Prognostic significance of absolute lymphocyte count in patients with metastatic renal cell carcinoma receiving first-line combination immunotherapies: results from the International Metastatic Renal Cell Carcinoma Database Consortium.

BACKGROUND: Lymphocytes are closely linked to mechanisms of action of immuno-oncology (IO) agents. We aimed to assess the prognostic significance of absolute lymphocyte count (ALC) in patients with metastatic renal cell carcinoma (mRCC). PATIENTS AND METHODS: Using the International mRCC Database Consortium (IMDC), patients receiving first-line IO-based combination therapy were analysed. Baseline patient characteristics, objective response rates (ORRs), time to next treatment (TTNT), and overall survival (OS) were compared. RESULTS: Of 966 patients included, 195 (20%) had lymphopenia at baseline, and they had a lower ORR (37% versus 45%; P < 0.001), shorter TTNT (10.1 months versus 24.3 months; P < 0.001), and shorter OS (30.4 months versus 48.2 months; P < 0.001). Among 125 patients with lymphopenia at baseline, 52 (42%) experienced ALC recovery at 3 months, and they had longer OS (not reached versus 30.4 months; P = 0.012). On multivariable analysis for OS, lymphopenia was an independent adverse prognostic factor (hazard ratio 1.68; P < 0.001). Incorporation of lymphopenia into the IMDC criteria improved OS prediction accuracy (C-index from 0.688 to 0.707). CONCLUSIONS: Lymphopenia was observed in one-fifth of treatment-naive patients with mRCC and may serve as an indicator of unfavourable oncologic outcomes in the contemporary IO era.

Fragmentomic analysis of circulating tumor DNA-targeted cancer panels.

BACKGROUND: The isolation of cell-free DNA (cfDNA) from the bloodstream can be used to detect and analyze somatic alterations in circulating tumor DNA (ctDNA), and multiple cfDNA-targeted sequencing panels are now commercially available for Food and Drug Administration (FDA)-approved biomarker indications to guide treatment. More recently, cfDNA fragmentation patterns have emerged as a tool to infer epigenomic and transcriptomic information. However, most of these analyses used whole-genome sequencing, which is insufficient to identify FDA-approved biomarker indications in a cost-effective manner. PATIENTS AND METHODS: We used machine learning models of fragmentation patterns at the first coding exon in standard targeted cancer gene cfDNA sequencing panels to distinguish between cancer and non-cancer patients, as well as the specific tumor type and subtype. We assessed this approach in two independent cohorts: a published cohort from GRAIL (breast, lung, and prostate cancers, non-cancer, n = 198) and an institutional cohort from the University of Wisconsin (UW; breast, lung, prostate, bladder cancers, n = 320). Each cohort was split 70%/30% into training and validation sets. RESULTS: In the UW cohort, training cross-validated accuracy was 82.1%, and accuracy in the independent validation cohort was 86.6% despite a median ctDNA fraction of only 0.06. In the GRAIL cohort, to assess how this approach performs in very low ctDNA fractions, training and independent validation were split based on ctDNA fraction. Training cross-validated accuracy was 80.6%, and accuracy in the independent validation cohort was 76.3%. In the validation cohort where the ctDNA fractions were all <0.05 and as low as 0.0003, the cancer versus non-cancer area under the curve was 0.99. CONCLUSIONS: To our knowledge, this is the first study to demonstrate that sequencing from targeted cfDNA panels can be utilized to analyze fragmentation patterns to classify cancer types, dramatically expanding the potential capabilities of existing clinically used panels at minimal additional cost.

COVID-19 vaccination and breakthrough infections in patients with cancer.

BACKGROUND: Vaccination is an important preventive health measure to protect against symptomatic and severe COVID-19. Impaired immunity secondary to an underlying malignancy or recent receipt of antineoplastic systemic therapies can result in less robust antibody titers following vaccination and possible risk of breakthrough infection. As clinical trials evaluating COVID-19 vaccines largely excluded patients with a history of cancer and those on active immunosuppression (including chemotherapy), limited evidence is available to inform the clinical efficacy of COVID-19 vaccination across the spectrum of patients with cancer. PATIENTS AND METHODS: We describe the clinical features of patients with cancer who developed symptomatic COVID-19 following vaccination and compare weighted outcomes with those of contemporary unvaccinated patients, after adjustment for confounders, using data from the multi-institutional COVID-19 and Cancer Consortium (CCC19). RESULTS: Patients with cancer who develop COVID-19 following vaccination have substantial comorbidities and can present with severe and even lethal infection. Patients harboring hematologic malignancies are over-represented among vaccinated patients with cancer who develop symptomatic COVID-19. CONCLUSIONS: Vaccination against COVID-19 remains an essential strategy in protecting vulnerable populations, including patients with cancer. Patients with cancer who develop breakthrough infection despite full vaccination, however, remain at risk of severe outcomes. A multilayered public health mitigation approach that includes vaccination of close contacts, boosters, social distancing, and mask-wearing should be continued for the foreseeable future.

Phase 2 trial of sunitinib and gemcitabine in patients with sarcomatoid and/or poor-risk metastatic renal cell carcinoma. Michaelson MD, McKay RR, Werner L, Atkins MB, Van Allen EM, Olivier KM, Song J, Signoretti S, McDermott DF, Choueiri TK.Cancer. 2015 Oct 1;121(19):3435-43. [Epub 2015 Jun 8]. doi: 10.1002/cncr.29503.

BACKGROUND: Sarcomatoid renal cell carcinoma (RCC) is associated with an aggressive biology and a poor prognosis. Poor-risk RCC is defined by clinical prognostic factors and demonstrates similarly aggressive behavior. No standard treatment exists for patients with sarcomatoid RCC, and treatment options for patients with poor-risk disease are of limited benefit. The objective of this study was to investigate the efficacy of antiangiogenic therapy in combination with cytotoxic chemotherapy in clinically aggressive RCC. METHODS: This was a phase 2, single-arm trial of sunitinib and gemcitabine in patients with sarcomatoid or poor-risk RCC. The primary end point was the objective response rate (ORR). Secondary end points included the time to progression (TTP), overall survival (OS), safety, and biomarker correlatives. RESULTS: Overall, 39 patients had sarcomatoid RCC, and 33 had poor-risk RCC. The ORR was 26% for patients with sarcomatoid RCC and 24% for patients with poor-risk RCC. The median TTP and OS for patients with sarcomatoid RCC were 5 and 10 months, respectively. For patients with poor-risk disease, the median TTP and OS were 5.5 and 15 months, respectively. Patients whose tumors had>10% sarcomatoid histology had a higher clinical benefit rate (ORR plus stable disease) than those with≤10% sarcomatoid histology (P = 0.04). The most common grade 3 or higher treatment-related adverse events included neutropenia (n = 20), anemia (n = 10), and fatigue (n = 7). CONCLUSIONS: These results suggest that antiangiogenic therapy and cytotoxic chemotherapy are an active and well-tolerated combination for patients with aggressive RCC. The combination may be more efficacious than either therapy alone and is currently under further investigation.

Predictors of duration of abiraterone acetate in men with castration-resistant prostate cancer.

BACKGROUND: Androgen receptor signaling remains important in castration-resistant prostate cancer (CRPC) as demonstrated by the efficacy of abiraterone acetate (henceforth abiraterone) in phase III trials. Given that heterogeneous patient responses are observed, we sought to identify clinical factors associated with duration of abiraterone. METHODS: We retrospectively identified patients with CRPC treated with abiraterone in our database. Patient characteristics and types and duration of prostate cancer (PC) therapies were analyzed. These parameters were analyzed with duration of abiraterone in univariate and multivariable analyses. RESULTS: We identified 161 patients who had received abiraterone. All had received primary androgen-deprivation therapy (ADT), 86% prior secondary hormone therapy (SHT) and 33% prior chemotherapy. The median duration of primary ADT was 23 months, duration of SHT (excluding abiraterone) was 17 months and duration of chemotherapy was 8 months. We demonstrated that lower PSA at abiraterone initiation, longer primary ADT duration, no prior ketoconazole, no prior chemotherapy and longer chemotherapy duration were associated with a longer duration on abiraterone in univariate analysis. In multivariable analysis, duration of primary ADT (duration of abiraterone 9 versus 13 months for ⩽12 versus >12 months, P=0.03) and no use of prior chemotherapy (duration of abiraterone 16 versus 7 months for no versus yes prior chemotherapy, P<0.01) were associated with duration of abiraterone. CONCLUSIONS: Several clinical parameters, including type and duration of prior therapy, are predictive of responsiveness to abiraterone. These parameters are logical and correlate with smaller disease burden or less exposure to PC therapies. This information can help physicians counsel patients about the potential durability of efficacy of abiraterone. Identifying predictive biomarkers that inform patient selection for therapy is critical to optimizing treatment outcomes.

Imaging, procedural and clinical variables associated with tumor yield on bone biopsy in metastatic castration-resistant prostate cancer.

BACKGROUND: Understanding the mechanisms driving disease progression is fundamental to identifying new therapeutic targets for the treatment of men with metastatic castration-resistant prostate cancer (mCRPC). Owing to the prevalence of bone metastases in mCRPC, obtaining sufficient tumor tissue for analysis has historically been a challenge. In this exploratory analysis, we evaluated imaging, procedural and clinical variables associated with tumor yield on image-guided bone biopsy in men with mCRPC. METHODS: Clinical data were collected prospectively from men with mCRPC enrolled on a phase II trial with serial metastasis biopsies performed according to standard clinical protocol. Imaging was retrospectively reviewed. We evaluated the percent positive biopsy cores (PPC), calculated as the number of positive cores divided by the total number of cores collected per biopsy. RESULTS: Twenty-nine men had 39 bone biopsies. Seventy-seven percent of bone biopsies had at least one positive biopsy core. We determined that lesion size and distance from the skin to the lesion edge correlated with tumor yield on biopsy (median PPC 75% versus 42% for lesions >8.8 cm(3) versus ⩽ 8.8 cm(3), respectively, P=0.05; median PPC 33% versus 71% for distance ⩾ 6.1 versus <6.1 cm, respectively, P = 0.02). There was a trend towards increased tumor yield in patients with increased uptake on radionuclide bone scan, higher calcium levels and shorter duration of osteoclast-targeting therapy, although this was not statistically significant. Ten men had 14 soft tissue biopsies. All soft tissue biopsies had at least one positive biopsy core. CONCLUSIONS: This exploratory analysis suggests that there are imaging, procedural and clinical variables that have an impact on image-guided bone biopsy yield. In order to maximize harvest of prostate cancer tissue, we have incorporated a prospective analysis of the metrics described here as part of a multi-institutional project aiming to use the molecular characterization of mCRPC tumors to direct individual therapy.

The application of Bayesian spectral analysis to optical sectioning using structured light imaging.

We describe significant improvements to a well-established method of obtaining optical sectioning in a conventional wide-field microscope. This method relies on the projection of a single-frequency grid pattern onto an object followed by mathematical manipulation of three images taken with the grid in different phases. Here, we present the use of Bayesian Spectral Analysis to determine accurate estimates of the phase of the grid pattern, permitting rapid and precise calibration of grid location. In common with previous algorithms, multiple images are combined to produce both an optical section and a wide-field image. We describe innovations such as the use of non-uniform phase or least-squares solutions involving more than three images, in conjunction with direct phase estimates obtained using Bayesian Spectral Analysis, to yield images substantially free of artefacts. Auxiliary results, such as a method for determining the tilt of the grid, are also presented.

Cloning and expression of the human transient receptor potential 4 (TRP4) gene: localization and functional expression of human TRP4 and TRP3.

Mammalian homologues of the Drosophila transient receptor potential (TRP) protein have been proposed to function as ion channels, and in some cases as store-operated or capacitative calcium entry channels. However, for each of the mammalian TRP proteins, different laboratories have reported distinct modes of cellular regulation. In the present study we describe the cloning and functional expression of the human form of TRP4 (hTRP4), and compare its activity with another well studied protein, hTRP3. When hTRP4 was transiently expressed in human embryonic kidney (HEK)-293 cells, basal bivalent cation permeability (barium) was increased. Whole-cell patch-clamp studies of hTRP4 expressed in Chinese hamster ovary cells revealed a constitutively active non-selective cation current which probably underlies the increased bivalent cation entry. Barium entry into hTRP4-transfected HEK-293 cells was not further increased by phospholipase C (PLC)-linked receptor activation, by intracellular calcium store depletion with thapsigargin, or by a synthetic diacylglycerol, 1-oleoyl-2-acetyl-sn-glycerol (OAG). In contrast, transient expression of hTRP3 resulted in a bivalent cation influx that was markedly increased by PLC-linked receptor activation and by OAG, but not by thapsigargin. Despite the apparent differences in regulation of these two putative channel proteins, green fluorescent protein fusions of both molecules localized similarly to the plasma-membrane, notably in discrete punctate regions suggestive of specialized signalling complexes. Our findings indicate that while both hTRP4 and hTRP3 can apparently function as cation channels, their putative roles as components of capacitative calcium entry channels are not readily demonstrable by examining their behaviour when exogenously expressed in cells.

Effects of elevated cytoplasmic calcium and protein kinase C on endoplasmic reticulum structure and function in HEK293 cells.

In human embryonic kidney (HEK) cells stably transfected with green fluorescent protein targeted to the endoplasmic reticulum (ER), elevation of intracellular Ca2+ ([Ca2+]i) altered ER morphology, making it appear punctate. Electron microscopy revealed that these punctate structures represented circular and branched rearrangements of the endoplasmic reticulum, but did not involve obvious swelling or pathological fragmentation. Activation of protein kinase C with phorbol 12-myristate 13-acetate (PMA), prevented the effects of ionomycin on ER structure without affecting the elevation of [Ca2+]i. These results suggest that protein kinase C activation alters cytoplasmic or ER components underlying the effects of high [Ca2+]i on ER structure. Treatment of HEK cells with PMA also reduced the size of the thapsigargin-sensitive Ca2+ pool and inhibited Ca2+ entry in response to thapsigargin. Thus, protein kinase C activation has multiple actions on the calcium storage and signalling function of the endoplasmic reticulum in HEK cells: (1) reduced intracellular Ca2+ storage capacity, (2) inhibition of capacitative Ca2+ entry, and (3) protection of the endoplasmic reticulum against the effects of high [Ca2+]i.

Capacitative calcium entry channels.

In the phospholipase C signaling system, Ca(2+) is mobilized from intracellular stores by an action of inositol 1,4,5-trisphosphate. The depletion of intracellular calcium stores activates a calcium entry mechanism at the plasma membrane called capacitative calcium entry. The signal for activating the entry is unknown but likely involves either the generation or release, or both, from the endoplasmic reticulum of some diffusible signal. Recent research has focused on mammalian homologues of the Drosophila TRP protein as potential candidates for capacitative calcium entry channels. This review summarizes current knowledge about the nature of capacitative calcium entry signals, as well as the potential role of mammalian TRP proteins as capacitative calcium entry channel molecules.

Invertebrate phosphatidylinositol-specific phospholipases C and their role in cell signaling.

Phosphatidylinositol-specific phospholipase C (PLC) is a family of enzymes that occupy a pivotal role in one of the largest classes of cellular signaling pathways known. Mammalian PLC enzymes have been divided into four major classes and a variety of subclasses based on their structural characteristics and immunological differences. There have been five invertebrate PLC-encoding genes cloned thus far and these fall within three of the four major classes used in categorizing mammalian PLC. Four of these invertebrate genes have been cloned from Drosophila melanogaster and one is from Artemia, a brine shrimp. Structural characteristics of the invertebrate enzymes include the presence of highly conserved Box X and Box Y domains found in major types of mammalian PLC as well as novel features. Two of the invertebrate PLC genes encode multiple splice-variant subtypes which is a newly emerging level of diversity observed in mammalian enzymes. Studies of the invertebrate PLCs have contributed to the identification of the physiological functions of individual isozymes. These identified roles include cellular processes such as phototransduction, olfaction, cell growth and differentiation.

Phospholipase C rescues visual defect in norpA mutant of Drosophila melanogaster.

Mutations in the norpA gene of Drosophila melanogaster severely affect the light-evoked photoreceptor potential with strong mutations rendering the fly blind. The norpA gene has been proposed to encode phosphatidylinositol-specific phospholipase C (PLC), which enzymes play a pivotal role in one of the largest classes of signaling pathways known. A chimeric norpA minigene was constructed by placing the norpA cDNA behind an R1-6 photoreceptor cell-specific rhodopsin promoter. This minigene was transferred into norpAP24 mutant by P-element-mediated germline transformation to determine whether it could rescue the phototransduction defect concomitant with restoring PLC activity. Western blots of head homogenates stained with norpA antiserum show that norpA protein is restored in heads of transformed mutants. Moreover, transformants exhibit a large amount of measurable PLC activity in heads, whereas heads of norpAP24 mutant exhibit very little to none. Immunohistochemical staining of tissue sections using norpA antiserum confirm that expression of norpA protein in transformants localizes in the retina, more specifically in rhabdomeres of R1-6 photoreceptor cells, but not R7 or R8 photoreceptor cells. Furthermore, electrophysiological analyses reveal that transformants exhibit a restoration of light-evoked photoreceptor responses in R1-6 photoreceptor cells, but not in R7 or R8 photoreceptor cells. This is the strongest evidence thus far supporting the hypothesis that the norpA gene encodes phospholipase C that is utilized in phototransduction.

Multiple subtypes of phospholipase C are encoded by the norpA gene of Drosophila melanogaster.

The norpA gene of Drosophila melanogaster encodes a phosphatidylinositol-specific phospholipase C that is essential for phototransduction. Besides being found abundantly in retina, norpA gene products are expressed in a variety of tissues that do not contain phototransduction machinery, implying that norpA is involved in signaling pathways in addition to phototransduction. We have identified a second subtype of norpA protein that is generated by alternative splicing of norpA RNA. The alternative splicing occurs at a single exon that is excluded from mature norpA transcripts when a substitute exon of equal size is retained. The net difference between the two subtypes of norpA protein is 14 amino acid substitutions occurring between amino acid positions 130 and 155 of the enzyme. Results from Northern analyses suggest that norpA subtype I transcripts are most abundantly expressed in adult retina, while subtype II transcripts are most abundant in adult body. Moreover, norpA subtype I RNA can be detected by the reverse transcription-polymerase chain reaction in extracts of adult head tissue but not adult body nor at earlier stages of Drosophila development. Conversely, norpA subtype II RNA can be detected by reverse transcription-polymerase chain reaction throughout development as well as in heads and bodies of adults. Furthermore, norpA subtype I RNA is easily detected in retina using tissue in situ hybridization analysis, while subtype II RNA is not detectable in retina but is found in brain. Since only norpA subtype I RNA is found in retina, we conclude that subtype I protein is utilized in phototransduction. Since norpA subtype II RNA is not found in retina but is expressed in a variety of tissues not known to contain phototransduction machinery, subtype II protein is likely to be utilized in signaling pathways other than phototransduction. The amino acid differences between the two subtypes of norpA protein may reflect the need for each subtype to interact with signaling components of different signal-generating pathways.

A Drosophila gene that encodes a member of the protein disulfide isomerase/phospholipase C-alpha family.

Screening of a Drosophila genomic DNA library at reduced stringency hybridization conditions using a rat PLC alpha cDNA probe yielded a gene which encodes a member of the protein disulfide isomerase/PLC alpha family. The gene has been localized to band 74C on the left arm of the third chromosome and has been designated dpdi. Northern analysis shows that the dpdi gene encodes a transcript that is 2.3 kb in length and is present throughout development as well as in both heads and bodies of adults. The deduced dpdi protein is 496 amino acids in length and contains two domains exhibiting high similarity to thioredoxin, two regions that are similar to the hormone binding domain of human estrogen receptor, and a sequence of four amino acids (KDEL) at the C-terminus which has been described by others as being responsible for retention of proteins in the endoplasmic reticulum. Overall, dpdi contains a higher similarity to rat protein disulfide isomerase (53% identical) than to rat PLC alpha (30% identical). However, it is unclear whether dpdi functions in vivo as a PDI or as a PLC, or both. Drosophila, with its well characterized genetics and the ability to generate mutants in a gene that has been cloned, provides an excellent system in which to resolve this issue.

Membrane association of phospholipase C encoded by the norpA gene of Drosophila melanogaster.

Severe mutations within the norpA gene of Drosophila abolish the photoreceptor potential and render the fly blind by deleting phospholipase C, an essential component of the phototransduction pathway. To study the membrane association of phospholipase C, we have utilized biochemical assays of phospholipase C activity, which predominant measurable phospholipase C activity in head homogenates has been shown to be encoded by norpA, as well as antisera generated against the major gene product of norpA to examine its subcellular distribution before and during phototransduction. We find that both phospholipase C activity and the norpA protein are predominantly associated with membrane fractions in heads of both light- and dark-adapted flies. Moreover, phospholipase C activity as well as norpA protein can be easily extracted from membrane preparations of light- or dark-adapted flies using high salt, indicating that the norpA protein is peripherally localized on the membrane. These data suggest that the norpA encoded phospholipase C of Drosophila is a permanent peripheral membrane protein. If this is indeed the case, then it would mean that the reversible redistribution of phospholipase C from the cytosol to the membrane, as observed in epidermal growth factor receptor stimulation of mammalian phospholipase C gamma, is not a universal mechanism utilized by all types of phosphatidylinositol-specific phospholipase C.

The rpa (receptor potential absent) visual mutant of the blowfly (Calliphora erythrocephala) is deficient in phospholipase C in the eye.

The rpa (receptor potential absent) mutation of the blowfly, Calliphora erythrocephala, reduces the light-evoked responses of photoreceptor cells and renders the fly blind. This phenotype is similar to the phenotype caused by norpA mutations in Drosophila which have been shown to occur within a gene encoding phospholipase C. In Western blots, norpA antiserum stains a protein in homogenates of wild-type Calliphora eye and head that is similar in molecular weight to the norpA protein. Very little staining of this protein is observed in similar homogenates of rpa mutant. Moreover, norpA antiserum strongly stains retina in immunohistochemical assays of wild-type adult head, but not in rpa mutant. Furthermore, eyes of rpa mutant have a reduced amount of phospholipase C activity compared to eye of wild-type Calliphora. These data suggest that the rpa mutation occurs in a phospholipase C gene of the blowfly that is homologous to the norpA gene of Drosophila.

Tissue-specific expression of phospholipase C encoded by the norpA gene of Drosophila melanogaster.

Mutations in the norpA gene of Drosophila melanogaster severely affect the light-evoked photoreceptor potential with strong mutations rendering the fly blind. Molecular cloning of the norpA gene revealed that it encodes phosphatidylinositol-specific phospholipase C, which enzymes play a pivotal role in one of the largest classes of signaling pathways known. We have used Northern analysis, Western blots, phospholipase C activity assays, and immunohistochemical staining of tissues to examine the tissue-specific expression of the norpA gene and found that it is expressed in a variety of tissues besides the eye. Hybridization of norpA cRNA probe to blots of poly(A+) RNA reveals that the gene encodes at least four transcripts: a 7.5-kilobase (kb) transcript that is expressed in eye and 6.5-, 5.5-, and 5.0-kb transcripts that are expressed in adult body or early stages of development. Antiserum generated against the major gene product of norpA recognizes a 130-kDa protein that is abundant in eyes but severely reduced or absent in norpA mutants, a result which is consistent with previous conclusions that the norpA gene encodes an essential component of the visual system. However, the norpA antiserum also recognizes a 130-kDa protein in adult legs, thorax, and male abdomen, but not female abdomen. These localizations are supported by results of phospholipase C activity assays which show that the norpA mutation reduces phospholipase C activity in each of the tissues in which norpA protein can be detected. Furthermore, immunohistochemical staining of tissue sections with the norpA antiserum demonstrates that the norpA protein is abundant in the retina and ocelli and is present to a lesser extent in the brain and thoracic nervous system. Since some of the above mentioned tissues that express norpA (such as thorax, legs, and abdomen) have no known photoreceptor tissue, we conclude that the norpA gene product is also likely to have a role in signaling pathways other than phototransduction.