SUMMARY KRAS is one of the driver oncogenes in non-small-cell lung cancer (NSCLC) but remains refractory to current modalities of targeted pathway inhibition, which include inhibiting downstream kinase MEK to circumvent KRAS activation. Here, we show that pulsatile, rather than continuous, treatment with MEK inhibitors (MEKis) maintains T cell activation and enables their proliferation. Two MEKis, selumetinib and trametinib, induce T cell activation with increased CTLA-4 expression and, to a lesser extent, PD-1 expression on T cells in vivo after cyclical pulsatile MEKi treatment. In addition, the pulsatile dosing schedule alone shows superior anti-tumor effects and delays the emergence of drug resistance. Furthermore, pulsatile MEKi treatment combined with CTLA-4 blockade prolongs survival in mice bearing tumors with mutant Kras. Our results set the foundation and show the importance of a combinatorial therapeutic strategy using pulsatile targeted therapy together with immunotherapy to optimally enhance tumor delay and promote long-term anti-tumor immunity.

Leiomyosarcoma (LMS) belongs to the class of genetically complex sarcomas and shows numerous, often non-recurrent chromosomal imbalances and aberrations. We investigated a group of LMS using NGS platform to identify recurrent genetic abnormalities and possible therapeutic targets. Targeted exome sequencing of 230 cancer-associated genes was performed on 35 primary soft tissue and visceral (extra-uterine) LMS. Sequence data were analyzed to identify single nucleotide variants, small insertions/deletions (indels), and copy number alterations. Key alterations were further investigated using FISH assay. The study group included patients with median age of 64 years and median tumor size of 7 cm. The primary sites included retroperitoneal/intra-abdominal, extremity, truncal and visceral. Thirty one tumors were high grade LMS, while 4 were low grade. Losses of chromosomal regions involving key tumor suppressor genes PTEN (10q), RB1 (13q), CDH1 (16q) and TP53 (17p) were the most frequent genetic events. Gains mainly involved chromosome regions 17p11.2 (MYOCD) and 15q25-26 (IGF1R). The most frequent mutations were identified in the TP53 gene in 13 of 35 (37%) cases. FISH analysis showed amplification of the myocardin (MYOCD) gene in 5 of 25 (20%) cases analyzed. None of the four low grade LMS showed losses or mutations of PTEN or TP53 genes. Genetic complexity is the hallmark of LMS with losses of important tumor suppressor genes being a common feature. MYOCD, a key gene associated with smooth muscle differentiation, is amplified in a subset of both retroperitoneal and extremity LMS. Further studies are necessary to investigate the significance of gains/amplifications in the development of these tumors.

Context.-Touch preparations (TP) of core needle biopsies (CNBs) are used at some institutions for on-site assessment of CNB adequacy. In our clinical practice, we have encountered instances in which TPs resulted in substantial depletion of CNB cellularity.Objective.-To examine the effect of increasingly vigorous TPs on cellularity and DNA content of CNBs.Design.-Ex vivo CNBs (n ¼ 56) were performed on resected lung and kidney tumor specimens. For each specimen, CNBs were performed in quadruplicate on tumor and nontumor tissue and subjected to 1 of 4 TP methods: imprint, 1-cm drag, 2-cm drag, or full-slide drag. Overall cellularity in TPs relative to corresponding CNBs was estimated semiquantitatively. DNA was extracted and quantified from 12 TPs and corresponding CNBs. Two cytopathologists performed a blinded diagnostic assessment of Diff-Quik-stained TPs.Results.-Cellularity of imprint, 1-cm, 2-cm, and fullslide TPs represented, on average, 19%, 33%, 41%, and 46% of total CNB cellularity, respectively (P ¼ .003). Average DNA content in imprint, 1-cm, and 2-cm TPs was 0.3 lg (range, 0.1-0.8 lg), 0.4 lg (range, 0.1-1 lg), and 0.6 lg (range, 0.2-1.3 lg), respectively, which represented on average 15%, 36%, and 50%, respectively, of total CNB DNA content. Diagnostic accuracy was not inferior for less-extensive TPs, compared with more-extensive TPs.Conclusions.-Vigorous TPs may contain a substantial fraction of CNB cellularity and DNA content, whereas more-limited TPs are less disruptive to CNBs but remain suitable for cytologic assessment. We suggest avoiding excessively forceful TPs and, whenever clinically feasible, obtaining additional samples to ensure sufficient cellularity for potential ancillary studies.

Context Targeted needle biopsies are increasingly performed for the genetic characterization of cancer. While the nucleic acid content of core needle biopsies after standard pathology processing (i.e., formalin fixation and paraffin embedding (FFPE)) has been previously reported, little is known about the potential yield for molecular analysis at the time of biopsy sample acquisition. Objectives Our objective was to improve the understanding of DNA and RNA yields from commonly used core needle biopsy techniques prior to sample processing. Methods We performed 552 ex vivo 18 and 20G core biopsies in the lungs, liver, and kidneys. DNA and RNA were extracted from fresh-frozen core samples and quantified for statistical comparisons based on needle gauge, biopsy site, and tissue type. Results Median tumor DNA yields from all 18G and 20G samples were 5880 ng and 2710 ng, respectively. Median tumor RNA yields from all 18G and 20G samples were 1100 ng and 230 ng, respectively. A wide range of DNA and RNA quantities (1060–13,390 ng and 370–6280 ng, respectively) were acquired. Median DNA and RNA yields from 18G needles were significantly greater than those from 20G needles across all organs (p < 0.001). Conclusions Core needle biopsy techniques for cancer diagnostics yield a broad range of DNA and RNA for molecular pathology, though quantities are greater than what has been reported for FFPE processed material. Since non-formalin-fixed DNA is advantageous for molecular studies, workflows that optimize core needle biopsy yield for molecular characterization should be explored.