Pathologic features of response to neoadjuvant anti-PD-1 in resected non-small-cell lung carcinoma: a proposal for quantitative immune-related pathologic response criteria (irPRC).

Background: Neoadjuvant anti-PD-1 may improve outcomes for patients with resectable NSCLC and provides a critical window for examining pathologic features associated with response. Resections showing major pathologic response to neoadjuvant therapy, defined as ≤10% residual viable tumor (RVT), may predict improved long-term patient outcome. However, %RVT calculations were developed in the context of chemotherapy (%cRVT). An immune-related %RVT (%irRVT) has yet to be developed. Patients and methods: The first trial of neoadjuvant anti-PD-1 (nivolumab, NCT02259621) was just reported. We analyzed hematoxylin and eosin-stained slides from the post-treatment resection specimens of the 20 patients with non-small-cell lung carcinoma who underwent definitive surgery. Pretreatment tumor biopsies and preresection radiographic 'tumor' measurements were also assessed. Results: We found that the regression bed (the area of immune-mediated tumor clearance) accounts for the previously noted discrepancy between CT imaging and pathologic assessment of residual tumor. The regression bed is characterized by (i) immune activation-dense tumor infiltrating lymphocytes with macrophages and tertiary lymphoid structures; (ii) massive tumor cell death-cholesterol clefts; and (iii) tissue repair-neovascularization and proliferative fibrosis (each feature enriched in major pathologic responders versus nonresponders, P < 0.05). This distinct constellation of histologic findings was not identified in any pretreatment specimens. Histopathologic features of the regression bed were used to develop 'Immune-Related Pathologic Response Criteria' (irPRC), and these criteria were shown to be reproducible amongst pathologists. Specifically, %irRVT had improved interobserver consistency compared with %cRVT [median per-case %RVT variability 5% (0%-29%) versus 10% (0%-58%), P = 0.007] and a twofold decrease in median standard deviation across pathologists within a sample (4.6 versus 2.2, P = 0.002). Conclusions: irPRC may be used to standardize pathologic assessment of immunotherapeutic efficacy. Long-term follow-up is needed to determine irPRC reliability as a surrogate for recurrence-free and overall survival.

Histopathological and immunophenotypic features of ipilimumab-associated colitis compared to ulcerative colitis.

BACKGROUND: Use of the immune checkpoint inhibitor ipilimumab is sometimes complicated by ipilimumab-associated colitis (Ipi-AC), an immune-mediated colitis that mimics inflammatory bowel disease. OBJECTIVE: We sought to characterize the histopathologic and immunophenotypic features of Ipi-AC and to directly compare these features to ulcerative colitis (UC). METHODS: This is a retrospective cohort study of 22 patients with Ipi-AC, 12 patients with treatment-naïve UC and five controls with diarrhoea but normal endoscopic findings. Immunohistopathologic features were described, and quantitative immunohistochemistry (IHC) was performed for CD4, CD8, CD20, CD138 and FOXP3. RESULTS: Endoscopic findings in both the Ipi-AC and UC groups included ulcerated, oedematous and erythematous mucosa. Involvement of the GI tract was more diffuse in Ipi-AC. As compared to UC, a smaller proportion of Ipi-AC biopsies had basal plasmacytosis (14% for Ipi-AC vs. 92% for UC, P < 0.0001) and crypt distortion (23% for Ipi-AC vs. 75% for UC, P = 0.003), whereas Ipi-AC biopsies had more apoptotic bodies in the left colon (17.6 ± 15.3 for Ipi-AC vs. 8.2 ± 4.2 for UC, P = 0.011). Cryptitis, ulcerations and crypt abscesses were common in both groups. Biopsy specimens from Ipi-AC had a lower density of CD20-positive lymphocytes than UC (275.8 ± 253.3 cells mm-2 for Ipi-AC vs. 1173.3 ± 1158.2 cells mm-2 for UC, P = 0.022) but had a similar density of CD4, CD8, CD138 and FOXP3-positive cells. CONCLUSIONS: Ipi-AC is a distinct pathologic entity with notable clinical and histopathological differences compared to UC. These findings provide insights into the pathophysiology of immune-related adverse events (iAEs) from ipilimumab therapy.

Long-term follow-up of hepatic adenoma and adenomatosis: analysis of size change on imaging with histopathological correlation.

AIM: To analyse the change in size on follow-up of hepatic adenomas (HAs) and adenomatosis, and to investigate the relationship of imaging features with size change. MATERIALS AND METHODS: The study included 44 patients (142 lesions) who underwent magnetic resonance imaging (MRI) or computed tomography (CT) for diagnosis and follow-up of HA. The imaging features and percentage change in maximum tumour dimension were observed over a follow-up duration of up to 139 months. RESULTS: With an average follow-up of 43 months, 37% lesions decreased in size, 58% were stable, 4% increased; one lesion regressed completely. Adenomas were stratified into size groups (<3, 3-5, and ≥5 cm). Size change among the three groups was similar (p>0.05). Percent size change was different for lesions followed for ≤12 months (-7.2%) compared with lesions followed for 13-60 months (-20.5%), and those followed for ≥60 months (-23.5%; p<0.05); there was no difference between lesions followed for 13-60 months and ≥60 months (p=0.523). Baseline size and percent size change was similar between the hepatocyte nuclear factor 1α-inactivated HA (HA-H) and inflammatory HA (HA-I) subtype (p>0.05). CONCLUSION: Most adenomas were either stable or regressed on follow-up. Size change was independent of baseline size. After an initial size decrease within 5 years, no further size reduction was noted on extended follow-up. The percent size change in the HA-H and HA-I subtype was similar.

Chronic diarrhea, ascites, and protein-losing enteropathy in an infant with hepatic venous outflow obstruction after liver transplantation.

An 18-month-old female status post-orthotopic liver transplant for biliary atresia presented nine months after transplant with severe diarrhea and intolerance of feeds. She was found to have a PLE as evidenced by a low serum albumin and a persistent elevation of fecal A1AT. Investigation eventually revealed that the cause of the PLE was a stricture at the anastomosis site between the hepatic vein and inferior cava, supported by resolution of the PLE after venoplasty of the stricture. The patient has subsequently required several repeat venoplasties for recurrence of her symptoms correlating with recurrence of the stricture. This is a very rare presentation of hepatic venous outflow obstruction. Moreover, normal duplex ultrasound imaging of liver vasculature and her unusual presentation led to a delay in her diagnosis highlighting the need for an increased index of suspicion.

Pneumocystis carinii inhibits cyclin-dependent kinase activity in lung epithelial cells.

Pneumocystis carinii remains an important cause of pneumonia in patients with AIDS. Attachment of the organism to epithelial cells is a central event in establishing infection, impairing the growth potential of lung epithelial cells and thereby slowing repair. In light of investigations documenting a central role for cyclin-dependent kinases in controlling the cell cycle, we addressed the hypothesis that P. carinii inhibits epithelial cell growth by interfering with host epithelial cyclin-dependent kinase (cdk) activity. We observed that P. carinii significantly impaired growth of cultured mink lung epithelial cells, with effects observed after 48-72 h of treatment. However, the kinase activity associated with p34cdc2 or p33cdk2 was maximally inhibited as early as 24 h after P. carinii exposure. The inhibitory effect on cyclin-dependent kinase activity was mediated by the trophozoite form of P. carinii, in that highly purified trophozoites exerted marked inhibition of p34cdc2 activity. Growth impairment was similarly preceded by P. carinii-induced alteration in the state of epithelial cell p34cdc2 phosphorylation, with no change in p34cdc2 or p33cdk2 protein levels. These data strongly suggest that the antiproliferative activity of P. carinii on respiratory epithelium is mediated in part through modulation of the host cell cycle machinery.

Distinct endocytic responses of heteromeric and homomeric transforming growth factor beta receptors.

Transforming growth factor beta (TGF beta) family ligands initiate a cascade of events capable of modulating cellular growth and differentiation. The receptors responsible for transducing these cellular signals are referred to as the type I and type II TGF beta receptors. Ligand binding to the type II receptor results in the transphosphorylation and activation of the type I receptor. This heteromeric complex then propagates the signal(s) to downstream effectors. There is presently little data concerning the fate of TGF beta receptors after ligand binding, with conflicting reports indicating no change or decreasing cell surface receptor numbers. To address the fate of ligand-activated receptors, we have used our previously characterized chimeric receptors consisting of the ligand binding domain from the granulocyte/macrophage colony-stimulating factor alpha or beta receptor fused to the transmembrane and cytoplasmic domain of the type I or type II TGF beta receptor. This system not only provides the necessary sensitivity and specificity to address these types of questions but also permits the differentiation of endocytic responses to either homomeric or heteromeric intracellular TGF beta receptor oligomerization. Data are presented that show, within minutes of ligand binding, chimeric TGF beta receptors are internalized. However, although all the chimeric receptor combinations show similar internalization rates, receptor down-regulation occurs only after activation of heteromeric TGF beta receptors. These results indicate that effective receptor down-regulation requires cross-talk between the type I and type II TGF beta receptors and that TGF beta receptor heteromers and homomers show distinct trafficking behavior.

Paucity of PD-L1 expression in prostate cancer: innate and adaptive immune resistance.

BACKGROUND: Primary prostate cancers are infiltrated with programmed death-1 (PD-1) expressing CD8+ T-cells. However, in early clinical trials, men with metastatic castrate-resistant prostate cancer did not respond to PD-1 blockade as a monotherapy. One explanation for this unresponsiveness could be that prostate tumors generally do not express programmed death ligand-1 (PD-L1), the primary ligand for PD-1. However, lack of PD-L1 expression in prostate cancer would be surprising, given that phosphatase and tensin homolog (PTEN) loss is relatively common in prostate cancer and several studies have shown that PTEN loss correlates with PD-L1 upregulation--constituting a mechanism of innate immune resistance. This study tested whether prostate cancer cells were capable of expressing PD-L1, and whether the rare PD-L1 expression that occurs in human specimens correlates with PTEN loss. METHODS: Human prostate cancer cell lines were evaluated for PD-L1 expression and loss of PTEN by flow cytometry and western blotting, respectively. Immunohistochemical (IHC) staining for PTEN was correlated with PD-L1 IHC using a series of resected human prostate cancer samples. RESULTS: In vitro, many prostate cancer cell lines upregulated PD-L1 expression in response to inflammatory cytokines, consistent with adaptive immune resistance. In these cell lines, no association between PTEN loss and PD-L1 expression was apparent. In primary prostate tumors, PD-L1 expression was rare, and was not associated with PTEN loss. CONCLUSIONS: These studies show that some prostate cancer cell lines are capable of expressing PD-L1. However, in human prostate cancer, PTEN loss is not associated with PD-L1 expression, arguing against innate immune resistance as a mechanism that mitigates antitumor immune responses in this disease.

Longitudinal changes in liver fibrosis in children with sickle cell disease undergoing chronic transfusion therapy.

BACKGROUND AND STUDY AIMS: The progression of liver injury from transfusional iron overload in sickle cell disease (SCD) is poorly understood. We sought to identify predictors liver fibrosis development over time. PATIENTS AND METHODS: We performed a retrospective cohort study of chronically transfused SCD patients who had > or = 2 serial liver biopsies. Core biopsies were scored for fibrosis in a blinded fashion. Primary analyses evaluated longitudinal changes in liver fibrosis and changes in surrogate markers. Secondary analyses determined the relationship between liver iron concentration (LIC) and serum biomarkers. RESULTS: 26 people had > or = 2 serial biopsies for evaluation (n = 70 biopsies total). Fibrosis was Ishak grade 0 or 1 in all biopsies. Evaluation of the first 2 biopsies showed fibrosis regression (n = 6), development (n = 2), persistence (n = 1), and absence (n = 17). There was no consistent association of fibrosis with LIC over time, or between changes in fibrosis status and surrogate markers. For predicting fibrosis on a cross-sectional basis, ALT and ferritin performed moderately (AUCs 0.80 and 0.63, respectively) but LIC performed poorly (AUC 0.30). The highest positive likelihood ratios for fibrosis were for ferritin cutoff of 5000 ng/mL (LR + 5.7) and ALT cutoff of 65 U/L (LR + 5.2). CONCLUSIONS: Liver fibrosis progression is minimal in chronically transfused SCD. LIC does not correlate well with fibrosis development. We propose routine liver biopsies are not necessary components in the standard monitoring of chronically transfused SCD patients.

Chimeric granulocyte/macrophage colony-stimulating factor/transforming growth factor-beta (TGF-beta) receptors define a model system for investigating the role of homomeric and heteromeric receptors in TGF-beta signaling.

Transforming growth factor-beta (TGF-beta) belongs to a family of ligands that regulate cell growth and differentiation. The most commonly observed receptors are referred to as the type I, type II, and type III (betaglycan) TGF-beta receptors. Two receptor models have been presented to account for the various cellular responses to TGF-beta. The first proposes that all TGF-beta signaling results from the formation of a heteromeric type I/type II complex, while the second suggests that distinct type I or type II TGF-beta receptor combinations mediate aspects of TGF-beta signaling. We have addressed this general question relating to TGF-beta signaling by constructing chimeric receptors consisting of the extracellular domain of the granulocyte/macrophage colony-stimulating factor (GM-CSF) alpha or beta receptor fused to the transmembrane and cytoplasmic domain of the type I or type II TGF-beta receptor. Since high affinity GM-CSF binding requires dimerization of the alpha and beta ligand binding subunits, the response elicited by defined type I and/or type II TGF-beta receptor cytoplasmic domain homomers or heteromers can be examined. We show in mesenchymal AKR-2B cells that while TGF-beta-dependent transient luciferase activity, endogenous gene activity, and long-term biological responses are similarly induced by activating the chimeric heteromeric receptors with GM-CSF as the endogenous TGF-beta receptor, chimeric homomeric type I/type I or type II/type II receptors are signaling-incompetent.

Conditional binding to and cell cycle-regulated inhibition of cyclin-dependent kinase complexes by p27Kip1.

Mammalian cultures primarily regulate cell cycle traverse during G1. For progression through G1 and commitment to DNA synthesis, the activity of a family of proteins, the cyclin-dependent kinases (cdks), is required. There are two primary regulatory portions of G1: (a) the G0-G1 transition, which allows entry into G1; and (b) the G1-S transition, promoting entry to DNA synthesis and commitment to cell division. In the present manuscript, we provide evidence for cross-talk between these two cell cycle transitions. Extracts prepared from quiescent mouse mammary epithelial cells are shown to act in a dominant manner to specifically inhibit the histone H1 kinase activity of preformed/active cdk2, cdk4, cyclin A, or cyclin E complexes from G1-S cell extracts. The inhibitory activity arises as cells enter quiescence and decreases once cultures are stimulated to begin G1 traverse and endogenous cdk activity becomes evident. This activity is associated with the regulated binding of the cdk inhibitor p27Kip1 to cyclin A/cdk2 kinase complexes upon mixing of the extracts. Removal of p27Kip1 from the quiescent cell extract specifically abolishes the inhibitory effect. The inhibitory activity and p27Kip1 binding in vitro depend on incubation of the extracts at physiological temperature or the presence of a reducing agent. The results suggest an interplay between the acquisition of quiescence, cdk activity, and G1 traverse.

Differential requirement for type I and type II transforming growth factor beta receptor kinase activity in ligand-mediated receptor endocytosis.

Transforming growth factor beta (TGFbeta) superfamily polypeptides regulate cell growth and differentiation by binding to single pass serine/threonine kinases referred to as TGFbeta type I and type II receptors. Signal propagation is dependent upon heteromeric (type I-type II) complex formation and transphosphorylation of the type I receptor by the type II receptor. While many of the phosphorylation events necessary for receptor signaling have recently been characterized, the role of TGFbeta receptor kinase activity in modulating receptor endocytosis has not been addressed. To that end, we have used chimeric receptors consisting of the extracellular domain of the granulocyte/macrophage colony-stimulating factor alpha and beta receptors spliced to the TGFbeta type I and type II transmembrane and cytoplasmic domains to address the specific role of type I and/or type II receptor kinase activity in TGFbeta receptor internalization, down-regulation, and signaling. To inactivate chimeric receptor kinase activity, point mutations in the ATP binding site were made at amino acids 232 and 277 in the type I and type II receptor, respectively. Either of these mutations abolished plasminogen activator inhibitor 1 protein expression stimulated by granulocyte/macrophage colony-stimulating factor activation of chimeric heteromeric type I-type II TGFbeta receptors. They did not, however, modulate TGFbeta signaling stimulated through the endogenous TGFbeta receptor. Although TGFbeta receptor signaling was dependent upon the kinase activity of both chimeric receptors, the initial endocytic response was distinctly regulated by type I and/or type II receptor kinase activity. For instance, while heteromeric receptor complexes containing a kinase-inactive type I receptor were endocytosed similarly to wild type complexes, the kinase activity of the type II TGFbeta receptor was necessary for optimal internalization and receptor down-regulation. Furthermore, these responses were shown to occur independently of type II receptor autophosphorylation but require a type II receptor capable of transphosphorylation.

Differential regulation of p34cdc2 and p33cdk2 by transforming growth factor-beta 1 in murine mammary epithelial cells.

Cyclin-dependent kinases (cdks) are a family of proteins whose function plays a critical role in cell cycle traverse. Transforming growth factor-beta 1 (TGF-beta 1) is a potent growth inhibitor of epithelial cells. Since cdks have been suggested as possible biochemical markers for TGF-beta growth inhibition, we investigated the effect of TGF-beta 1 on cdc2 and cdk2 in a normal mouse mammary epithelial cell line (MME) and a TGF-beta-resistant MME cell line (BG18.2). TGF-beta 1 decreases newly synthesized cdc2 protein levels within 6 h after addition. Coincident with this decrease in newly synthesized cdc2 protein was a marked reduction in its ability to phosphorylate histone H1. This decrease in kinase activity is not due to a change in steady-state levels of cdc2 protein, since mRNA and total protein levels of cdc2 are not reduced until 12 h after TGF-beta 1 addition. This suggests that the kinase activity of cdc2 is dependent on newly synthesized cdc2 protein. Moreover, the protein synthesis of another cyclin-dependent kinase, cdk2, is not effected by TGF-beta 1 addition, but its kinase activity is substantially reduced. Thus, it appears that TGF-beta decreases the kinase activity of both cdc2 and cdk2 by distinct mechanisms.

Pneumocystis carinii contains a functional cell-division-cycle Cdc2 homologue.

Pneumocystis carinii causes life-threatening pneumonia in immunocompromised patients. The inability to culture P. carinii has hampered basic investigations of the organism's life cycle, limiting the development of new therapies directed against it. Recent investigations indicate that P. carinii is a fungus phylogenetically related to other ascomycetes such as Schizosaccharomyces pombe. The cell cycles of S. pombe and homologous fungi are carefully regulated by cell-division-cycle molecules (cdc), particularly cell-division-cycle 2 (Cdc2), a serine-threonine kinase with essential activity at the G1 restriction point and for entry into mitosis. Antibodies to the proline-serine-threonine-alanine-isoleucine-arginine (PSTAIR) amino-acid sequence conserved in Cdc2 proteins specifically precipitated, from P. carinii extracts, a molecule with kinase activity consistent with a Cdc2-like protein. Cdc2 molecules exhibit differential activity throughout the life cycle of the organisms in which they occur. In accord with this, the P. carinii Cdc2 showed greater specific activity in P. carinii trophic forms (trophozoites) than in spore-case forms (cysts). In addition, complete genomic and complementary DNA (cDNA) sequences of P. carinii Cdc2 were cloned and found to be most closely homologus to the corresponding sequences of other pathogenic fungi. The function of P. carinii cdc2 cDNA was further documented through its ability to complement the DNA of mutant strains of S. pombe with temperature-sensitive deficiencies in Cdc2 activity. The P. carinii cdc2 cDNA restored normal Cdc2 function in these mutant strains of S. pombe, and promoted fungal proliferation. These studies represent the first molecular analysis of the cell-cycle-regulatory machinery in P. carinii. Further understanding of P. carinii's life cycle promises novel insights for preventing and treating the intractable infection it causes in immunocompromised patients.