Characterization of Non-Small-Cell Lung Cancers With MET Exon 14 Skipping Alterations Detected in Tissue or Liquid: Clinicogenomics and Real-World Treatment Patterns.

PURPOSE: MET exon 14 (METex14) skipping alterations are oncogenic drivers in non-small-cell lung cancer (NSCLC). We present a comprehensive overview of METex14 samples from 1,592 patients with NSCLC, associated clinicogenomic characteristics, potential mechanisms of acquired resistance, treatment patterns, and outcomes to MET inhibitors. METHODS: Hybrid capture-based comprehensive genomic profiling (CGP) was performed on samples from 69,219 patients with NSCLC. For treatment patterns and outcomes analysis, patients with advanced METex14-altered NSCLC were selected from the Flatiron Health-Foundation Medicine clinicogenomic database, a nationwide deidentified electronic health record-derived database linked to Foundation Medicine CGP for patients treated between January 2011 and March 2020. RESULTS: A total of 1,592 patients with NSCLC (2.3%) were identified with 1,599 METex14 alterations spanning multiple functional sites (1,458 of 60,244 tissue samples and 134 of 8,975 liquid samples). Low tumor mutational burden and high programmed death ligand 1 expression were enriched in METex14-altered samples. MDM2, CDK4, and MET coamplifications and TP53 mutations were present in 34%, 19%, 11%, and 42% of tissue samples, respectively. Comparing tissue and liquid cohorts, coalteration frequency and acquired resistance mechanisms, including multiple MET mutations, EGFR, ERBB2, KRAS, and PI3K pathway alterations, were generally similar. Positive percent agreement with the tissue was 100% for METex14 pairs collected within 1 year (n = 7). Treatment patterns showed increasing adoption of MET inhibitors in METex14-altered NSCLC after receipt of CGP results; the real-world response rate to MET inhibitors was 45%, and time to treatment discontinuation was 4.4 months. CONCLUSION: Diverse METex14 alterations were present in 2%-3% of NSCLC cases. Tissue and liquid comparisons showed high concordance and similar coalteration profiles. Characterizing common co-occurring alterations and immunotherapy biomarkers, including those present before or acquired after treatment, may be critical for predicting responses to MET inhibitors and informing rational combination strategies.

Targetable BRAF and RAF1 Alterations in Advanced Pediatric Cancers.

RAF family protein kinases signal through the MAPK pathway to orchestrate cellular proliferation, survival, and transformation. Identifying BRAF alterations in pediatric cancers is critically important as therapeutic agents targeting BRAF or MEK may be incorporated into the clinical management of these patients. In this study, we performed comprehensive genomic profiling on 3,633 pediatric cancer samples and identified a cohort of 221 (6.1%) cases with known or novel alterations in BRAF or RAF1 detected in extracranial solid tumors, brain tumors, or hematological malignancies. Eighty percent (176/221) of these tumors had a known-activating short variant (98, 55.7%), fusion (72, 40.9%), or insertion/deletion (6, 3.4%). Among BRAF altered cancers, the most common tumor types were brain tumors (74.4%), solid tumors (10.8%), hematological malignancies (9.1%), sarcomas (3.4%), and extracranial embryonal tumors (2.3%). RAF1 fusions containing intact RAF1 kinase domain (encoded by exons 10-17) were identified in seven tumors, including two novel fusions TMF1-RAF1 and SOX6-RAF1. Additionally, we highlight a subset of patients with brain tumor with positive clinical response to BRAF inhibitors, demonstrating the rationale for incorporating precision medicine into pediatric oncology. IMPLICATIONS FOR PRACTICE: Precision medicine has not yet gained a strong foothold in pediatric cancers. This study describes the landscape of BRAF and RAF1 genomic alterations across a diverse spectrum of pediatric cancers, primarily brain tumors, but also encompassing melanoma, sarcoma, several types of hematologic malignancy, and others. Given the availability of multiple U.S. Food and Drug Administration-approved BRAF inhibitors, identification of these alterations may assist with treatment decision making, as described here in three cases of pediatric cancer.

Characterization of Clinical Cases of Malignant PEComa via Comprehensive Genomic Profiling of DNA and RNA.

PURPOSE: Perivascular epithelioid cell tumor (PEComa) is a rare mesenchymal soft tissue neoplasm often linked to mTOR pathway activation via TSC2 mutation. We analyzed a series of 31 consecutive metastatic PEComa (mPEComa) cases using a combined DNA/RNA hybrid capture-based comprehensive genomic profiling (CGP) assay to assess the genomic landscape of mPEComa. PATIENTS AND METHODS: Formalin-fixed, paraffin-embedded (FFPE) blocks or slides were obtained from tumors from 31 unique patients with mPEC-oma. DNA and RNA were extracted and CGP was performed on 405 genes using a targeted next-generation sequencing (NGS) assay in a CLIA-certified lab. RESULTS: All cases had locally advanced or metastatic disease, and 58% of patients were female with a median age of 50 years (range 8-76), and 17 and 14 specimens were from primary and metastatic sites, respectively. One hundred genomic alterations were identified in the cohort, with an average of 3.2 genomic alterations/case including alterations in TSC2 32.3% of cases (10), TSC1 9.6% (3), TFE3 16.1% (5, all fusions), and folliculin (FLCN) 6.4% (2), with all occurring in mutually exclusive fashion. Of TSC2 mutant cases, 70% had biallelic inactivation of this locus, as were 100% of TSC1 mutant cases. Two TSC1/2 wildtype cases harbored truncating mutations in FLCN, both of which were under LOH. Five TFE3 fusion cases were identified including the novel 5' fusion partner ZC3H4. CONCLUSIONS: We describe for the first time mPEComa cases with FLCN mutations under LOH, further characterizing dysregulation of the mTOR pathway as a unifying theme in mPEC-oma. Cumulatively, we demonstrate the feasibility and potential utility of segregating mPEComa by TSC, TFE3, and FLCN status via CGP in clinical care.

Patients with NSCLCs Harboring Internal Inversions or Deletion Rearrangements of the ALK Gene Have Durable Responses to ALK Kinase Inhibitors.

BACKGROUND: ALK fusions are targetable drivers in non-small-cell lung cancer (NSCLC). However, patients with NSCLC harboring ALK rearrangements without a fusion partner identified in DNA have also been shown to respond to ALK inhibitors. We aimed to characterize complex ALK variants that may predict sensitivity to multiple approved ALK inhibitors. METHODS: Comprehensive genomic profiling (CGP) of DNA isolated from formalin-fixed paraffin-embedded (FFPE) tumor tissue or blood-based circulating tumor DNA was performed for 39,159 NSCLC patients during routine clinical care. For a subset of cases, RNA sequencing was performed, and prior ALK test results and clinical treatment information were collected from treating physicians. RESULTS: We queried the Foundation Medicine NSCLC database and identified ALK internal inversions, as well as internal deletions, as the sole ALK rearrangements in 6 (0.02%) and 3 (0.01%) of cases, respectively. In cases with ALK internal inversions, RNA testing identified an EML4-ALK fusion in 2/2 cases evaluated, and 3/3 patients treated with ALK inhibitors had durable responses. A single patient with an ALK internal deletion and clinical data available responded to multiple ALK inhibitors. RNA data available for a subset of non-NSCLC cases suggest that ALK internal deletions removing a portion of the N-terminus are drivers themselves and do not result in ALK fusions. Fluorescence in situ hybridization (FISH) results were inconsistent for both classes of DNA events. CONCLUSION: Rare internal inversions of ALK appear to be indicative of ALK fusions, which can be detected in RNA, and response to ALK inhibitors in patients with NSCLC. In contrast, ALK internal deletions are not associated with ALK fusions in RNA but likely represent targetable drivers themselves. These data suggest that CGP of DNA should be supplemented with immunohistochemistry or RNA-based testing to further resolve these events and match patients to effective therapies.

Novel PDGFRB rearrangement in multifocal infantile myofibromatosis is tumorigenic and sensitive to imatinib.

Infantile myofibromatosis (IM) is an aggressive neoplasm composed of myofibroblast-like cells in children. Although typically localized, it can also present as multifocal disease, which represents a challenge for effective treatment. IM has previously been linked to activating somatic and germline point mutations in the PDGFRß tyrosine kinase encoded by the PDGFRB gene. Clinical panel-based targeted tumor sequencing of a tumor from a newborn with multifocal IM revealed a novel PDGFRB rearrangement, which was reported as being of unclear significance. Additional sequencing of cDNA from tumor and germline DNA confirmed a complex somatic/mosaic PDGFRB rearrangement with an apparent partial tandem duplication disrupting the juxtamembrane domain. Ectopic expression of cDNA encoding the mutant form of PDGFRB markedly enhanced cell proliferation of mouse embryo fibroblasts (MEFs) compared to wild-type PDGFRB and conferred tumor-forming capacity on nontumorigenic 10T1/2 fibroblasts. The mutated protein enhanced MAPK activation and retained sensitivity to the PDGFRß inhibitor imatinib. Our findings reveal a new mechanism by which PDGFRB can be activated in IM, suggest that therapy with tyrosine kinase inhibitors including imatinib may be beneficial, and raise the possibility that this receptor tyrosine kinase might be altered in a similar fashion in additional cases that would similarly present annotation challenges in clinical DNA sequencing analysis pipelines.

BRAF in Lung Cancers: Analysis of Patient Cases Reveals Recurrent BRAF Mutations, Fusions, Kinase Duplications, and Concurrent Alterations.

PURPOSE: Dabrafenib and trametinib are approved for the management of advanced non-small-cell lung cancers (NSCLCs) that harbor BRAF V600E mutations. Small series and pan-cancer analyses have identified non-V600 alterations as therapeutic targets. We sought to examine a large genomic data set to comprehensively characterize non-V600 BRAF alterations in lung cancer. PATIENTS AND METHODS: A total of 23,396 patients with lung cancer provided data to assay with comprehensive genomic profiling. Data were reviewed for predicted pathogenic BRAF base substitutions, short insertions and deletions, copy number changes, and rearrangements. RESULTS: Adenocarcinomas represented 65% of the occurrences; NSCLC not otherwise specified (NOS), 15%; squamous cell carcinoma, 12%; and small-cell lung carcinoma, 5%. BRAF was altered in 4.5% (1,048 of 23,396) of all tumors; 37.4% (n = 397) were BRAF V600E, 38% were BRAF non-V600E activating mutations, and 18% were BRAF inactivating. Rearrangements were observed at a frequency of 4.3% and consisted of N-terminal deletions (NTDs; 0.75%), kinase domain duplications (KDDs; 0.75%), and BRAF fusions (2.8%). The fusions involved three recurrent fusion partners: ARMC10, DOCK4, and TRIM24. BRAF V600E was associated with co-occurrence of SETD2 alterations, but other BRAF alterations were not and were instead associated with CDKN2A, TP53, and STK11 alterations (P < .05). Potential mechanisms of acquired resistance to BRAF V600E inhibition are demonstrated. CONCLUSION: This series characterized the frequent occurrence (4.4%) of BRAF alterations in lung cancers. Recurrent BRAF alterations in NSCLC adenocarcinoma are comparable to the frequency of other NSCLC oncogenic drivers, such as ALK, and exceed that of ROS1 or RET. This work supports a broad profiling approach in lung cancers and suggests that non-V600E BRAF alterations represent a subgroup of lung cancers in which targeted therapy should be considered.

Characterization of Clinical Cases of Advanced Papillary Renal Cell Carcinoma via Comprehensive Genomic Profiling.

BACKGROUND: Papillary renal cell carcinoma (PRCC) is a rare subset of RCC. The Cancer Genome Atlas (TCGA) data largely reflect localized disease, and there are limited data for advanced PRCC. OBJECTIVE: To characterize the frequency of genomic alterations (GAs) in patients with advanced PRCC for whom comprehensive genomic profiling (CGP) was performed in the context of routine clinical care. DESIGN, SETTING, AND PARTICIPANTS: Formalin-fixed, paraffin-embedded tissue was obtained for 169 consecutive patients with confirmed PRCC. DNA was extracted and comprehensive genomic profiling was performed in a certified central laboratory. MEASUREMENTS: Hybrid-capture, adaptor ligation-based libraries of up to 315 genes were sequenced to a median coverage of 648×. All classes of GAs were identified, including substitutions, insertions/deletions, copy number alterations, and rearrangements. RESULTS AND LIMITATIONS: From 169 patients, either primary tumor tissue (102 patients, 60%) or metastatic tissue (67 patients, 40%) was collected. In patients with type 1 PRCC, commonly altered genes were MET (33%; 8 activating mutations, 5 amplifications at >6 copies), TERT (30%), CDKN2A/B (13%), and EGFR (8%). In patients with type 2 PRCC, commonly altered genes were CDKN2A/B (18%), TERT (18%), NF2 (13%), and FH (13%); MET GAs (5 mutations, 3 amplifications) were observed in 7% of type 2 cases. Notable differences from TCGA data include higher frequencies of MET, NF2, and CDKN2A/B GAs, association of alterations in SWI/SNF complex genes with type 2 PRCC, and observation of frequent CDKN2A/B alterations in both type 1 and type 2 disease. CONCLUSIONS: Both the current study and the TCGA experience represent similarly sized cohorts of patients with PRCC. Key differences in GA frequency probably underscore the marked difference in stage distribution between these data sets. These results may inform planned precision medicine trials for metastatic PRCC. PATIENT SUMMARY: Papillary renal cell carcinoma (PRCC) is a rare subtype of kidney cancer, and understanding of the biology of advanced PRCC is limited. This report highlights some of the unique biologic features of PRCC that may inform on future use of targeted therapies for the treatment of metastatic disease.

Identification of NTRK fusions in pediatric mesenchymal tumors.

BACKGROUND: NTRK fusions are known oncogenic drivers and have recently been effectively targeted by investigational agents in adults. We sought to assess the frequency of NTRK fusions in a large series of pediatric and adolescent patients with advanced cancers. PROCEDURE: Genomic profiles from 2,031 advanced cancers from patients less than 21 years old who were assayed with comprehensive genomic profiling were reviewed to identify NTRK fusions. RESULTS: Total of nine cases (0.44%) harbored NTRK fusions, including novel partners. Four of these cases were in children less than 2 years old for which infantile fibrosarcoma was considered as a diagnosis, and two harbored the canonical ETV6-NTRK3. The remaining cases carried other diagnoses, at least one that carried the diagnosis of inflammatory myofibroblastic tumor. CONCLUSIONS: NTRK fusions occur in a subset of young patients with mesenchymal or sarcoma-like tumors at a low frequency, and are eminently druggable targets via either investigational agents or approved drugs.

Characterization of 298 Patients with Lung Cancer Harboring MET Exon 14 Skipping Alterations.

BACKGROUND: The hepatocyte growth factor receptor gene (MET) exon 14 skipping (METex14) has recently been described a potential driver alteration in lung cancer targetable by mesenchymal-to-epithelial transition factor (MET) tyrosine kinase inhibitors (TKIs). METHODS: Well-validated hybrid capture-based comprehensive genomic profiling was performed at the request of individual treating physicians. RESULTS: Of 11,205 lung cancers profiled by comprehensive genomic profiling, 298 (2.7%) carcinomas harbored alterations predicted to cause METex14, including adenosquamous (8.2%), sarcomatoid (7.7%), histologic subtype not otherwise specified (3.0%), adenocarcinoma (2.9%), squamous cell (2.1%), large cell (0.8%), and SCLC (0.2%). Acinar features were present in 24% of the METex14 samples. Six cases (2%) harbored MET Y1003X mutations affecting binding of the MET-negative regulator, E3 ubiquitin protein ligase. The median age of all patients with METex14 was 73 years (range 43-95) and 60% were female. Concurrent, murine double minute gene (MDM2) amplification, cyclin-dependent kinase 4 gene (CDK4) amplification, and EGFR amplification were observed in 35%, 21%, and 6.4% of patients with METex14, respectively. KRAS mutation was observed in 3% of cases. Concurrent MET amplification (METamp) (median copy number 10) was identified in 15% of METex14 samples. Significant differences in tumor mutational burden and type of the METex14 alterations were observed between the METamp and non-METamp samples. Response to MET TKI was observed in both in patients with METamp and in patients without METamp METex14. CONCLUSION: Diverse targetable METex14 alterations were identified in patients with NSCLC across age groups, including elderly patients, and in all major NSCLC histologic subtypes with an overall frequency of 2.7%. These findings support the use of hybrid capture-based molecular profiling across NSCLC subtypes to identify patients who will potentially benefit from MET TKIs.

Integrated genomic DNA/RNA profiling of hematologic malignancies in the clinical setting.

The spectrum of somatic alterations in hematologic malignancies includes substitutions, insertions/deletions (indels), copy number alterations (CNAs), and a wide range of gene fusions; no current clinically available single assay captures the different types of alterations. We developed a novel next-generation sequencing-based assay to identify all classes of genomic alterations using archived formalin-fixed paraffin-embedded blood and bone marrow samples with high accuracy in a clinically relevant time frame, which is performed in our Clinical Laboratory Improvement Amendments-certified College of American Pathologists-accredited laboratory. Targeted capture of DNA/RNA and next-generation sequencing reliably identifies substitutions, indels, CNAs, and gene fusions, with similar accuracy to lower-throughput assays that focus on specific genes and types of genomic alterations. Profiling of 3696 samples identified recurrent somatic alterations that impact diagnosis, prognosis, and therapy selection. This comprehensive genomic profiling approach has proved effective in detecting all types of genomic alterations, including fusion transcripts, which increases the ability to identify clinically relevant genomic alterations with therapeutic relevance.

Activation of MET via diverse exon 14 splicing alterations occurs in multiple tumor types and confers clinical sensitivity to MET inhibitors.

UNLABELLED: Focal amplification and activating point mutation of the MET gene are well-characterized oncogenic drivers that confer susceptibility to targeted MET inhibitors. Recurrent somatic splice site alterations at MET exon 14 (METex14) that result in exon skipping and MET activation have been characterized, but their full diversity and prevalence across tumor types are unknown. Here, we report analysis of tumor genomic profiles from 38,028 patients to identify 221 cases with METex14 mutations (0.6%), including 126 distinct sequence variants. METex14 mutations are detected most frequently in lung adenocarcinoma (3%), but also frequently in other lung neoplasms (2.3%), brain glioma (0.4%), and tumors of unknown primary origin (0.4%). Further in vitro studies demonstrate sensitivity to MET inhibitors in cells harboring METex14 alterations. We also report three new patient cases with METex14 alterations in lung or histiocytic sarcoma tumors that showed durable response to two different MET-targeted therapies. The diversity of METex14 mutations indicates that diagnostic testing via comprehensive genomic profiling is necessary for detection in a clinical setting. SIGNIFICANCE: Here we report the identification of diverse exon 14 splice site alterations in MET that result in constitutive activity of this receptor and oncogenic transformation in vitro. Patients whose tumors harbored these alterations derived meaningful clinical benefit from MET inhibitors. Collectively, these data support the role of METex14 alterations as drivers of tumorigenesis, and identify a unique subset of patients likely to derive benefit from MET inhibitors.

Human Capital Investment and the Gender Division of Labor in a Brawn-Based Economy.

We use a model of human capital investment and activity choice to explain facts describing gender differentials in the levels and returns to human capital investments. These include the higher return to and level of schooling, the small effect of healthiness on wages, and the large effect of healthiness on schooling for females relative to males. The model incorporates gender differences in the level and responsiveness of brawn to nutrition in a Roy-economy setting in which activities reward skill and brawn differentially. Empirical evidence from rural Bangladesh provides support for the model and the importance of the distribution of brawn.

TrpA1 regulates thermal nociception in Drosophila.

Pain is a significant medical concern and represents a major unmet clinical need. The ability to perceive and react to tissue-damaging stimuli is essential in order to maintain bodily integrity in the face of environmental danger. To prevent damage the systems that detect noxious stimuli are therefore under strict evolutionary pressure. We developed a high-throughput behavioral method to identify genes contributing to thermal nociception in the fruit fly and have reported a large-scale screen that identified the Ca²âº channel straightjacket (stj) as a conserved regulator of thermal nociception. Here we present the minimal anatomical and neuronal requirements for Drosophila to avoid noxious heat in our novel behavioral paradigm. Bioinformatics analysis of our whole genome data set revealed 23 genes implicated in Ca²âº signaling that are required for noxious heat avoidance. One of these genes, the conserved thermoreceptor TrpA1, was confirmed as a bona fide "pain" gene in both adult and larval fly nociception paradigms. The nociceptive function of TrpA1 required expression within the Drosophila nervous system, specifically within nociceptive multi-dendritic (MD) sensory neurons. Therefore, our analysis identifies the channel TRPA1 as a conserved regulator of nociception.

A genome-wide Drosophila screen for heat nociception identifies α2δ3 as an evolutionarily conserved pain gene.

Worldwide, acute, and chronic pain affects 20% of the adult population and represents an enormous financial and emotional burden. Using genome-wide neuronal-specific RNAi knockdown in Drosophila, we report a global screen for an innate behavior and identify hundreds of genes implicated in heat nociception, including the α2δ family calcium channel subunit straightjacket (stj). Mice mutant for the stj ortholog CACNA2D3 (α2δ3) also exhibit impaired behavioral heat pain sensitivity. In addition, in humans, α2δ3 SNP variants associate with reduced sensitivity to acute noxious heat and chronic back pain. Functional imaging in α2δ3 mutant mice revealed impaired transmission of thermal pain-evoked signals from the thalamus to higher-order pain centers. Intriguingly, in α2δ3 mutant mice, thermal pain and tactile stimulation triggered strong cross-activation, or synesthesia, of brain regions involved in vision, olfaction, and hearing.

Navigational decision making in Drosophila thermotaxis.

A mechanistic understanding of animal navigation requires quantitative assessment of the sensorimotor strategies used during navigation and quantitative assessment of how these strategies are regulated by cellular sensors. Here, we examine thermotactic behavior of the Drosophila melanogaster larva using a tracking microscope to study individual larval movements on defined temperature gradients. We discover that larval thermotaxis involves a larger repertoire of strategies than navigation in smaller organisms such as motile bacteria and Caenorhabditis elegans. Beyond regulating run length (i.e., biasing a random walk), the Drosophila melanogaster larva also regulates the size and direction of turns to achieve and maintain favorable orientations. Thus, the sharp turns in a larva's trajectory represent decision points for selecting new directions of forward movement. The larva uses the same strategies to move up temperature gradients during positive thermotaxis and to move down temperature gradients during negative thermotaxis. Disrupting positive thermotaxis by inactivating cold-sensitive neurons in the larva's terminal organ weakens all regulation of turning decisions, suggesting that information from one set of temperature sensors is used to regulate all aspects of turning decisions. The Drosophila melanogaster larva performs thermotaxis by biasing stochastic turning decisions on the basis of temporal variations in thermosensory input, thereby augmenting the likelihood of heading toward favorable temperatures at all times.

Distinct TRP channels are required for warm and cool avoidance in Drosophila melanogaster.

The ability to sense and respond to subtle variations in environmental temperature is critical for animal survival. Animals avoid temperatures that are too cold or too warm and seek out temperatures favorable for their survival. At the molecular level, members of the transient receptor potential (TRP) family of cation channels contribute to thermosensory behaviors in animals from flies to humans. In Drosophila melanogaster larvae, avoidance of excessively warm temperatures is known to require the TRP protein dTRPA1. Whether larval avoidance of excessively cool temperatures also requires TRP channel function, and whether warm and cool avoidance use the same or distinct TRP channels has been unknown. Here we identify two TRP channels required for cool avoidance, TRPL and TRP. Although TRPL and TRP have previously characterized roles in phototransduction, their function in cool avoidance appears to be distinct, as neither photoreceptor neurons nor the phototransduction regulators NORPA and INAF are required for cool avoidance. TRPL and TRP are required for cool avoidance; however they are dispensable for warm avoidance. Furthermore, cold-activated neurons in the larvae are required for cool but not warm avoidance. Conversely, dTRPA1 is essential for warm avoidance, but not cool avoidance. Taken together, these data demonstrate that warm and cool avoidance in the Drosophila larva involves distinct TRP channels and circuits.

An internal thermal sensor controlling temperature preference in Drosophila.

Animals from flies to humans are able to distinguish subtle gradations in temperature and show strong temperature preferences. Animals move to environments of optimal temperature and some manipulate the temperature of their surroundings, as humans do using clothing and shelter. Despite the ubiquitous influence of environmental temperature on animal behaviour, the neural circuits and strategies through which animals select a preferred temperature remain largely unknown. Here we identify a small set of warmth-activated anterior cell (AC) neurons located in the Drosophila brain, the function of which is critical for preferred temperature selection. AC neuron activation occurs just above the fly's preferred temperature and depends on dTrpA1, an ion channel that functions as a molecular sensor of warmth. Flies that selectively express dTrpA1 in the AC neurons select normal temperatures, whereas flies in which dTrpA1 function is reduced or eliminated choose warmer temperatures. This internal warmth-sensing pathway promotes avoidance of slightly elevated temperatures and acts together with a distinct pathway for cold avoidance to set the fly's preferred temperature. Thus, flies select a preferred temperature by using a thermal sensing pathway tuned to trigger avoidance of temperatures that deviate even slightly from the preferred temperature. This provides a potentially general strategy for robustly selecting a narrow temperature range optimal for survival.