Borsu, Laetitia

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.

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.