Y=Yes, N=No, U=Unknown Citation Format: Zimmerman BS, Cascetta KP, Ru M, Eggert L, Molot MC, Nayak A, Bleiweiss I, Tiersten A. Retrospective analysis of oncotype DX recurrence score (RS) and discordance in patients with node-negative, ER+ breast cancer with recurrence [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-07-16.

Clinical relevance and concordance of HER2 status in local and central testing-an analysis of 1581 HER2-positive breast carcinomas over 12 years.

Human epidermal growth factor receptor 2 (HER2) is a central predictive biomarker in breast cancer. Inaccurate HER2 results in different laboratories could be as high as 20%. However, this statement is based on data generated more than 13 years ago and may not reflect the standards of modern diagnostic pathology. We compared central and local HER2 testing in a total of 1581 HER2-positive tumors from five clinical trials. We evaluated the clinical relevance for pathological complete response (pCR) and disease-free survival in a subgroup of 677 tumors, which received an anti-HER2 therapy. Over the period of 12 years, the discordance rate for HER2 decreased from 52.4 (GeparTrio) to 8.4% (GeparSepto). Discordance rates were significantly higher in hormone receptor (HR)-positive tumors (26.6%), compared to HR-negative tumors (16.3%, P<0.0001), which could be explained by a different distribution of HER2 mRNA levels in HR-positive and HR-negative tumors. pCR rates were significantly lower in discordant tumors (13.7%) compared to concordant tumors (32.2%, GeparQuattro and GeparQuinto, P<0.001). In survival analysis, tumors with discordant HER2 testing had a reduced overall survival (OS) in the HR-negative group (P=0.019) and a trend for improved OS in the HR-positive group (P=0.125). The performance of local HER2 testing was considerably improved over time and has reached a 92% concordance, which shows that quality initiatives in diagnostic pathology are working. Tumors with discordant HER2 testing had a reduced therapy response and different survival rates.

Discordance of the estrogen receptor and HER-2/neu in breast cancer from primary lesion to first and second metastatic site.

BackgroundHormone receptor and HER-2/neu discordance between the primary lesion and first metastasis has been reported. This study was performed to determine further biomarker discordance rates between the first and subsequent metastatic breast cancer lesions.MethodsWe performed a retrospective review of paired biomarkers from primary breast cancers compared to first reported and subsequent metastases from 103 patients with breast cancer. The estrogen receptor (ER), progesterone receptor (PR), and HER-2/neu status were reported at all three time points. In addition, hormone, cytotoxic, and targeted treatments were recorded for primary and metastatic disease, and survival was determined.ResultsBetween the primary and first metastases, discordance rates for ER, PR, and HER-2/neu were 15.8%, 33.7%, and 14.3%, respectively. There was discordance between the first and second metastases for the ER receptor in 18.8%, PR receptor in 19.8%, and HER-2/neu in 10.7%. Overall, there was discordance between the primary tumor and either the first or second metastases for ER in 27.7%, PR receptor in 40.7%, and HER-2/neu in 19.6% of cases. Discordance of either ER or PR affected survival, with worse survival experienced by those patients with all three hormone receptors remaining negative, and intermediate survival reported for those with discordant tumors (ER χ2=14.27, p=0.0008; PR χ2=11.31, p=0.0035). There was no difference in survival for patients whose HER-2/neu tumors were discordant.ConclusionThis study demonstrated that continued metastatic disease evolution may be associated with different tumor biology and that studies of metastatic lesions appear warranted, especially if targeted therapy is an option.

Genomic alterations driving breast cancer (BC) metastases and their relationship with the subtype switch in the GEICAM ConvertHER study.

1017 Background: To understand the mechanisms underlying the evolution of tumors in the process of metastasis, we studied 61 paired primary-relapse BC from the GEICAM ConvertHER study. While some of the metastases maintained the clinical (ER/PR and HER2 status) and/or intrinsic subtype (defined by expression arrays) of the original tumor (concordant), others exhibited a subtype shift (discordant). We aimed to identify the genomic alterations driving the metastases and, particularly, their relationship with the subtype switch. Methods: We detected the somatic variants (mutations and copy number alterations (CNAs)) affecting 202 genes across the 61 sample pairs via targeted sequencing. We employed the Cancer Genome Interpreter (cancergenomeinterpreter.org), a bioinformatics approach to identify the alterations most likely driving tumorigenesis, and subsequently identified those whose cancer cell fraction markedly changed in the metastases. We explored the clonal remodeling in metastasis comparing the cell fractions of driver mutations in both concordant and discordant tumors. Results: We found that 156 genes had 747 somatic mutations and 171 genes suffered 1042 somatic CNAs in the 61 studied tumor pairs. We identified a median of 11 and 9 mutations in primaries and metastases, respectively. Several frequent BC mutational drivers, such as TP53, PIK3CA, MLL3, MAP3K1, and NOTCH2 were amongst the more frequently changed their cancer cell fraction in metastases with respect to primaries. We found that driver mutations of discordant tumors exhibited a significantly higher increase of clonal cell fraction. Moreover, whether the clonal status of a driver mutation was conserved in the metastasis was significantly associated to whether the tumor maintains its clinical subtype but not its intrinsic subtype. Conclusions: Our results suggest that a shift in the clinical subtype of BC undergoing metastasis is accompanied by more significant changes at the genomic level than those suffered by tumors that maintain their clinical subtype. This remodeling of the landscape of drivers could open new therapeutic opportunities to specifically target discordant BC.

Reflex Repeat HER2 Testing of Grade 3 Breast Carcinoma at Excision Using Immunohistochemistry and In Situ Analysis

The updated American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines (2013) for human epidermal growth factor receptor 2 (HER2) testing in breast cancer recommend repeat testing at excision of HER2-negative grade 3 breast tumors. This study aimed to identify the rate of HER2 discordance in this cohort of cases. All HER2-negative grade 3 tumors diagnosed at a single institution over a 15-month period had reflex repeat HER2 testing at excision : HER2 testing was performed in accordance with ASCO/CAP guidelines using immunohistochemistry (IHC) and dual in situ hybridization (ISH). One hundred cases were identified over the study period. HER2 was amplified at excision in three cases. The discordant tumors showed equivocal IHC at excision with low-level amplification on dual ISH. All discordant cases showed equivocal IHC on core needle biopsy (CNB) specimens and/or tumor upgrade at excision. Our series demonstrated a high concordance rate (97%) for HER2 at excision in grade 3 breast tumors with a negative core biopsy result. These findings suggest that reflex repeat HER2 testing of all these cases, which has significant cost and workload implications, may not be justified. Features that may indicate HER2 heterogeneity, such as equivocal IHC on CNB specimens or tumor upgrade at excision, may help refine selection of cases for repeat testing.

215 Using ALK IHC test to identify potential responders to ALK inhibitors outside of NSCLC

Background: In addition to anaplastic lymphomas and NSCLC, other solid malignancies can exhibit aberration in ALK expression potentially amenable to benefit from TKI ALK inhibitors. ALK expression by immunohistochemistry (IHC) has been extensively studied in NSCLC as a cost-effective and accurate screening tool for ALK rearrangement, and may identify patients (pts) with other mechanisms of ALK over-expression; however it has not been systematically studied outside of NSCLC. Methods: To validate ALK IHC test in a CLIA-certified lab, 86 cancer cases with documented ALK rearrangement by FISH (Abbott, Vysis break apart) results were tested by IHC using anti-ALK antibody D5F3 (Ventana) and evaluated by two pathologists (threshold: 3+ in >1% of cells). 43 NSCLC and 43 tumors of 23 other cancer types were included. Follow-up with treating physicians on ALK positive (pos) pts was attempted. Additionally, a prospective dataset of >2000 routinely profiled tumors tested for ALK IHC was analyzed. Results: In 43 NSCLC, ALK IHC was concordant with FISH in 93% (sensitivity = 90%, specificity = 95%). In non-NSCLC, all ALK-FISH pos tumors were also pos for ALK IHC (N = 4), and all IHC negative (neg) tumors were neg by FISH (N = 35) (sensitivity = 100%, specificity = 90%). Of the 4 IHC+/FISH+ tumors, 1 was myxoid leiomyosarcoma of the uterus (Pt A), 1 was inflammatory myofibroblastic tumor (IMT) (B), 1 was neuroendocrine tumor of the lung (C) and 1 was neuroblastoma (D). Of the 4 discordant (IHC +/FISH −) tumors, 1 was melanoma (E), 2 were serous ovarian cancers (F and G) and 1 was small cell cancer of the cervix (H). Follow-up was available for 5 IHC pos pts (A, B, F, G and H). A and B were treated with crizotinib: A deceased within 2 months and B has ongoing partial response to crizotinib for >2 years to date. G will start on a phase 1b crizotinib trial and outcome will be presented. F and H were not treated with ALK inhibitors. Prospective collection of ALK IHC on a large cohort of tumors (N > 2000) from >24 cancer types and additional follow up will be presented. Conclusions: Using FISH for comparison, our pilot study of 43 tumors of 23 cancer types outside of NSCLC shows the sensitivity and specificity for ALK IHC in non-NSCLC to be 100% and 90%. ALK IHC therefore carries great potential as a screening tool for ALK rearrangement and could potentially identify other ALK aberrations, thus identifying potential responders for ALK inhibitors in various cancers. No conflict of interest.

BRAF Testing in Multifocal Papillary Thyroid Carcinoma.

Background. BRAF V600E mutation is associated with poor prognosis in patients with papillary thyroid carcinoma (PTC). PTC is often multifocal, and there are no guidelines on how many tumors to test for BRAF mutation in multifocal PTC. Methods. Fifty-seven separate formalin-fixed and paraffin-embedded PTCs from twenty-seven patients were manually macrodissected and tested for BRAF mutation using a commercial allele-specific real-time polymerase chain reaction-based assay (Entrogen, Woodland Hills, CA). Data related to histologic characteristics, patient demographics, and clinical outcomes were collected. Results. All mutations detected were BRAF V600E. Seventeen patients (63%) had concordant mutation status in the largest and second-largest tumors (i.e., both were positive or both were negative). The remaining ten patients (37%) had discordant mutation status. Six of the patients with discordant tumors (22% overall) had a BRAF-negative largest tumor and a BRAF-positive second-largest tumor. No histologic feature was found to help predict which cases would be discordant. Conclusions. Patients with multifocal PTC whose largest tumor is BRAF-negative can have smaller tumors that are BRAF-positive. Therefore, molecular testing of more than just the dominant tumor should be considered. Future studies are warranted to establish whether finding a BRAF mutation in a smaller tumor has clinical significance.

Heterogeneity in hormone-receptor status and survival outcomes among women with synchronous and metachronous bilateral breast cancers

To examine whether discordance in the hormone-receptor status predicts clinical outcomes in patients with bilateral synchronous (SBC) or metachronous breast cancer (MBC), we analyzed data from the Surveillance, Epidemiology, and End Results program (1998–2011) using Cox models. After excluding 10,231 patients with missing data on hormone receptors in at least one tumor, 4403 SBC and 7159 MBC were included in the study. Among SBC cases, patients with estrogen receptor (ER)-discordant tumors had higher mortality risk (multivariable-adjusted hazard ratio [HR] = 1.96, 95% confidence interval [CI] 1.60–2.40) than patients with ER concordant-positive tumors, whereas patients with ER concordant-negative tumors had the highest risk (HR = 2.49, 95% CI 2.03–3.07). Among MBC cases, patients with a positive-to-negative change in ER status (HR = 1.32, 95% CI: 1.08–1.62) or ER concordant-negative tumors (HR = 1.48, 95% CI: 1.19–1.85) had worse survival than patients with ER concordant-positive tumors. In conclusion, discordance in the hormone-receptor status was an independent predictor of survival outcomes.

Relative cerebral blood volume from dynamic susceptibility contrast perfusion in the grading of pediatric primary brain tumors.

The aim of this study is to evaluate the utility of relative cerebral blood volume (rCBV) data from dynamic susceptibility contrast (DSC) perfusion in grading pediatric primary brain tumors. A retrospective blinded review of 63 pediatric brain tumors with DSC perfusion was performed independently by two neuroradiologists. A diagnosis of low- versus high-grade tumor was obtained from conventional imaging alone. Maximum rCBV (rCBVmax) was measured from manual ROI placement for each reviewer and averaged. Whole-tumor CBV data was obtained from a semi-automated approach. Results from all three analyses were compared to WHO grade. Based on conventional MRI, the two reviewers had a concordance rate of 81% (k = 0.62). Compared to WHO grade, the concordant cases accurately diagnosed high versus low grade in 82%. A positive correlation was demonstrated between manual rCBVmax and tumor grade (r = 0.30, P = 0.015). ROC analysis of rCBVmax (area under curve 0.65, 0.52-0.77, P = 0.03) gave a low-high threshold of 1.38 with sensitivity of 92% (74-99%), specificity of 40% (24-57%), NPV of 88% (62-98%), and PPV of 50% (35-65%) Using this threshold on 12 discordant tumors between evaluators from conventional imaging yielded correct diagnoses in nine patients. Semi-automated analysis demonstrated statistically significant differences between low- and high-grade tumors for multiple metrics including average rCBV (P = 0.027). Despite significant positive correlation with tumor grade, rCBV from pediatric brain tumors demonstrates limited specificity, but high NPV in excluding high-grade neoplasms. In selective patients whose conventional imaging is nonspecific, an rCBV threshold may have further diagnostic value.

Loss of Human Epidermal Growth Factor Receptor 2 (HER2) Expression in Metastatic Sites of HER2-Overexpressing Primary Breast Tumors

We evaluated whether patients with human epidermal growth factor receptor 2 (HER2) -positive primary breast tumors had metastatic tumors that were HER2 positive (concordant) or HER2 negative (discordant). We then evaluated whether treatment with trastuzumab or chemotherapy before biopsy of the metastasis had any effect on the rate of HER2 discordance. We also compared the overall survival durations of patients with HER2-concordant and -discordant tumors. We retrospectively identified all patients who initially had been diagnosed with HER2-positive (immunohistochemistry 3+ and/or fluorescent in situ hybridization positive) primary breast cancer between 1997 and 2008 at MD Anderson Cancer Center who also had metastatic tumor biopsy results available for review. We included 182 patients who met our criteria. Forty-three (24%) of the 182 patients with HER2-positive primary tumors had HER2-negative metastatic tumors. The HER2 discordance rates differed significantly on the basis of whether patients received chemotherapy (P = .022) but not on the basis of whether patients received trastuzumab (P = .296). Patients with discordant HER2 status had shorter overall survival than did patients with concordant HER2 status (hazard ratio [HR], 0.43; P = .003). A survival difference remained among the 67 patients who received trastuzumab (HR, 0.56; P = .083) and 101 patients who did not (HR, 0.53; P = .033) before their metastasis biopsies. We confirmed that loss of HER2-positive status in metastatic tumors can occur in patients with primary HER2-positive breast cancer. Our data strongly support the need for biopsies of metastatic lesions to accurately determine patient prognosis and appropriate use of targeted therapy.

Detection of BRAF kinase mutations in melanoma, ovarian, and prostate carcinomas: Evidence for tumor heterogeneity in clinical samples

11031 Background: Several studies have provided evidence that solid tumors are polyclonal malignancies, an observation which may contribute to difficulties in achieving durable treatment responses. In some patients, molecularly targeted therapies may be compromised due to heterogeneity among tumor subclones. In this study we compared conventional DNA sequencing with a fluorescent-based mutant-specific PCR (MS-PCR) assay to detect the BRAF hotspot mutation V600E in a large panel of patient tumors, including paired primary and metastatic tumors from individual patients. Methods: BRAF MS-PCR and conventional sequencing were performed on DNA from 304 tumors (112 melanoma, 110 ovarian, 82 prostate) to determine the presence of the BRAFV600E hot-spot mutation. Among the melanomas were 18 matched primary and metastatic specimens, and 40 metastatic specimens from 19 patients, each of whom had 2 or more metastases. Results: DNA sequencing detected mutations in 5/110 (4.5%) ovarian tumors, 1/82 (1.2%) prostate tumors, and 36/112 (32%) melanomas. In contrast, the MS-PCR assay detected mutations in 12/110 (11%) ovarian tumors, 15/82 (18%) prostate tumors and 85/112 (76%) melanomas. The presence of contaminating normal tissue was scored for each melanoma sample, but excess normal tissue did not influence the results using either methodology. In all cases mutations detected by sequencing were also detected by MSPCR. Among 18 patients with matched primary and metastatic melanoma, 8/18 (44%) had discordant results including 2 patients with mutant primary tumors and wild-type metastases; among the 19 patients with multiple metastases 5/19 (26%) had discordant (both wild-type and mutant) tumors. Conclusions: Using a highly sensitive BRAF mutation detection method, we observed substantial evidence for heterogeneity within clinical tumor specimens. This was especially true in melanoma samples, where multiple specimens from individual patients differed with respect to the presence of the mutant BRAF allele. These results suggest that failures of molecularly targeted therapies, such as those directed against mutant BRAF, may be due in part to a lack of clonality among the tumors under treatment. No significant financial relationships to disclose.