Incidental germline findings during molecular profiling of tumor tissues for precision oncology: molecular survey and methodological obstacles.

BACKGROUND: A fraction of patients referred for complex molecular profiling of biopsied tumors may harbor germline variants in genes associated with the development of hereditary cancer syndromes (HCS). Neither the bioinformatic analysis nor the reporting of such incidental germline findings are standardized. METHODS: Data from Next-Generation Sequencing (NGS) of biopsied tumor samples referred for complex molecular profiling were analyzed for germline variants in HCS-associated genes. Analysis of variant origin was performed employing bioinformatic algorithms followed by manual curation. When possible, the origin of the variant was validated by Sanger sequencing of the sample of normal tissue. The variants' pathogenicity was assessed according to ACMG/AMP. RESULTS: Tumors were sampled from 183 patients (Males: 75 [41.0%]; Females: 108 [59.0%]; mean [SD] age, 57.7 [13.3] years) and analysed by targeted NGS. The most common tumor types were colorectal (19%), pancreatic (13%), and lung cancer (10%). A total of 56 sequence variants in genes associated with HCS were detected in 40 patients. Of them, 17 variants found in 14 patients were predicted to be of germline origin, with 6 variants interpreted as pathogenic (PV) or likely pathogenic (LPV), and 9 as variants of uncertain significance (VUS). For the 41 out of 42 (97%) missense variants in HCS-associated genes, the results of computational prediction of variant origin were concordant with that of experimental examination. We estimate that Sanger sequencing of a sample of normal tissue would be required for ~ 1-7% of the total assessed cases with PV or LPV, when necessity to follow with genetic counselling referral in ~ 2-15% of total assessed cases (PV, LPV or VUS found in HCS genes). CONCLUSION: Incidental findings of pathogenic germline variants are common in data from cancer patients referred for complex molecular profiling. We propose an algorithm for the management of patients with newly detected variants in genes associated with HCS.

Effects of subanesthetic ketamine and (2R,6R) hydroxynorketamine on working memory and synaptic transmission in the nucleus reuniens in mice.

RATIONALE: Acute cognitive impairment and abuse potential of ketamine incentivizes the search for alternatives to ketamine for clinical management of treatment-resistant depression. Recently, (2R,6R) hydroxynorketamine ((2R,6R)-HNK), a metabolite of ketamine, has shown promise due to its reported lack of ketamine-like reinforcing properties. Nonetheless, the effect of (2R,6R)-HNK on cognition has not been reported. METHOD: Adult male mice were placed in a Y-maze to measure spatial working memory (SWM) 24 h after treatment with either a single or repeated subanesthetic dose of (2R,6R)-HNK or ketamine. To determine the effect of the drug regimens on synaptic mechanisms in neural circuits deemed critical for SWM, we conducted patch-clamp electrophysiological recordings from neurons in the midline thalamic nucleus reuniens (RE) in response to optogenetic stimulation of medial prefrontal cortex (mPFC) inputs in acutely prepared brain slices. RESULTS: Single or repeated treatment with a 10 mg/kg dose of either drug did not impact performance in a Y-maze. However, single administration of a ½-log higher dose (32 mg/kg) of ketamine significantly reduced SWM. The same dose of (2R,6R)-HNK did not produce SWM deficits. Interestingly, repeated administration of either drugs at the 32 mg/kg had no effect on SWM performances. Concomitant to these effects on SWM, only single injection of 32 mg/kg of ketamine was found to increase the mPFC-driven action potential firing activity in the RE neurons. Conversely, both single and repeated administration of the 32 mg/kg dose of (2R,6R)-HNK but not ketamine, increased the input resistance of the RE neurons. CONCLUSION: Our results indicate that acute treatment of ketamine at 32 mg/kg increases mPFC-driven firing activity of RE neurons, and this contributes to the ketamine-mediated cognitive deficit. Secondly, sub-chronic treatment with the same dose of ketamine likely induces tolerance. Although single or repeated administration of the 32 mg/kg dose of (2R,6R)-HNK can alter intrinsic properties of RE neurons, this dose does not produce cognitive deficit or changes in synaptic mechanism in the RE. This article is part of the special Issue on 'Stress, Addiction and Plasticity'.

Opioid receptor system contributes to the acute and sustained antidepressant-like effects, but not the hyperactivity motor effects of ketamine in mice.

In 2000, a subanesthetic dose (0.5 mg/kg i.v.) of the dissociative anesthetic ketamine was reported to have both rapid and robust antidepressant effects in patients diagnosed with major depressive disorder and later, ketamine also was shown to be effective in treatment-resistant depressed patients. However, the mechanisms responsible for ketamine's antidepressant effects remain unclear. In 2018, a clinical study reported that pretreatment with the nonselective opioid antagonist naltrexone attenuated the rapid antidepressant effect of ketamine in depressed patients. The current study investigated the potential role of the opioid receptor system in the acute and sustained antidepressant-like and hyperactive effects of ketamine. Mice were tested in the tail suspension test (TST) and differential-reinforcement-of-low-rate responding (DRL) 72 s task which are behavioral screens for antidepressant-like properties. Additionally, open field locomotor activity also was measured. In all behavioral assays, mice were pretreated with the nonselective opioid receptor antagonist naltrexone or saline prior to ketamine administration. The current study found that ketamine (10 mg/kg) produced acute (30 min) and sustained (24 h) antidepressant-like effects in TST, which were attenuated by pretreatment of 2 mg/kg naltrexone. Ketamine (32 mg/kg) also produced an acute antidepressant-like effect in the DRL 72 s task that was attenuated by pretreatment of 2 mg/kg naltrexone. Finally, ketamine (10 and 32 mg/kg) produced hyperactivity in the open field; however, pretreatment with 2 mg/kg naltrexone failed to block the hyperactivity effects ketamine. These results, along with recent clinical findings, suggest that ketamine's antidepressant effects, but not its hyperactive effects, involve activation of the opioid system.