Charcot arthropathy outcomes after early referral to a regional tertiary care foot clinic.

BACKGROUND: Community physicians may not encounter Charcot arthropathy frequently, and its symptoms and signs may be nonspecific. Patients often have a delay of several months before receiving a formal diagnosis and referral for specialty care. However, limited Canadian data are available. We evaluated the clinical history, treatment and outcomes of patients treated for Charcot arthropathy after prompt referral and diagnosis. METHODS: We performed a retrospective chart review of 76 patients with diabetes (78 feet) who received nonoperative treatment for Charcot arthropathy in a specialty foot clinic between Jan. 20, 2009, and Mar. 26, 2018. Patients were referred to the foot clinic by community physicians for evaluation or were pre-existing patients at the foot clinic with new-onset Charcot arthropathy. RESULTS: Of the 78 feet included in our analyses, 52 feet (67%) were evaluated initially by a community physician and referred to the foot clinic, where they were seen within 3 ± 5 weeks. The remaining 26 feet (33%) were already being treated at the foot clinic. Most feet had swelling, erythema, warmth, a palpable pulse and loss of protective sensation. Ulcers were present initially in 23 feet (29%). Sixty-four feet (82%) with Charcot arthropathy were in Eichenholtz classification stage 1 and most had midfoot involvement. Nonoperative treatment included total contact casting (60 feet, 77%). Mean duration of nonoperative treatment until resolution for 55 feet (71%) was 6 ± 5 months. Surgery was performed on 20 feet (26%) for the treatment of infection and recurrent ulcer associated with deformity, including 6 (8%) lower limb amputations. CONCLUSION: Charcot arthropathy may resolve in most feet with early referral and nonoperative treatment, but remains a limb-threatening condition.

Subcellular Propagation of Cardiomyocyte ß-Adrenergic Activation of Calcium Uptake Involves Internal ß-Receptors and AKAP7.

ß-adrenergic receptor (ß-AR) signaling in cardiac myocytes is central to cardiac function, but spatiotemporal activation within myocytes is unresolved. In rabbit ventricular myocytes, ß-AR agonists or high extracellular [Ca] were applied locally at one end, to measure ß-AR signal propagation as Ca-transient (CaT) amplitude and sarcoplasmic reticulum (SR) Ca uptake. High local [Ca]o, increased CaT amplitude under the pipette faster than did ISO, but was also more spatially restricted. Local isoproterenol (ISO) or norepinephrine (NE) increased CaT amplitude and SR Ca uptake, that spread along the myocyte to the unexposed end. Thus, local [Ca]i decline kinetics reflect spatio-temporal progression of ß-AR end-effects in myocytes. To test whether intracellular ß-ARs contribute to this response, we used ß-AR-blockers that are membrane permeant (propranolol) or not (sotalol). Propranolol completely blocked NE-dependent CaT effects. However, blocking surface ß-ARs only (sotalol) suppressed only ∼50% of the NE-induced increase in CaT peak and rate of [Ca]i decline, but these changes spread more gradually than NE alone. We also tested whether A-kinase anchoring protein 7γ (AKAP7γ; that interacts with phospholamban) is mobile, such that it might contribute to intracellular spatial propagation of ß-AR signaling. We found AKAP7γ to be highly mobile using fluorescence recovery after photobleach of GFP tagged AKAP7γ, and that PKA activation accelerated AKAP7γ-GFP wash-out upon myocyte saponin-permeabilization, suggesting increased AKAP7γ mobility. We conclude that local ß-AR activation can activate SR Ca uptake at remote myocyte sites, and that intracellular ß-AR and AKAP7γ mobility may play a role in this spread of activation.

Prevalence, management, and outcomes of nerve injury after shoulder arthroplasty: a case-control study and review of the literature.

Background: Neurologic injury is a rare and potentially devastating complication of shoulder arthroplasty. Patients typically present with a mixed plexopathy or mononeuropathy, most commonly affecting the axillary and radial nerves. Given the paucity of studies available on the topic, our goal was to elucidate the prevalence of nerve injury after shoulder arthroplasty and to describe the treatment course and outcomes of neurologic injuries. Methods: This is a retrospective case-control study performed at a single, urban, academic institution. Consecutive patients who underwent anatomic total shoulder arthroplasty (TSA) or reverse shoulder arthroplasty (RSA) by a single surgeon from 2014 to 2020 were reviewed, and patients with a documented nerve injury were identified. A control group of patients without nerve injury were selected in a 2:1 ratio controlling for age and procedure type (TSA vs. RSA; primary vs. revision). Data collected included demographics, comorbidities as per the Charlson Comorbidity Index, radiographic evaluations, surgical and implant details, patient-reported outcome measures, and perioperative complications. Results: Of 923 patients, 33 (3.6%) sustained an iatrogenic nerve injury: 10 (2.1%) after TSA, 23 (5.0%) after RSA, and 3 (7.8%) after revision arthroplasty. Axillary mononeuropathy was most common (42%), followed by brachial plexopathies (18%). There was no significant difference in age, sex, race, body mass index, and preoperative diagnoses between groups. Patients with nerve injury had fewer comorbidities (Charlson Comorbidity Index <3, 33 vs. 65%, P<.001). Patients with nerve injury had higher rates of cervical spine pathology (15 vs. 6%; P = .15) and increased postoperative lateralization (8.9 mm [7.2] vs. 5.5 mm [7.3]; P<.06). The majority (91%) were managed with observation alone. Three (9%) underwent an additional procedure: carpal tunnel release (1, 3%), ulnar nerve decompression (1, 3%), and ulnar nerve transposition (1, 3%) for peripheral compressive neuropathies. At the final follow-up, 19 (57%) nerves fully recovered, and 14 (43%) showed mild residual sensorimotor dysfunction. The mean time to first sign of recovery and ultimate recovery were 11 (7.2) and 36 (23.5) weeks, respectively. At the final follow-up, patients with nerve injury performed worse on patient-reported outcomes, including visual analog score pain (2.2 vs. 1.0, P<.001), American Shoulder and Elbow Surgeons score (67.8 vs. 84.8, P<.001), and Single Assessment Numeric Evaluation scores (62 vs. 77, P = .009). Discussion: Nerve injury after shoulder arthroplasty is rare, occurring in 3.6% of our patient population. Axillary mononeuropathy and brachial plexopathies are the most common. Most patients can be managed expectantly with observation and will recover at least partial nerve function, although clinical outcomes remain inferior to those without nerve complication.

Outcome of arthroscopic pectoralis minor release and scapulopexy for the management of scapulothoracic abnormal motion.

BACKGROUND: Management of persistent symptomatic scapulothoracic abnormal motion (STAM) in the absence of periscapular muscle paralysis may be challenging. This study reports the outcomes of arthroscopic pectoralis minor release and scapulopexy for the management of symptomatic STAM secondary to pectoralis minor hyperactivity and serratus anterior hypoactivity in the absence of periscapular paralysis. METHODS: This was a retrospective cohort study with prospectively collected data of patients with symptomatic STAM secondary to pectoralis minor hyperactivity and serratus anterior hypoactivity. Surgery was indicated if patients failed 6 months of conservative management. Patient outcomes were assessed with shoulder range of motion (ROM) measurements, numerical pain scale, shoulder subjective value (SSV), and Constant score. Data were analyzed with Fischer's exact test for categorical variables and Student's t-test of unequal variance for continuous and categorical variables. RESULTS: Thirty-one consecutive patients were included in the study period between 2017 and 2020. Average age at the time of surgery was 24 years (range, 14-44 years), 80% of patients were female, and average follow-up after surgery was 23 months (range, 15-39 months). Thirteen patients also had a diagnosis of recurrent posterior instability. At final follow-up, 81% reported significant improvements in their STAM, as demonstrated by improved mean pain scale, ROM, SSV, and Constant scores. Pain improved from 6 (range, 4-10) to 2 (range, 1-4), SSV from 30% (range, 10%-40%) to 75% (range, 60%-100%), and Constant score from 49 (range, 43-61) preoperatively to 79 (range, 51-100) postoperatively (P < .01). All 13 patients with recurrent associated posterior instability had resolution of their instability. Flexion ROM improved from average 100° (range, 60°-150°) to 140° (range, 120°-160°). One patient had traumatic rupture of her scapulopexy 7 weeks postoperatively and underwent revision scapulopexy. Thirteen percent had minimal improvement after surgery and experienced recurrence 3 months postoperatively. CONCLUSION: In patients with symptomatic STAM secondary to pectoralis minor hyperactivity and serratus anterior hypoactivity, arthroscopic pectoralis minor release and scapulopexy is an effective surgical option.

Precisely timed theta oscillations are selectively required during the encoding phase of memory.

Brain oscillations have been hypothesized to support cognitive function by coordinating spike timing within and across brain regions, yet it is often not known when timing is either critical for neural computations or an epiphenomenon. The entorhinal cortex and hippocampus are necessary for learning and memory and exhibit prominent theta oscillations (6-9 Hz), which are controlled by pacemaker cells in the medial septal area. Here we show that entorhinal and hippocampal neuronal activity patterns were strongly entrained by rhythmic optical stimulation of parvalbumin-positive medial septal area neurons in mice. Despite strong entrainment, memory impairments in a spatial working memory task were not observed with pacing frequencies at or below the endogenous theta frequency and only emerged at frequencies ≥10 Hz, and specifically when pacing was targeted to maze segments where encoding occurs. Neural computations during the encoding phase were therefore selectively disrupted by perturbations of the timing of neuronal firing patterns.

mAb Das-1 identifies pancreatic ductal adenocarcinoma and high-grade pancreatic intraepithelial neoplasia with high accuracy.

Pancreatic intraepithelial neoplasia (PanIN) is a microscopic precursor lesion to pancreatic ductal adenocarcinoma (PDAC); however, there are few biomarkers that segregate high-grade PanIN/PDAC from low-grade PanIN lesions. mAb Das-1 is a monoclonal antibody against a colonic epithelial antigen that is reactive to premalignant conditions of the upper gastrointestinal tract including Barrett's esophagus, incomplete-type gastric intestinal metaplasia, and intraductal papillary mucinous neoplasm of the pancreas at high risk of malignancy. We sought to examine a role for Das-1 expression in differentiating high-grade PanIN/PDAC from low-grade PanIN lesions. We examined surgical specimens from 86 patients and 2 autopsied pancreata (74 with and 14 without PDAC) with 107 distinct PanIN lesions, 74 PDAC cases, and 32 associated lymph node metastases, with internal controls of normal pancreatic ducts observed in 56 cases. All of the normal pancreatic duct controls (0/56) and low-grade PanIN (0/95) lesions were nonreactive to Das-1. Das-1 expression among high-grade PanIN (7/12, 58%), PDAC (55/74, 74%), and lymph node metastasis (21/32, 66%) cases was significantly higher (p < 0.0001). Clinicopathologically, Das-1 reactivity was significantly correlated with nodal metastasis (p = 0.021). Overall, the sensitivity, specificity, and accuracy of Das-1 in segregating high-grade PanIN/PDAC from low-grade PanIN lesions and normal ducts were 72%, 100%, and 90%, respectively. Thus, mAb Das-1 reacts with high specificity with high-grade PanIN and PDAC and may help in preoperative diagnosis and/or clinical risk stratification.

Thoracic nuclear protein in testis (NUT) carcinoma: expanded pathological spectrum with expression of thyroid transcription factor-1 and neuroendocrine markers.

AIMS: Nuclear protein in testis (NUT) carcinoma, an aggressive tumour driven by NUTM1 rearrangements, often involves the lung/mediastinum and shows squamous differentiation. We encountered an index patient with a thoracic NUT carcinoma diagnosed by molecular testing, showing extensive pleural involvement and diffuse thyroid transcription factor-1 (TTF-1) expression, initially suggestive of lung adenocarcinoma with pseudomesotheliomatous growth. We thus gathered an institutional series of thoracic NUT carcinomas to examine their pathological spectrum. METHODS AND RESULTS: We searched for thoracic NUT carcinomas in our surgical pathology files and in 2289 consecutive patients with primary thoracic tumours investigated with RNA-based assays. We performed NUT immunohistochemistry on 425 additional lung adenocarcinomas. Collectively, we identified six patients (five men and one woman; age 31-80 years; four never-smokers) with thoracic NUT carcinomas confirmed by molecular testing (including five with positive NUT immunohistochemistry). They died at 2.3-12.9 months (median, 2.8 months) after presentation. Two patients were diagnosed by histopathological assessment, and the remaining four (including the index patient) were diagnosed by molecular testing. Analysis of the index case revealed expression of multiple neuroendocrine markers and TTF-1; no ultrastructural evidence of neuroendocrine differentiation was noted. No additional NUT-positive cases were found by immunohistochemical screening. CONCLUSIONS: Although NUT carcinoma classically shows squamous differentiation, it can rarely express TTF-1 (even diffusely) and/or multiple neuroendocrine markers. This immunophenotypic spectrum may lead to diagnostic confusion with pulmonary adenocarcinoma, neuroendocrine tumour, and others. To circumvent this pitfall, NUT immunohistochemistry and/or NUTM1 molecular testing should be considered in primitive-appearing tumours, regardless of their immunophenotypic features.

Growth-Dependent Activation of Protein Kinases Suggests a Mechanism for Measuring Cell Growth.

In all cells, progression through the cell cycle occurs only when sufficient growth has occurred. Thus, cells must translate growth into a proportional signal that can be used to measure and transmit information about growth. Previous genetic studies in budding yeast suggested that related kinases called Gin4 and Hsl1 could function in mechanisms that measure bud growth; however, interpretation of the data was complicated by the use of gene deletions that cause complex terminal phenotypes. Here, we used the first conditional alleles of Gin4 and Hsl1 to more precisely define their functions. We show that excessive bud growth during a prolonged mitotic delay is an immediate consequence of inactivating Gin4 and Hsl1 Thus, acute loss of Gin4 and Hsl1 causes cells to behave as though they cannot detect that bud growth has occurred. We further show that Gin4 and Hsl1 undergo gradual hyperphosphorylation during bud growth that is dependent upon growth and correlated with the extent of growth. Moreover, gradual hyperphosphorylation of Gin4 during bud growth requires binding to anionic phospholipids that are delivered to the growing bud. While alternative models are possible, the data suggest that signaling lipids delivered to the growing bud generate a growth-dependent signal that could be used to measure bud growth.

Implementation and assessment of a pilot, community pharmacy-based, opioid pain medication management program.

OBJECTIVES: This study aimed to evaluate care gaps in risk- and harm-reduction strategies for patients prescribed opioids and to describe the implementation of a community pharmacy-based, pilot pain-management program. SETTING: The pilot program was established in a community pharmacy within an academic medical center. Patients enrolled were prescribed opioids for chronic pain by a rheumatology clinic. PRACTICE DESCRIPTION: The patients enrolled met 1 or more of the following criteria: they were prescribed more than 1 short-acting opioid; more than 90 morphine milligram equivalents/d; and more than 7 days' supply of medications for acute pain, including high-risk medication combinations. Comprehensive pain-medication assessments and pharmacist interventions were communicated to providers and implemented at follow-up. Data were analyzed using descriptive statistics. PRACTICE INNOVATION: A gap analysis was conducted by including 23 patients seen at the clinic over a 22-month period. The care gaps identified served as the basis for the pilot-program design. EVALUATION: Patients referred to the program were seen over a span of 1 to 2 visits; a total of 19 visits were documented. Pharmacists identified unaddressed issues with mood (68%). Recommendations made to the providers included additional adjuvant therapy (84%), dose adjustment (58%), and laboratory tests (74%). Naloxone was provided (58%), and education on naloxone use was provided at every visit. DISCUSSION: Untreated depression, anxiety, and insomnia were the most common problems identified by pharmacists. Pharmacists implemented and documented risk-reduction strategies and coprescribed naloxone more frequently compared with clinic providers. The program enhanced the pharmacists' ability to make safe and clinically appropriate decisions with regard to filling opioid prescriptions. CONCLUSION: The pilot program identified care gaps and provided an approach for engaging with patients and providers to optimize pain management, implement opioid risk-reduction strategies, and expand naloxone access.

PTEN Loss Mediates Clinical Cross-Resistance to CDK4/6 and PI3Kα Inhibitors in Breast Cancer.

The combination of CDK4/6 inhibitors with antiestrogen therapies significantly improves clinical outcomes in ER-positive advanced breast cancer. To identify mechanisms of acquired resistance, we analyzed serial biopsies and rapid autopsies from patients treated with the combination of the CDK4/6 inhibitor ribociclib with letrozole. This study revealed that some resistant tumors acquired RB loss, whereas other tumors lost PTEN expression at the time of progression. In breast cancer cells, ablation of PTEN, through increased AKT activation, was sufficient to promote resistance to CDK4/6 inhibition in vitro and in vivo. Mechanistically, PTEN loss resulted in exclusion of p27 from the nucleus, leading to increased activation of both CDK4 and CDK2. Because PTEN loss also causes resistance to PI3Kα inhibitors, currently approved in the post-CDK4/6 setting, these findings provide critical insight into how this single genetic event may cause clinical cross-resistance to multiple targeted therapies in the same patient, with implications for optimal treatment-sequencing strategies. SIGNIFICANCE: Our analysis of serial biopsies uncovered RB and PTEN loss as mechanisms of acquired resistance to CDK4/6 inhibitors, utilized as first-line treatment for ER-positive advanced breast cancer. Importantly, these findings have near-term clinical relevance because PTEN loss also limits the efficacy of PI3Kα inhibitors currently approved in the post-CDK4/6 setting.This article is highlighted in the In This Issue feature, p. 1.

Outcomes and Complications After Repair of Complete Distal Biceps Tendon Rupture with the Cortical Button Technique.

Numerous surgical techniques have been described for the repair of complete distal biceps tendon ruptures. However, the outcome of repair with cortical button fixation has not been extensively evaluated. The hypothesis of the present study was that elbow strength and range of motion would be less than normal after repair but that ongoing disability would be minimal as measured with use of the Disabilities of the Arm, Shoulder and Hand (DASH) score. METHODS: We performed a retrospective cohort study of patients with complete distal biceps tendon rupture that was repaired with cortical button fixation via a 1-incision anterior approach. Outcome was assessed on the basis of elbow range-of-motion and strength measurements, DASH scores, and radiographs of the operatively treated elbow. Descriptive statistics were generated for patient demographics and outcome variables. Strength was assessed with limb-symmetry index, and range of motion was evaluated with paired t tests. RESULTS: Sixty male patients consented to this study. The average age at the time of follow-up was 49.6 ± 7.8 years, and the average time from injury to follow-up was 3.7 ± 1.7 years. The mechanism of injury included lifting heavy objects (62%) and sporting activities (25%). Elbow flexion and supination range of motion were not different between the operatively treated and contralateral arms. The operatively treated elbow demonstrated decreased flexion strength (96% of that on the contralateral side) and supination strength (91% of that on the contralateral side). The findings did not change when controlling for hand dominance. The mean DASH score was 7.9 ± 11.4, which is not significantly different from the normative value for the general population. Postoperative complications included heterotopic ossification (Brooker class I [29 patients] or II [5 patients]), neurapraxia (7 patients), and rerupture (3 patients). CONCLUSIONS: The repair of complete distal biceps tendon ruptures with cortical button fixation was associated with decreased strength in elbow flexion and forearm supination compared with the contralateral arm, although the differences were small and likely were not clinically important. The complication rate was relatively high; however, most complications were minor and were associated with minimal disability, as reflected by the DASH scores. LEVEL OF EVIDENCE: Therapeutic Level IV. See Instructions for Authors for a complete description of levels of evidence.

Cross Validation of the Monoclonal Antibody Das-1 in Identification of High-Risk Mucinous Pancreatic Cystic Lesions.

BACKGROUND & AIMS: Although pancreatic cystic lesions (PCLs) are frequently and incidentally detected, it is a challenge to determine their risk of malignancy. In immunohistochemical and enzyme-linked immunosorbent assay (ELISA) analyses of tissue and cyst fluid from pancreatic intraductal papillary mucinous neoplasms, the monoclonal antibody Das-1 identifies those at risk for malignancy with high levels of specificity and sensitivity. We aimed to validate the ability of Das-1 to identify high-risk PCLs in comparison to clinical guidelines and clinical features, using samples from a multicenter cohort. METHODS: We obtained cyst fluid samples of 169 PCLs (90 intraductal papillary mucinous neoplasms, 43 mucinous cystic neoplasms, and 36 non-mucinous cysts) from patients undergoing surgery at 4 tertiary referral centers (January 2010 through June 2017). Histology findings from surgical samples, analyzed independently and centrally re-reviewed in a blinded manner, were used as the reference standard. High-risk PCLs were those with invasive carcinomas, high-grade dysplasia, or intestinal-type intraductal papillary mucinous neoplasms with intermediate-grade dysplasia. An ELISA with Das-1 was performed in parallel using banked cyst fluid samples. We evaluated the biomarker's performance, generated area under the curve values, and conducted multivariate logistic regression using clinical and pathology features. RESULTS: The ELISA for Das-1 identified high-risk PCLs with 88% sensitivity, 99% specificity, and 95% accuracy, at a cutoff optical density value of 0.104. In 10-fold cross-validation analysis with 100 replications, Das-1 identified high-risk PCLs with 88% sensitivity and 98% specificity. The Sendai, Fukuoka, and American Gastroenterological Association guideline criteria identified high-risk PCLs with 46%, 52%, and 74% accuracy (P for comparison to Das-1 ELISA <.001). When we controlled for Das-1 in multivariate regression, main pancreatic duct dilation >5 mm (odds ratio, 14.98; 95% confidence interval, 2.63-108; P < .0012), main pancreatic duct dilation ≥1 cm (odds ratio, 47.9; 95% confidence interval, 6.39-490; P < .0001), and jaundice (odds ratio, 6.16; 95% confidence interval, 1.08-36.7; P = .0397) were significantly associated with high-risk PCLs. CONCLUSIONS: We validated the ability of an ELISA with the monoclonal antibody Das-1 to detect PCLs at risk for malignancy with high levels of sensitivity and specificity. This biomarker might be used in conjunction with clinical guidelines to identify patients at risk for malignancy.

Differential expression of PD-L1 and IDO1 in association with the immune microenvironment in resected lung adenocarcinomas.

Like programmed cell death ligand 1 (PD-L1), indoleamine 2,3-dioxygenase 1 (IDO1) is known to exert immunosuppressive effects and be variably expressed in human lung cancer. However, IDO1 expression has not been well studied in lung adenocarcinoma. PD-L1 and IDO1 expression was evaluated in 261 resected lung adenocarcinomas using tissue microarrays and H-scores (cutoff: 5). We compared IDO1 and PD-L1 expression with clinical features, tumor-infiltrating lymphocytes, HLA class I molecule expression, molecular alterations, and patient outcomes. There was expression of PD-L1 in 89 (34%) and IDO1 in 74 (29%) cases, with co-expression in 49 (19%). Both PD-L1 and IDO1 were significantly associated with smoking, aggressive pathologic features, and abundant CD8+ and T-bet+ (Th1 marker) tumor-infiltrating lymphocytes. PD-L1 expression was also associated with preserved HLA class I molecule expression (p = 0.002). Compared to PD-L1+/IDO1+ and PD-L1+ only cases, significantly fewer IDO1+ only cases had abundant CD8+ and T-bet+ tumor-infiltrating lymphocytes (p < 0.001, respectively). PD-L1 expression was significantly associated with EGFR wild-type (p < 0.001) and KRAS mutants (p = 0.021), whereas isolated IDO1 expression was significantly associated with EGFR mutations (p = 0.007). As for survival, PD-L1 was a significant predictor of decreased progression-free and overall survival by univariate but not multivariate analysis, while IDO1 was not associated with progression-free or overall survival. Interestingly, there was a significant difference in the 5-year progression-free and overall survival (p = 0.004 and 0.038, respectively), where cases without PD-L1 or IDO1 expression had the longest survival, and those with PD-L1 alone had the shortest survival. While PD-L1+/-IDO1 expression is observed in association with HLA class I expression, cytotoxic T lymphocyte/Th1 microenvironments, EGFR wild-type, and KRAS mutations, isolated IDO1 expression does not demonstrate these associations, suggesting that IDO1 may serve a distinct immunosuppressive role in lung adenocarcinomas. Thus, further investigation of IDO1 may demonstrate its role as a potential biomarker for patients who undergo anti-PD-1/PD-L1 therapy.

PD-L1 expression and CD8+ infiltration shows heterogeneity in juvenile recurrent respiratory papillomatosis.

INTRODUCTION: Tumor immunotherapy have broadened therapeutic options for tumor treatment. The role of immune function in juvenile recurrent respiratory papillomatosis (JRRP) has not been investigated. Applying immunoblockade inhibitors as a novel disease treatment is unclear. Our study, for the first time, evaluates immune infiltration and immuno-suppressive molecule expression in JRRP. Our study provides insights in possibly treating this disease with tumor immunotherapies. We aimed to determine expression of programmed death-ligand 1 (PD-L1), a cancer escape protein, and presence of CD8+ T cell infiltration in tumor microenvironment. MATERIAL AND METHODS: Seven patients with JRRP (mean age: 7.43; age range 3-17) in this study routinely have their tumors surgical debulked at Massachusetts Eye and Ear Infirmary. Following surgery, samples were de-identified and sent to pathology where they were stained and analyzed. RESULTS: Six out of seven patients expressed PD-L1 on tumor cells to various extents. Three patients showed concurrent PD-L1 expression on tumor cells and abundant CD8+ tumor infiltrating lymphocytes as well as PD-L1+ stromal lymphocytes, while PD-L1 expression on tumor cells were not associated with CD8+ tumor infiltrating T cells nor PD-L1+ stromal lymphocytes in the other three patients. HPV 6/11 and p16 was detected in all the patients. There appeared to be no correlation between either PD-L1 expression and CD8+ infiltration and clinical severity as measured by both the number of surgeries per year or Derkay score. CONCLUSIONS: Despite a small cohort, the expression of p16 and HPV 6/11 in all of the patients confirms the tissues were HPV tumor cells. PD-L1 expression was detected in the vast majority of tumor samples, while inflammatory cell compartments showed a higher degree of variation. Expression of PD-L1 on tumor cells but not inflammatory cells raises the possibility of a tumor cell intrinsic manner of PD-L1 expression. In contrast, a group of patients showed PD-L1 positivity in both tumor and inflammatory cells along with abundant CD8+ tumor infiltrating lymphocytes, suggesting adoptive immune resistance in these tumors and potential benefits from tumor immunotherapy.

Successful Treatment of an Aggressive Tracheal Malignancy With Immunotherapy.

Immune checkpoint inhibitors are emerging as therapeutic options for oncology patients in whom conventional treatment regimens have failed. These immunotherapies counteract tumor-induced tolerance and have been shown to be effective in thoracic malignancies, including non-small cell lung cancer (NSCLC). This report highlights the successful use of nivolumab-an immunotherapeutic agent that binds to proteins involved in T-cell proliferation-for the management of recurrent tracheal squamous cell cancer after exhaustion of conventional surgical, chemotherapeutic, and radiation therapy options. Observations provide a strong indication of the potential value of checkpoint inhibitors for managing a wide array of thoracic malignancies.

Programmed Cell Death Ligand (PD-L1) Expression in Stage II and III Lung Adenocarcinomas and Nodal Metastases.

INTRODUCTION: Programmed death ligand 1 (PD-L1) expression determined by immunohistochemistry (IHC) may serve as a predictive biomarker for anti-PD-1/PD-L1 therapies; however, little is known about intertumoral heterogeneity of PD-L1 expression determined by IHC in lung adenocarcinomas (ADCs), and there have been conflicting results on the prognostic role of PD-L1 expression in ADCs. METHODS: PD-L1 expression was evaluated in resected stage II and III ADCs by using various cutoffs and correlated with clinicopathologic parameters and survival. PD-L1 expression was also compared between the primary tumor and lymph node metastases. RESULTS: There were 109 study cases. PD-L1 expression was seen in 56 (51%), 43 (39%), and 19 (17%) when cutoffs of at least 1%, at least 5%, and at least 50%, respectively, were used. Abundant intratumoral CD8-positive T cells were a significant predictor of the expression in the primary tumor, with cutoffs of 1% and 5% (p < 0.001 for both) by multivariate analysis, whereas they were a nonsignificant predictor of the expression with a 50% cutoff (p = 0.076). PD-L1 expression was concordant between the primary tumor and nodal metastasis in most of the cases, but it was discrepant in up to 38%. The discrepancy was attributed in part to different predominant histologic patterns between the primary and metastatic tumors. In the entire cohort, PD-L1 expression with all cutoffs had no bearing on 5-year recurrence-free survival. CONCLUSIONS: PD-L1 expression is associated with abundant intratumoral CD8-positive T cells in resected ADCs, suggesting a predictive role of PD-L1 expression in anti-PD-1/PD-L1 therapies; however, the possible intertumoral heterogeneity of PD-L1 expression raises a concern about selecting the most appropriate sample for PD-L1 IHC.

Programmed Cell Death Ligand 1 Expression in Resected Lung Adenocarcinomas: Association with Immune Microenvironment.

INTRODUCTION: Programmed cell death ligand 1 (PD-L1) expression on tumor cells can be upregulated via activation of CD8+ cytotoxic T lymphocytes (CTLs) or the T helper cell (Th1) pathway, counterbalancing the CTL/Th1 microenvironment. However, PD-L1 expression in association with subtypes of tumor-associated lymphocytes and molecular alterations has not been well characterized in lung adenocarcinomas. METHODS: PD-L1 expression was evaluated in 261 resected lung adenocarcinomas using tissue microarrays and various scoring systems, and was correlated with clinicopathologic/molecular features, including the extent/subtype of tumor-associated lymphocytes (i.e., CD8, T-bet [Th1 transcription factor], and GATA3 [Th2 transcription factor]), and patient outcomes. RESULTS: PD-L1 expression was present in 129 (49%), 95 (36.5%), and 62 (24%) cases using cutoffs of ≥1%, ≥5%, and ≥50%, respectively, 98 (38%) by H score and 72 (28%) by immune score. PD-L1 expression was associated with abundant CD8+ and/or T-bet+ tumor-infiltrating lymphocytes and EGFR wild-type, significant smoking history, and aggressive pathologic features. In addition, concurrent PD-L1 expression and abundant CD8+ tumor-associated lymphocytes were seen in 25% of KRAS mutants or cases with no alterations by clinical molecular testing as opposed to only 7.4% of EGFR mutants. PD-L1 expression was significantly associated with decreased progression-free and overall survival rates by univariate analysis, but not by multivariate analysis. CONCLUSION: PD-L1 expression in resected lung adenocarcinomas is frequently observed in the presence of CTL/Th1 microenvironment, in particular in those with KRAS mutations or no common molecular alterations, suggesting that blockade of the PD-1/PD-L1 axis may be a promising treatment strategy to reinstitute active immune response for at least a subset of such patient populations.

Molecular Mechanisms of Resistance to First- and Second-Generation ALK Inhibitors in ALK-Rearranged Lung Cancer.

Advanced, anaplastic lymphoma kinase (ALK)-positive lung cancer is currently treated with the first-generation ALK inhibitor crizotinib followed by more potent, second-generation ALK inhibitors (e.g., ceritinib and alectinib) upon progression. Second-generation inhibitors are generally effective even in the absence of crizotinib-resistant ALK mutations, likely reflecting incomplete inhibition of ALK by crizotinib in many cases. Herein, we analyzed 103 repeat biopsies from ALK-positive patients progressing on various ALK inhibitors. We find that each ALK inhibitor is associated with a distinct spectrum of ALK resistance mutations and that the frequency of one mutation, ALKG1202R, increases significantly after treatment with second-generation agents. To investigate strategies to overcome resistance to second-generation ALK inhibitors, we examine the activity of the third-generation ALK inhibitor lorlatinib in a series of ceritinib-resistant, patient-derived cell lines, and observe that the presence of ALK resistance mutations is highly predictive for sensitivity to lorlatinib, whereas those cell lines without ALK mutations are resistant. SIGNIFICANCE: Secondary ALK mutations are a common resistance mechanism to second-generation ALK inhibitors and predict for sensitivity to the third-generation ALK inhibitor lorlatinib. These findings highlight the importance of repeat biopsies and genotyping following disease progression on targeted therapies, particularly second-generation ALK inhibitors. Cancer Discov; 6(10); 1118-33. ©2016 AACRSee related commentary by Qiao and Lovly, p. 1084This article is highlighted in the In This Issue feature, p. 1069.

Napsin A/p40 antibody cocktail for subtyping non-small cell lung carcinoma on cytology and small biopsy specimens.

BACKGROUND: Subtyping non-small cell lung carcinomas (NSCLC) into adenocarcinoma (ACA) or squamous cell carcinoma (SQCC) is important for treatment and specimen triage for molecular studies. To preserve tissue for molecular studies in cytology/small biopsy specimens, a 2-antibody cocktail for NSCLC subtyping was developed. METHODS: Markers for lung ACA (thyroid transcription factor 1 and napsin A) and SQCC (cytokeratin 5/6 and p40) were evaluated on tissue microarrays (TMAs) with 143 ACA and 98 SQCC specimens. The napsin A/p40 combination was selected for NSCLC subtyping and validated on the TMA as well as on a cohort of cell block/small biopsy specimens from 80 poorly differentiated NSCLCs. RESULTS: Using TMA analysis, the napsin A-positive (+)/p40± immunophenotype identified ACA with 94% sensitivity and 100% specificity, whereas the napsin A (negative)-/p40+ immunophenotype identified SQCC with 100% sensitivity and specificity. On the validation cohort of 80 cell block and small biopsy specimens, the napsin A/p40 cocktail accurately subtyped 63 of 70 NSCLC (90%) as ACA or SQCC using the subsequent surgical resection as reference histology. Of the remaining 17 cases, 15 were classified as NSCLC-not otherwise specified based on a napsin A-/p40- immunophenotype; their corresponding resections were diagnosed as ACA (7 cases), large cell carcinoma (7 cases), or pleomorphic carcinoma (1 case). Two additional large cell carcinoma cases showed a napsin A-/p40+ or napsin A+/p40+ profile in the preoperative cell block/small biopsy sample. CONCLUSIONS: A napsin A/p40 cocktail can accurately subtype NSCLC into ACA and SQCC in most cell block/small biopsy specimens of poorly differentiated NSCLC. In the minority of cases in which the napsin A/p40 immunophenotype is indeterminate, additional stains may be necessary for precise classification. Cancer Cytopathol 2016;124:472-84. © 2016 American Cancer Society.

EGFR Mutations and ALK Rearrangements Are Associated with Low Response Rates to PD-1 Pathway Blockade in Non-Small Cell Lung Cancer: A Retrospective Analysis.

PURPOSE: PD-1 inhibitors are established agents in the management of non-small cell lung cancer (NSCLC); however, only a subset of patients derives clinical benefit. To determine the activity of PD-1/PD-L1 inhibitors within clinically relevant molecular subgroups, we retrospectively evaluated response patterns among EGFR-mutant, anaplastic lymphoma kinase (ALK)-positive, and EGFR wild-type/ALK-negative patients. EXPERIMENTAL DESIGN: We identified 58 patients treated with PD-1/PD-L1 inhibitors. Objective response rates (ORR) were assessed using RECIST v1.1. PD-L1 expression and CD8(+) tumor-infiltrating lymphocytes (TIL) were evaluated by IHC. RESULTS: Objective responses were observed in 1 of 28 (3.6%) EGFR-mutant or ALK-positive patients versus 7 of 30 (23.3%) EGFR wild-type and ALK-negative/unknown patients (P = 0.053). The ORR among never- or light- (≤10 pack years) smokers was 4.2% versus 20.6% among heavy smokers (P = 0.123). In an independent cohort of advanced EGFR-mutant (N = 68) and ALK-positive (N = 27) patients, PD-L1 expression was observed in 24%/16%/11% and 63%/47%/26% of pre-tyrosine kinase inhibitor (TKI) biopsies using cutoffs of ≥1%, ≥5%, and ≥50% tumor cell staining, respectively. Among EGFR-mutant patients with paired, pre- and post-TKI-resistant biopsies (N = 57), PD-L1 expression levels changed after resistance in 16 (28%) patients. Concurrent PD-L1 expression (≥5%) and high levels of CD8(+) TILs (grade ≥2) were observed in only 1 pretreatment (2.1%) and 5 resistant (11.6%) EGFR-mutant specimens and was not observed in any ALK-positive, pre- or post-TKI specimens. CONCLUSIONS: NSCLCs harboring EGFR mutations or ALK rearrangements are associated with low ORRs to PD-1/PD-L1 inhibitors. Low rates of concurrent PD-L1 expression and CD8(+) TILs within the tumor microenvironment may underlie these clinical observations. Clin Cancer Res; 22(18); 4585-93. ©2016 AACRSee related commentary by Gettinger and Politi, p. 4539.