Ladanyi, Marc

Abstract BACKGROUND Accurate molecular diagnosis of infiltrating brainstem tumors, including diffuse midline gliomas and IDH mutant astrocytomas, is essential for prognostication and optimal therapy. Surgical biopsy of the brainstem carries risk of permanent neurological deficits and may yield insufficient or non-diagnostic tissue. We hypothesized that minimally invasive “liquid biopsy” analyzing circulating tumor cell-free DNA (cfDNA) in cerebrospinal fluid (CSF) could represent an alternative molecular diagnostic modality. METHODS We performed MSK-IMPACT, a New York State Department of Health authorized hybridization capture-based next-generation panel DNA sequencing clinical assay on 44 CSF samples from 37 unique patients with brainstem tumors. Ages at time of CSF collection ranged from 1–47 years (median: 29 years). For samples in which diagnostic alterations could not be detected, we performed a series of secondary analyses for known hotspot mutations such as H3F3A, IDH1/2, and BRAF, including manual review for mutant reads below the official clinical reporting threshold, as well as droplet digital PCR (ddPCR). RESULTS Among 44 samples included in this study, 10/44 (23%) samples had mutations detected by MSK-IMPACT using standard clinical calling criteria and 14 of the remaining 34 samples (41%) had supporting evidence of a diagnostically relevant mutation based on manual review. Testing by ddPCR was performed on 11 samples (based on DNA availability), uncovering 4 additional mutations. Overall positive diagnostic yield was 64% (28/44). In biopsied patients, CSF confirmed disease-defining mutations in 80% (16/20) of CSF samples, including H3F3A K28M (9/10; 90%) and IDH1/2 (5/6; 83%). Among 24 unbiopsied patients, a disease-defining mutation was identified in 60% (15/25) of CSF samples. CONCLUSIONS Analysis of CSF cfDNA in patients with brainstem tumors has a high diagnostic yield and obviates the need for tissue biopsy in a majority of patients. A tiered testing approach using next-generation sequencing and ddPCR assays helps maximize diagnostic information and sensitivity.

Abstract Intrinsic brainstem tumors arising in pediatric, adolescent, and young adult patients comprise a spectrum of entities, predominantly diffuse midline gliomas (DMG) and IDH mutant astrocytomas. Accurate molecular diagnosis is essential for prognostication and optimal therapy. Therapeutic considerations include inclusion (in IDH mutant) or exclusion (in H3K27M) of adjuvant Temozolomide post radiotherapy and use of molecular targeted therapy (IDH inhibitors). Surgical biopsy, however, is associated with increased risk of permanent neurological deficits and may yield insufficient or non-diagnostic tissue. We hypothesized that minimally-invasive “liquid biopsy” of cerebrospinal fluid (CSF) could represent a superior diagnostic modality in this patient population, employing molecular analysis of cell free DNA (cfDNA). We analyzed 44 CSF samples from 38 unique patients for recurrent driver mutations in brainstem gliomas, including H3K27M, IDH1, and IDH2. MSK-IMPACT, a NY state-authorized hybridization capture-based next-generation panel DNA sequencing assay, was used for analysis. Samples without a detectable driver mutation by MSK-IMPACT testing were further subjected to gene targeted testing by droplet digital PCR. In all, 10/44 (22.7%) samples had mutations detected by MSK-IMPACT using standard calling criteria and 13 of the remaining 34 samples (38.2%) had supporting evidence of a mutation based on manual review. Further testing by ddPCR was performed on a subset of cases (based on DNA availability) which confirmed 7 of the previous low-level mutations and uncovered 4 additional mutations. Overall, 27/44 (61.4%) of cases had detectable evidence of a mutation, and of the 23 patients without a known tissue diagnosis, a driver in the CSF was identified for 47.8% of patients (11/23). Minimally-invasive analysis of CSF cfDNA in patients with intrinsic brainstem tumors has a high diagnostic yield and may obviate the need for tissue biopsy in a majority of patients. Testing with high sensitivity assays is valuable to maximize the rate of detection.

Abstract PURPOSE: A subset of pediatric, adolescent and young adult (AYA) gliomas are located in the brainstem, eloquent locations, or present with diffuse/leptomeningeal disease, and are associated with high risk and low yield of biopsy. At the same time, accurate molecular diagnosis is necessary to direct optimal therapy. In such cases, analysis of cell free DNA (cfDNA) form cerebral spinal fluid (CSF) may represent a diagnostic alternative to biopsy. METHODS: We investigated the utility of CSF cfDNA sequencing through a stepwise approach, using clinically validated, targeted molecular assays. Testing was performed using a broad hybrid capture next generation sequencing assay (MSK-IMPACT) and subsequent targeted digital droplet PCR in a subset of cases. RESULTS: We analyzed 17 CSF samples from 17 pediatric (n=6) and AYA (n=11) glioma patients with primary or recurrent disease. Thirteen had tumors located within the brainstem, and four had leptomeningeal involvement. Somatic alterations were detected in 12/17 samples (71%). In 3/4 patients with leptomeningeal involvement, cfDNA testing revealed a BRAF fusion consistent with the diagnosis of diffuse leptomeningeal glioneuronal tumor (DLGNT). Among the 13 patients with brainstem involvement, four had somatic H3 K27M mutations, three had IDH mutations, and one had TP53 and ATRX mutations; five patients had no detectable mutations. CONCLUSION: In our analysis, we found that established glioma hotspot mutations were able to be detected within the CSF. We propose that in patients for whom tissue biopsy is high risk, not feasible, or tissue was nondiagnostic, CSF cfDNA sequencing has a substantial diagnostic yield and should be considered as a valuable novel diagnostic tool. Ongoing research is aiming to further increase the sensitivity of cfDNA testing, especially in patients with very low levels of CSF cfDNA.

BACKGROUND: Immune checkpoint inhibitors are an important therapeutic option for urothelial carcinoma, but durable responses are achieved in a minority of patients. Identifying pre-treatment biomarkers that may predict response to these therapies or who exhibit intrinsic resistance, is of paramount importance. OBJECTIVE: To explore the prevalence of PD-L1 copy number alteration in urothelial carcinoma and correlate with response to immune checkpoint inhibitors. METHODS: We analyzed a cohort of 1050 carcinomas of the bladder and upper urinary tract that underwent targeted next generation sequencing, prospectively. We assessed PD-L1 protein expression, copy number status (next generation sequencing/FISH), and detailed treatment response. RESULTS: We identified 9 tumors with PD-L1 amplification and 9 tumors with PD-L1 deletion. PD-L1 protein expression was the highest in PD-L1 amplified tumors. Of the 9 patients whose tumors harbored PD-L1 amplification, 6 received immunotherapy with 4 deriving clinical benefit, and 2 achieving durable response. Of the 9 patients whose tumors had PD-L1 copy number losses, 4 received immunotherapy with 3 experiencing disease progression. CONCLUSIONS: PD-L1 copy number alterations may serve as potential biomarkers of response to immunotherapy in urothelial carcinoma patients, if validated in larger cohorts.

11085 Background: The College of American Pathologists (CAP), the International Association for the Study of Lung Cancer (IASLC), and the Association for Molecular Pathology (AMP) jointly initiated an effort to establish evidence-based recommendations for the molecular analysis of lung cancers required to guide EGFR- and ALK-directed therapies, addressing which patients and samples should be tested, and when and how testing should be performed. Methods: Three co-chairs without relevant conflicts of interest were selected, one from each of the sponsoring societies: CAP (P.T.C.), IASLC (M.L.), and AMP (N.I.L.). Writing and advisory panels were formed from additional experts from these societies. Unbiased literature searches were performed to capture articles up to February 2012, yielding 1,533 articles whose abstracts were screened to identify 521 pertinent articles that were then reviewed in detail for relevance. Evidence was formally graded for each of the recommendations first formulated by the co-chairs and panel members at a public meeting. Each guideline section was assigned to at least two panelists. Successive drafts were circulated for comments to the writing panel, the advisory panel, the public (online posting), and the three professional societies. Results: We generated 37 guideline items addressing 14 areas of EGFR and ALK testing. The major, evidence-based recommendations are to test for EGFR mutations and ALK fusions in all patients with advanced stage adenocarcinoma, regardless of sex, race, or smoking history, and to prioritize EGFR and ALK testing over other molecular predictive tests. Recommendations and expert consensus opinions were generated for all other key aspects of EGFR and ALK testing in lung cancer related to oncology and pathology practice and technical issues in molecular testing. Conclusions: As scientific discoveries and clinical practice outpace the completion of randomized clinical trials, evidence-based guidelines developed by expert practitioners are vital for communicating emerging clinical standards and thereby improving patient outcomes.

We have identified 25 lesions involving alveolar lung parenchyma characterized by nodular proliferation of bland bilayered bronchiolar-type epithelium containing a continuous layer of basal cells. These lesions shared some histologic features with the recently described entity of ciliated muconodular papillary tumor (CMPT); however, the majority did not fit all diagnostic criteria in that they exhibited only focal or absent papillary architecture, and they had variable number of ciliated and mucinous cells, with some lesions entirely lacking 1 or both of these components. The morphologic and immunohistochemical features ranged from those resembling proximal bronchioles (proximal-type: moderate to abundant mucinous and ciliated cells; negative or weak TTF1 in luminal cells; n=8) to those resembling respiratory bronchioles (distal-type: scant or absent mucinous and ciliated cells; positive TTF1 in luminal cells; n=17). The hallmark of all lesions was a continuous layer of basal cells (p40 and CK5/6-positive). We provisionally designated these lesions as bronchiolar adenomas (BAs) and analyzed their clinicopathologic and molecular features. All BAs were discrete, sharply circumscribed lesions with a median size of 0.5 cm (range, 0.2 to 2.0 cm). Most lesions were either entirely flat (n=14) or contained focal papillary architecture (n=7); only 4 lesions, all proximal-type, were predominantly papillary, fitting the classic description of CMPT. Notably, of 9 lesions submitted for frozen section evaluation, 7 were diagnosed as adenocarcinoma. No postsurgical recurrences were observed for any lesions (median follow-up, 11 mo). Twenty-one BAs underwent next-generation sequencing and/or immunohistochemistry for BRAF V600E, revealing mutation profiles similar to those previously described for CMPTs, including BRAF V600E mutations (n=8, 38%), unusual EGFR exon 19 deletions (n=2, 10%), EGFR exon 20 insertions (n=2, 10%), KRAS mutations (n=5, 24%), and HRAS mutations (n=1, 5%). The mutation profiles were similar in proximal-type and distal-type lesions. In conclusion, we describe a family of putatively benign clonal proliferations with a spectrum of morphology recapitulating various levels of the bronchiolar tree, of which only a minor subset fits the classic description of CMPT. Comparable mutation profiles and partially overlapping morphologic features across the spectrum of these lesions support their nosological relationship. We propose designating this entire family of lesions as BAs, and that lesions currently designated CMPTs represent a subgroup of this family.

Abstract Introduction. For patients with non-small cell lung cancer (NSCLC) to benefit from ALK inhibitors, sensitive and specific detection of ALK genomic rearrangements is needed. ALK break-apart fluorescence in situ hybridization (FISH) is the U.S. Food and Drug Administration approved and standard-of-care diagnostic assay, but identification of ALK rearrangements by other methods reported in NSCLC cases that tested negative for ALK rearrangements by FISH suggests a significant false-negative rate. We report here a large series of NSCLC cases assayed by hybrid-capture-based comprehensive genomic profiling (CGP) in the course of clinical care. Materials and Methods. Hybrid-capture-based CGP using next-generation sequencing was performed in the course of clinical care of 1,070 patients with advanced lung cancer. Each tumor sample was evaluated for all classes of genomic alterations, including base-pair substitutions, insertions/deletions, copy number alterations and rearrangements, as well as fusions/rearrangements. Results. A total of 47 patients (4.4%) were found to harbor ALK rearrangements, of whom 41 had an EML4-ALK fusion, and 6 had other fusion partners, including 3 previously unreported rearrangement events: EIF2AK-ALK, PPM1B-ALK, and PRKAR1A-ALK. Of 41 patients harboring ALK rearrangements, 31 had prior FISH testing results available. Of these, 20 were ALK FISH positive, and 11 (35%) were ALK FISH negative. Of the latter 11 patients, 9 received crizotinib based on the CGP results, and 7 achieved a response with median duration of 17 months. Conclusion. Comprehensive genomic profiling detected canonical ALK rearrangements and ALK rearrangements with noncanonical fusion partners in a subset of patients with NSCLC with previously negative ALK FISH results. In this series, such patients had durable responses to ALK inhibitors, comparable to historical response rates for ALK FISH-positive cases.