HDAC6 selective inhibition of melanoma patient T-cells augments anti-tumor characteristics.

BACKGROUND: Therapies targeting anti-tumor T-cell responses have proven successful in the treatment of a variety of malignancies. However, as most patients still fail to respond, approaches to augment immunotherapeutic efficacy are needed. Here, we investigated the ability of histone deacetylase 6 (HDAC6)-selective inhibitors to decrease immunosuppression and enhance immune function of melanoma patient T-cells in ex vivo cultures. METHODS: T-cells were harvested from peripheral blood or tumor biopsies of metastatic melanoma patients and cultured in the presence of pan-, class-specific or class-selective histone deacetylase (HDAC) inhibitors. Changes in cytokine production were evaluated by Luminex and intracellular flow cytometry staining. Expression of surface markers, transcription factors, protein phosphorylation, and cell viability were assessed by flow cytometry. Changes in chromatin structure were determined by ATAC-seq. RESULTS: T-cell viability was impaired with low doses of pan-HDAC inhibitors but not with specific or selective HDAC inhibitors. The HDAC6-selective inhibitors ACY-1215 (ricolinostat) and ACY-241 (citarinostat) decreased Th2 cytokine production (i.e. IL-4, IL-5, IL-6, IL-10 and IL-13). Expansion of peripheral blood T-cells from melanoma patients in the presence of these inhibitors resulted in downregulation of the Th2 transcription factor GATA3, upregulation of the Th1 transcription factor T-BET, accumulation of central memory phenotype T-cells (CD45RA-CD45RO + CD62L + CCR7+), reduced exhaustion-associated phenotypes (i.e. TIM3 + LAG3 + PD1+ and EOMES+PD1+), and enhanced killing in mixed lymphocyte reactions. The frequency, FOXP3 expression, and suppressive function of T regulatory cells (Tregs) were decreased after exposure to ACY-1215 or ACY-241. Higher frequencies of T-cells expressing CD107a + IFNγ+ and central memory markers were observed in melanoma tumor-infiltrating lymphocytes (TIL), which persisted after drug removal and further expansion. After ACY-1215 treatment, increased chromatin accessibility was observed in regions associated with T-cell effector function and memory phenotypes, while condensed chromatin was found in regions encoding the mTOR downstream molecules AKT, SGK1 and S6K. Decreased phosphorylation of these proteins was observed in ACY-1215 and ACY-241-treated T-cells. AKT- and SGK1-specific inhibition recapitulated the increase in central memory frequency and decrease in IL-4 production, respectively, similar to the observed effects of HDAC6-selective inhibition. CONCLUSIONS: HDAC6-selective inhibitors augmented melanoma patient T-cell immune properties, providing a rationale for translational investigation assessing their potential clinical efficacy.

Five-year survival outcomes for patients with advanced melanoma treated with pembrolizumab in KEYNOTE-001.

BACKGROUND: Pembrolizumab demonstrated robust antitumor activity and safety in the phase Ib KEYNOTE-001 study (NCT01295827) of advanced melanoma. Five-year outcomes in all patients and treatment-naive patients are reported herein. Patients whose disease progressed following initial response and who received a second course of pembrolizumab were also analyzed. PATIENTS AND METHODS: Patients aged ≥18 years with previously treated or treatment-naive advanced/metastatic melanoma received pembrolizumab 2 mg/kg every 3 weeks, 10 mg/kg every 3 weeks, or 10 mg/kg every 2 weeks until disease progression, intolerable toxicity, or patient/investigator decision to withdraw. Kaplan-Meier estimates of overall survival (OS) and progression-free survival (PFS) were calculated. Objective response rate and PFS were based on immune-related response criteria by investigator assessment (data cut-off, September 1, 2017). RESULTS: KEYNOTE-001 enrolled 655 patients with melanoma; median follow-up was 55 months. Estimated 5-year OS was 34% in all patients and 41% in treatment-naive patients; median OS was 23.8 months (95% CI, 20.2-30.4) and 38.6 months (95% CI, 27.2-not reached), respectively. Estimated 5-year PFS rates were 21% in all patients and 29% in treatment-naive patients; median PFS was 8.3 months (95% CI, 5.8-11.1) and 16.9 months (95% CI, 9.3-35.5), respectively. Median response duration was not reached; 73% of all responses and 82% of treatment-naive responses were ongoing at data cut-off; the longest response was ongoing at 66 months. Four patients [all with prior response of complete response (CR)] whose disease progressed during observation subsequently received second-course pembrolizumab. One patient each achieved CR and partial response (after data cut-off). Treatment-related AEs (TRAEs) occurred in 86% of patients and resulted in study discontinuation in 7.8%; 17% experienced grade 3/4 TRAE. CONCLUSIONS: This 5-year analysis of KEYNOTE-001 represents the longest follow-up for pembrolizumab to date and confirms the durable antitumor activity and tolerability of pembrolizumab in advanced melanoma. CLINICAL TRIAL REGISTRY: ClinicalTrials.gov, NCT01295827.

Predictors of responses to immune checkpoint blockade in advanced melanoma.

Immune checkpoint blockers (ICB) have become pivotal therapies in the clinical armamentarium against metastatic melanoma (MMel). Given the frequency of immune related adverse events and increasing use of ICB, predictors of response to CTLA-4 and/or PD-1 blockade represent unmet clinical needs. Using a systems biology-based approach to an assessment of 779 paired blood and tumor markers in 37 stage III MMel patients, we analyzed association between blood immune parameters and the functional immune reactivity of tumor-infiltrating cells after ex vivo exposure to ICB. Based on this assay, we retrospectively observed, in eight cohorts enrolling 190 MMel patients treated with ipilimumab, that PD-L1 expression on peripheral T cells was prognostic on overall and progression-free survival. Moreover, detectable CD137 on circulating CD8+ T cells was associated with the disease-free status of resected stage III MMel patients after adjuvant ipilimumab + nivolumab (but not nivolumab alone). These biomarkers should be validated in prospective trials in MMel.The clinical management of metastatic melanoma requires predictors of the response to checkpoint blockade. Here, the authors use immunological assays to identify potential prognostic/predictive biomarkers in circulating blood cells and in tumor-infiltrating lymphocytes from patients with resected stage III melanoma.

Clinical outcomes of melanoma brain metastases treated with stereotactic radiation and anti-PD-1 therapy.

BACKGROUND: The anti-programmed death-1 (anti-PD-1) therapy nivolumab has significant clinical activity in patients with metastatic melanoma. However, little is known about the safety and outcomes in patients receiving anti-PD-1 therapy and stereotactic radiation for the treatment of brain metastases (BMs). PATIENTS AND METHODS: Data were analyzed retrospectively from two prospective nivolumab protocols enrolling 160 patients with advanced resected and unresectable melanoma at a single institution. Patients were included if BMs were diagnosed and treated with stereotactic radiation within 6 months of receiving nivolumab. The primary end point of this study was neurotoxicity; secondary end points included BM control and survival. RESULTS: Twenty-six patients with a total of 73 BMs treated over 30 sessions were identified. Radiation was administered before, during and after nivolumab in 33 lesions (45%), 5 lesions (7%), and 35 lesions (48%), respectively. All BMs were treated with stereotactic radiosurgery (SRS) in a single session except 12 BMs treated with fractionated stereotactic radiation therapy, nine of which were in the postoperative setting. One patient experienced grade 2 headaches following SRS with symptomatic relief with steroid treatment. No other treatment-related neurologic toxicities or scalp reactions were reported. Eight (11%) local BM failures with a ≥20% increase in volume were noted. Of these lesions, hemorrhage was noted in 4, and edema was noted in 7. Kaplan-Meier estimates for local BM control following radiation at 6 and 12 months were 91% and 85%, respectively. Median overall survival (OS) from the date of stereotactic radiation and nivolumab initiation was 11.8 and 12.0 months, respectively, in patients receiving nivolumab for unresected disease (median OS was not reached in patients treated in the resected setting). CONCLUSIONS: In our series, stereotactic radiation to melanoma BMs is well tolerated in patients who received nivolumab. BM control and OS appear prolonged compared with standard current treatment. Prospective evaluation is warranted.

Antipsychotic agents used to augment clozapine during long-term inpatient hospitalizations.

INTRODUCTION: Literature assessing effective clozapine augmentation strategies is limited. The aim of this retrospective evaluation was to examine antipsychotics used to augment clozapine and assess whether an augmentation antipsychotic would continue at discharge. METHODS: Demographic, clinical and pharmacy data were collected retrospectively if patients had received clozapine plus an antipsychotic used for augmentation. The dose of the augmentation agent, length of augmentation therapy, and concomitant medications were collected. RESULTS: Of the 49 patients (mean age 45.3±12.1 years), 27 (55%) were male. The mean clozapine dose at discharge was 406.1±121.8 mg. When a first generation antipsychotic (FGA) was selected initially to augment clozapine there was a greater likelihood it would be continued until discharge compared to a second generation antipsychotic (SGA) (78 vs. 50%, OR=3.6, 95% CI 1.03-12.6). FGAs (3.2%) compared to SGAs (35%) were less likely to be discontinued due to a documented lack of benefit when first selected to augment clozapine (OR=16.2, 95% CI 2-131.3). Electroconvulsive therapy plus clozapine was found to be beneficial in several patients (n=14) who failed at least 1 augmentation strategy. DISCUSSION: This real world data suggests that adding an antipsychotic to clozapine is a reasonable approach to those who do not fully respond to clozapine monotherapy. While comparisons of all agents could not be conducted from this small retrospective study, these data suggest FGAs should be investigated in future studies as potential agents to successfully augment clozapine therapy.

Survival follow-up and ipilimumab retreatment of patients with advanced melanoma who received ipilimumab in prior phase II studies.

BACKGROUND: This report provides a survival update at a follow-up of >5 years (5.5-6 years) for patients with advanced melanoma who previously received ipilimumab in phase II clinical trials. Safety and efficacy data following ipilimumab retreatment are also reported. PATIENTS AND METHODS: Patients who previously received ipilimumab 0.3, 3, or 10 mg/kg in one of six phase II trials (CA184-004, CA184-007, CA184-008, CA184-022, MDX010-08, and MDX010-15) were eligible to enroll in the companion study, CA184-025. Upon enrollment, patients initially received ipilimumab retreatment, extended maintenance therapy, or were followed for survival only. Overall survival (OS) rates were evaluated in patients from studies CA184-004, CA184-007, CA184-008, and CA184-022. Safety and best overall response during ipilimumab retreatment at 10 mg/kg were assessed in study CA184-025. RESULTS: Five-year OS rates for previously treated patients who received ipilimumab induction at 0.3, 3, or 10 mg/kg were 12.3%, 12.3%-16.5%, and 15.5%-28.4%, respectively. Five-year OS rates for treatment-naive patients who received ipilimumab induction at 3 or 10 mg/kg were 26.8% and 21.4%-49.5%, respectively. Little to no change in OS was observed from year 5 up to year 6. The objective response rate among retreated patients was 23%. Grade 3/4 immune-related adverse events occurred in 25%, 5.9%, and 13.2% of retreated patients who initially received ipilimumab 0.3, 3, and 10 mg/kg, with the most common being observed in the skin (4.2%, 2.9%, 3.8%) and gastrointestinal tract (12.5%, 2.9%, 3.8%), respectively. CONCLUSIONS: At a follow-up of 5-6 years, ipilimumab continues to demonstrate durable, long-term survival in a proportion of patients with advanced melanoma. In some patients, ipilimumab retreatment can re-establish disease control with a safety profile that is comparable with that observed during ipilimumab induction. Further studies are needed to determine the contribution of ipilimumab retreatment to OS. CLINICALTRIALSGOV: NCT00162123.

Four-year survival rates for patients with metastatic melanoma who received ipilimumab in phase II clinical trials.

BACKGROUND: This analysis was carried out to evaluate the long-term survival of patients with metastatic melanoma who received ipilimumab, a fully human monoclonal antibody that binds to cytotoxic T-lymphocyte antigen-4, in clinical trials. PATIENTS AND METHODS: Patients received ipilimumab in one of three completed phase II clinical trials (CA184-008, CA184-022, and CA184-007). Previously treated patients were enrolled in all studies, and treatment-naïve patients were also included in study CA184-007. Patients received ipilimumab at a dose of 10 mg/kg in studies CA184-008 and CA184-007, and at doses of 0.3, 3, or 10 mg/kg in study CA184-022. Ipilimumab was given every 3 weeks for four doses, and eligible patients could receive ipilimumab maintenance therapy every 12 weeks. In study CA184-022, patients could cross over to be retreated with ipilimumab at 10 mg/kg upon disease progression. Ongoing survival follow-up is conducted in a companion study, CA184-025. RESULTS: Four-year survival rates [95% confidence interval (95% CI)] for previously treated patients who received ipilimumab at 0.3, 3, or 10 mg/kg were 13.8% [6.1-22.5], 18.2% [9.5-27.6], and 19.7% [13.4-26.5] to 28.4% [13.9-44.2], respectively. In treatment-naïve patients who received ipilimumab at 10 mg/kg, 4-year survival rates were 37.7% [18.6-57.4] to 49.5% [23.8-75.4]. CONCLUSIONS: These results demonstrate durable survival in a significant proportion of patients with metastatic melanoma who received ipilimumab therapy.

Phase I clinical trial of the Src inhibitor dasatinib with dacarbazine in metastatic melanoma.

BACKGROUND: Src inhibitors sensitise melanoma cells to chemotherapy in preclinical models. The combination of dasatinib and dacarbazine was tested in a phase I trial in melanoma. METHODS: Patients had ECOG performance status 0-2 and normal organ function. Dacarbazine was administered on day 1 and dasatinib on day 2 through 19 of each 21-day cycle. Both were escalated from 50 mg b.i.d. of dasatinib and 800 mg m(-2) of dacarbazine. Available pre-treatment biopsies were sequenced for BRAF, NRAS, and C-Kit mutations. RESULTS: Dose-limiting toxicity was reached at dasatinib 70 mg b.i.d./dacarbazine 1000 mg m(-2), and was predominantly haematological. In 29 patients receiving dasatinib 70 mg b.i.d., the objective response rate (ORR) was 13.8%, the clinical benefit rate (ORR+SD) was 72.4%, the 6-month progression-free survival (PFS) was 20.7%, and the 12-month overall survival (OS) was 34.5%. Two out of three patients who were wild type for BRAF, NRAS, and c-KIT mutations had confirmed partial responses, and one had a minor response. CONCLUSION: The recommended phase II dose is dasatinib 70 mg b.i.d with dacarbazine 800 mg m(-2). PFS and OS data for dasatinib at 70 mg b.i.d. with dacarbazine compared favourably with historical controls. Preliminary data support evaluating tumour mutation status further as a biomarker of response.

Assessing oncologic benefit in clinical trials of immunotherapy agents.

BACKGROUND: USA Food and Drug Administration approval for cancer therapy requires demonstration of patient benefit as a marker of clinical efficacy. Prolonged survival is the gold standard for demonstration of efficacy, but other end points such as antitumor response, progression-free survival, quality of life, or surrogate end points may be used. DESIGN: This study was developed based on discussion during a roundtable meeting of experts in the field of immunotherapy. RESULTS: In most clinical trials involving cytotoxic agents, response end points use RECIST based on the premise that 'effective' therapy causes tumor destruction, target lesion shrinkage, and prevention of new lesions. However, RECIST may not be appropriate in trials of immunotherapy. Like other targeted agents, immunotherapies may mediate cytostatic rather than direct cytotoxic effects, and these may be difficult to quantify with RECIST. Furthermore, significant time may elapse before clinical effects are quantifiable because of complex response pathways. Effective immunotherapy may even mediate transient lesion growth secondary to immune cell infiltration. CONCLUSIONS: RECIST may not be an optimal indicator of clinical benefit in immunotherapy trials. This article discusses alternative clinical trial designs and end points that may be more relevant for immunotherapy trials and may offer more effective prediction of survival in pivotal phase III studies.

A phase II study of Hsp-7 (SGN-00101) in women with high-grade cervical intraepithelial neoplasia.

OBJECTIVE: Approximately 2 million women worldwide are infected with high-risk human papillomaviruses (HPV), resulting in a substantial risk for the development of invasive lower genital malignancies. This study was undertaken to determine the effects of vaccination with a protein encoding a bacterial heat shock protein fused to sequences from the oncogenic E7 protein of HPV-16 in women with high-grade cervical intraepithelial neoplasia. Endpoints included lesion regression, immune response, and viral clearance. METHODS: Twenty-one women were prospectively entered into an IRB-approved Phase II study. All women had biopsy-proven high-grade cervical intraepithelial neoplasia and persistent post-biopsy lesions visible by colposcopy. Four injections of HPV-16 Hsp E7 fusion protein at a dose of 500 mug were given 3 weeks apart after which Loop Electrosurgical Excision of the Transformation Zone (LLETZ) was performed. Immune parameters were evaluated pre-vaccine and at the time of LLETZ, and HPV testing was performed at intervals before and after LLETZ. Study subjects were followed for 1 year after LLETZ. RESULTS: Seven of 20 women (35%) evaluable for response had complete regression of their intraepithelial neoplasia at the time of LLETZ, 1 (5%) had regression to CIN I, 11 (55%) had stable disease and 1 (5%) had progression due to enlargement of her lesion. Immune responses were seen in 9 of the 17 women tested; 5 of the 7 complete responders had an immune response. Only 5 of 21 women had HPV-16 or -18. HPV clearance was not associated with lesion regression. CONCLUSION: Hsp-7 (SGN-00101), at this dose and schedule induced lesion regression in women with high-grade intraepithelial neoplasia. The fact that regression was correlated with immune response suggests that enhancing the immunogenicity of this vaccine may lead to improvement in the rate of lesion eradication.

Biological responses to porcine respiratory and reproductive syndrome virus in pigs of two genetic populations.

One hundred pigs from the NE Index Line (NEI) and 100 Hampshire-Duroc cross pigs (HD) were inoculated intranasally with porcine respiratory and reproductive syndrome virus (PRRSV 97-7895 strain) at 26 d of age to determine whether genetic variation in response to PRRSV exists. An uninfected littermate to each infected pig served as a control. Pigs were from 163 dams and 83 sires. Body weight and rectal temperature were recorded, and blood samples were drawn from each pig on d 0 before inoculation and on d 4, 7, and 14 after inoculation. Pigs were sacrificed on d 14. Lung and bronchial lymph nodes were collected, placed in optimal cutting temperature compound, and frozen at -80 degrees C. The presence of PRRSV in serum and in lung tissue and bronchial lymph nodes was determined by isolation in cell culture. The presence of antibodies in serum collected on d 14 was determined by a commercial ELISA test. Lung tissue was examined microscopically and scored for incidence and severity of lesions (score of 1 to 3; 1 = no or few lesions, and 3 = severe interstitial pneumonia). Data were analyzed with a mixed model that included random sire and dam effects. The interaction of line x treatment was significant (P < 0.001) for weight change and rectal temperature. Un-infected HD pigs gained 0.67 kg more from d 0 to 14 and averaged 0.32 degrees C higher rectal temperature than uninfected NEI pigs (P < 0.001), whereas infected NEI pigs gained 0.34 kg more and had -0.54 degrees C lower temperature than infected HD pigs (P < 0.001). Viremic titer (cell culture infectious dose 50%/mL) was greater (P < 0.05) in HD than NEI at d 4 (10(4.52) vs. 10(4.22)), 7 (10(4.47) vs. 10(3.99)), and 14 (10(3.49) vs. 10(3.23)). Viral titer loads in lung (P = 0.11) and bronchial lymph nodes tended (P = 0.07) to be greater in HD than NEI pigs. Antibody signal-to-positive (S/P) ELISA ratios in infected pigs ranged from 0.18 to 3.38, and 88% had levels > or = 0.40, which is the positive threshold for this ELISA. The S/P range in uninfected pigs was 0 to 1.11, and 99% had levels < or = 0.40. Mean S/P ratio for infected pigs was 0.23 units higher in HD than in NEI (P < 0.001). The HD pigs had a greater incidence of interstitial pneumonia and 0.65 higher mean lesion scores than NEI pigs (P < 0.001). In summary, responses of pigs of the two lines to infection with PRRSV differed, indicating that underlying genetic variation existed.

Phase I trial of continuous infusion 5-aza-2'-deoxycytidine.

PURPOSE: To identify a dose of the demethylating agent 5-aza-2'-deoxycytidine (DAC) with acceptable side effects, and to study its effect on the methylation patterns of relevant genes in tumor biopsies before and after treatment with a novel methylation assay using real-time PCR. METHODS: A group of 19 patients with metastatic solid tumors were treated with DAC by continuous intravenous infusion over 72 h, days 1-3 of a 28-day cycle. Tumor biopsies were taken before and 7 days after starting DAC. RESULTS: The dose levels studied were 20, 30 and 40 mg/m(2). Grade 4 neutropenia was found in two of five patients at 40 mg/m(2) and one of six patients at 30 mg/m(2). No objective responses were seen in this study. Steady-state DAC levels of 0.1 to 0.2 microM were achieved in the 30 and 40 mg/m(2) cohorts. Changes in methylation were observed, but no single gene consistently demonstrated evidence of demethylation. CONCLUSIONS: DAC was tolerated at a dose of 30 mg/m(2) per day for a 72-h intravenous infusion. Changes in gene methylation were observed.

Resposta incremental do crescimento do ramo mandibular com o uso dos aparelhos de Fränkel e Herbst, segundo a análise de Björk e Jarabak / Mandibular ramus incremental growth response using Fränkel and Herbst appliances according to the analysis by Björk and Jarabak

Este trabalho destinou-se a avaliar a resposta de crescimento do ramo mandibular, frente ao uso dos aparelhos funcionais de Fränkel e Herbst, quando comparados com os valores padräo de incrementos anuais descritos por Jarabak. Utilizaram-se, como amostra, os traçados cefalométricos pré e pós-tratamento da análise de Björk e Jarabak de 16 pacientes tratados com aparelhos de Herbst e 16 pacientes tratados com aparelhos de Fränkel. Estes foram selecionados previamente, através de critérios específicos. Observou-se uma melhor resposta incremental anualizada do ramo mandibular nos pacientes que apresentaram, no traçado inicial, a porcentagem de Jarabak, indicativa de crescimento anti-horário. Do mesmo modo, uma resposta positiva em relaçäo ao crescimento foi observada com uso dos aparelhos funcionais descritos

Novel immunologic approaches to the management of malignant melanoma.

In the past decade, the discovery of tumor antigens recognized by T cells has revolutionized the tumor vaccine field. The appreciation that peptides are bound to and restricted by major histocompatibility class I and II molecules for immune recognition has encouraged a number of early-phase clinical trials of peptide vaccines. I summarize herein the rationale for and the results of a number of clinical trials of peptide vaccines for melanoma, suggesting that immune and clinical responses can be seen in those with metastatic and resected disease using a variety of surrogate assays. The challenge for the future is to correlate the results of immunologic assays with clinical benefit in patients with advanced cancer.

Effects of ENU dosage on mouse strains.

The germline supermutagen, N-ethyl-N-nitrosourea (ENU), has a variety of effects on mice. ENU is a toxin and carcinogen as well as a mutagen, and strains differ in their susceptibility to its effects. Therefore, it is necessary to determine an appropriate mutagenic, non-toxic dose of ENU for strains that are to be used in experiments. In order to provide some guidance, we have compiled data from a number of laboratories that have exposed male mice from inbred and non-inbred strains or their F(1) hybrids to ENU. The results show that most F(1) hybrid animals tolerate ENU well, but that inbred strains of mice vary in their longevity and in their ability to recover fertility after treatment with ENU.

Optimal N-ethyl-N-nitrosourea (ENU) doses for inbred mouse strains.

ENU is a powerful germline mutagen in the mouse, providing the opportunity to analyze the functions of large numbers of genes in the mammalian genome. In many mutagenesis experiments, it would be beneficial to exploit the advantages of inbred mouse strains. To perform an effective ENU mutagenesis screen using inbred mice, a dosage regimen is required to determine the optimal dose of ENU for that inbred strain, a time-consuming preliminary process. We have carried out dosage regimens for mutagenizing doses of ENU in ten inbred strains of mouse: 129X1/SvJ, 129S6/SvEv, A/J, BALB/cJ, BTBR/N, C3He/J, C3HeB/FeJ, C57BL/6J, C57BR/cdJ, and CBA/CaJ, and determined an optimal dose for each strain, defined by length of sterile period and number of males to survive treatment. Three strains: A/J, BALB/cJ and C57BL/6J, are able to tolerate high doses, up to 300 mg/kg body weight, and are highly recommended for mutagenesis studies.

A comparative transcript map and candidates for mutant phenotypes in the Tyrp1 (brown) deletion complex homologous to human 9p21-23.

The mouse Tyrp1 deletion complex is a valuable resource for high-resolution mapping of genes and phenotypes to the central region of Chromosome (Chr) 4. The distal part of the complex is homologous to human Chr 9p21-23, and we have used the available radiation hybrid maps to identify human transcripts in the region. We localize seven genes to a human YAC contig that spans the full extent of the distal deletion complex and show that the mouse homologs of four of these, including Cer1, map within the complex. On the basis of location and/or expression, we exclude genes as candidates for several known phenotypes in the region and identify a candidate transcript for the neonatal lethal phenotype l(4)Rn2.