Important stuff floating in our blood - tumor DNA, micro RNA, cytokines - can determine tumor burden, predict response, and side effects for melanoma patients!!!

Continuing from yesterday.....  Our blood is a fairly accessible information rich soup that can tell us all sorts of things about our bodies!!!  One important ingredient for melanoma patients is circulating tumor DNA.  Here's a load of prior reports: Circulating tumor DNA - to determine diagnosis, disease burden, response to therapy, etc  Here are a couple more reports:

Correlation between circulating tumour DNA and metabolic tumour burden in metastatic melanoma patients.  McEvoy, Warburton, Al-Ogaili, et al. BMC Cancer. 2018 Jul 9.

Circulating tumour DNA (ctDNA) may serve as a measure of tumour burden and a useful tool for non-invasive monitoring of cancer. However, ctDNA is not always detectable in patients at time of diagnosis of metastatic disease. Therefore, there is a need to understand the correlation between ctDNA levels and the patients' overall metabolic tumour burden (MTB).  Thirty-two treatment naïve metastatic melanoma patients were included in the study. MTB and metabolic tumour volume (MTV) was measured by 18F-fluoro-D-glucose positron emission tomography/computed tomography (FDG PET/CT). Plasma ctDNA was quantified using droplet digital PCR (ddPCR).  CtDNA was detected in 23 of 32 patients. Overall, a significant correlation was observed between ctDNA levels and MTB. CtDNA was not detectable in patients with an MTB of greater than/= to 10, defining this value as the lower limit of tumour burden that can be detected through ctDNA analysis by ddPCR.  We showed that ctDNA levels measured by ddPCR correlate with MTB in treatment naïve metastatic melanoma patients and observed a limit in tumour size for which ctDNA cannot be detected in blood. Nevertheless, our findings support the use of ctDNA as a non-invasive complementary modality to functional imaging for monitoring tumour burden.

From validity to clinical utility: the influence of circulating tumor DNA on melanoma patient management in a real-world setting. Rose, Luber, Makell, et al. Mol Oncol. 2018 Aug 16.

Melanoma currently lacks a reliable blood-based biomarker of disease activity, although circulating tumor DNA (ctDNA) may fill this role. We investigated the clinical utility (i.e., impact on clinical outcomes and interpretation of radiographic data) of measuring ctDNA in patients with metastatic or high-risk resected melanoma. Patients were prospectively accrued into greater than/= to 1 of 3 cohorts, as follows. Cohort A: patients with radiographically-measurable metastatic melanoma who underwent comparison of ctDNA measured by a BEAMing digital PCR assay to tissue mutational status and total tumor burden; when appropriate, determinations about initiation of targeted therapy were based on ctDNA data. Cohorts B and C: patients with BRAF- or NRAS-mutant melanoma who had either undergone surgical resection of high-risk disease (cohort B) or were receiving or had received medical therapy for advanced disease (cohort C). Patients were followed longitudinally with serial ctDNA measurements with contemporaneous radiographic imaging to ascertain times to detection of disease activity and progressive disease, respectively. The sensitivity and specificity of the ctDNA assay was 86.8% and 100%, respectively. Higher tumor burden and visceral metastases were found to be associated with detectable ctDNA. In 2 patients in cohort A, ctDNA test results revealed a targetable mutation where tumor testing had not; both patients experienced a partial response to targeted therapy. In 4 of 30 patients with advanced melanoma, ctDNA assessments indicated evidence of melanoma activity that predicted radiographic evidence of disease progression by 8, 14, 25 and 38 weeks, respectively. CtDNA was detectable in 3 of these 4 patients coincident with radiographic evaluations that alone were interpreted as showing no evidence of neoplastic disease. Our findings provide evidence for the clinical utility of integrating ctDNA data in managing patients with melanoma in a real-world setting.

Among the many bio-markers present in our blood, there's also circulating RNA.   Prior reports:  RNA and biomarkers generally  Now, this:

Extracellular microvesicle microRNAs as predictive biomarkers for targeted therapy in metastastic cutaneous malignant melanoma.  Svedman, Loncharoenkal, Bottaj, et al. PLoS One. 2018 Nov.

Mitogen activated-protein kinase pathway inhibitors (MAPKis) improve treatment outcome in patients with disseminated BRAFV600 mutant cutaneous malignant melanoma (CMM) but responses are of limited duration due to emerging resistance. Although extensive research in mechanisms of resistance is being performed, predictive biomarkers for durable responses are still lacking. We used miRNA qPCR to investigate if different levels of extracellular microvesicle microRNA (EV miRNA) in matched plasma samples collected from patients with metastatic IV BRAFV600 mutated CMM before, during and after therapy with MAPKis could serve as predictive biomarkers.  EV miRNAs were extracted from plasma samples from 28 patients collected before and during therapy, measured by quantitative PCR-array and correlated to therapy outcome. Increased levels of EV let-7g-5p during treatment compared to before treatment were associated with better disease control with MAPKis. Elevated levels of EV miR-497-5p during therapy were associated with prolonged progression free survival (PFS).  EV miRNAs let-7g-5p and miR-497-5p were identified as putative novel predictive biomarkers of MAPKi treatment benefit in metastatic CMM patients highlighting the potential relevance of assessing EV miRNA during and after treatment to unravel novel mechanisms of resistance.

Circulating cytokines are substances like interleukin, interferon, and growth factors, that cells in our immune system secrete in order to affect other cells. Here's a report for a little background:  Eosinophilia with Nivo and Pembro - A predictor of success?!!
Here they are being examined as predictors of toxicity to immunotherapy:

Circulating cytokines predict immune-related toxicity in melanoma patients receiving anti-PD-1-based immunotherapy. Lim, Lee, Gide, et al. Clin Cancer Res. 2018 Nov 8.  

Combination PD-1 and CTLA-4 inhibitor therapy has dramatically improved the survival of patients with advanced melanoma but is also associated with significant immune-related toxicities. This study sought to identify circulating cytokine biomarkers of treatment response and immune-related toxicity.  The expression of 65 cytokines was profiled longitudinally in 98 melanoma patients treated with PD-1 inhibitors, alone or in combination with anti-CTLA-4, and in an independent validation cohort of 49 patients treated with combination anti-PD-1 and anti-CTLA-4. Cytokine expression was correlated with RECIST response and immune-related toxicity, defined as toxicity that warranted permanent discontinuation of treatment and administration of high dose steroids.

Eleven cytokines were significantly upregulated in patients with severe immune-related toxicities at baseline and early during treatment. The expression of these eleven cytokines was integrated into a single toxicity score, the CYTOX score, and the predictive utility of this score was confirmed in the discovery and validation cohorts. 

The CYTOX score is predictive of severe immune-related toxicity in melanoma patients treated with combination anti-CTLA-4 and anti-PD-1 immunotherapy. This score, which includes pro-inflammatory cytokines such as IL-1a, IL-2 and IFNa2, may help in the early management of severe, potentially life-threatening immune-related toxicity.

Hoping for much more consistency and use of these relatively simple tests in order to reap better treatment outcomes for melanoma patients in 2019!!! - c

Circulating Tumor Cells...how they may eventually impact melanoma diagnosis and response to therapy

Circulating Tumor Cells, DNA, and mRNA: Potential for Clinical Utility in Patients With Melanoma.

Xu, Dorsey, Amaravadi, et al.  Oncologist. 2015 Nov 27.

Circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), and messenger RNA (mRNA), collectively termed circulating tumor products (CTPs), represent areas of immense interest from scientists' and clinicians' perspectives. In melanoma, CTP analysis may have clinical utility in many areas, from screening and diagnosis to clinical decision-making aids, as surveillance biomarkers or sources of real-time genetic or molecular characterization. In addition, CTP analysis can be useful in the discovery of new biomarkers, patterns of treatment resistance, and mechanisms of metastasis development. Here, we compare and contrast CTCs, ctDNA, and mRNA, review the extent of translational evidence to date, and discuss how future studies involving both scientists and clinicians can help to further develop this tool for the benefit of melanoma patients.

This article doesn't exactly provide any new or particularly useful data.  However, it does make one hopeful that work in this area will continue to advance so that we can have simple, standardized and reliable tests to measure and diagnose tumor status.  What a boon it would be to be able to diagnosis and follow tumor load and type, in order to better understand current status in patients, as well as response to therapy, without scans and the radiation, dye exposure and red herrings inherent in them.  Mostly, it is a call for more studies like the one discussed here: PCR Testing for melanoma

Best - c

Biomarkers - blood components, circulating tumor cells AND of the tumor itself

Biomarkers.  Sounds important.  What are they?  What can they really tell us? 

Researchers have been seeking to find a connection between nonspecific biomarkers (things we ALL have circulating in our blood) like LDH, various white blood cells (neutrophils and monocytes - whether as an absolute count or as a neutrophil to lymphocyte ratio), myeloid-derived suppressor cells (MDSCs) and T-regs (See post with links about all that here:   Blood markers associated with clinical outcomes).  And while many meaningful correlations have been drawn between these components and response to treatment (or lack thereof); the fact that they are all affected by numerous circumstances other than melanoma and its treatment, create limitations in the clarity with which they can be used to PREDICT responses.

With recent scientific advances, PCR testing (Polymerase chain reaction) has become more efficient and cost-effective, allowing labs to copy or "amplify" small segments of DNA or RNA though screening blood or other biological specimens.  This ability allows the cellular identification of whatever may be floating in that blood sample....whether it is a virus or fungus, or actual bits and pieces of tumor cells themselves, to allow a determination of  disease burden, prognosis, and response to treatment.  Here is an earlier post:  Circulating tumor cells: how they may eventually impact melanoma diagnosis and evaluation of response

Now there are these reports:

Liquid biopsy utility for the surveillance of cutaneous malignant melanoma patients.  Haung and Hoon.  Mol Oncol. 2015 Dec 17.

Cutaneous melanoma is one of the highest incident-rate cancers with increasing prevalence in Western societies. Despite the advent of new approved therapeutics, the 5-year overall survival rate of stage IV melanoma patients remains below 15%. Current treatments for late stage disease have shown higher efficacy when treated at a lower disease burden. Thus, blood-based biomarkers capable of detecting melanoma prior to clinically evident distant metastasis, will improve the treatment and outcomes for melanoma patients. To that end, effective treatment of melanoma necessitates identification of patients at risk for developing distant metastases. Furthermore, employing blood biomarkers that monitor cancer progression over the course of treatment is a promising solution to post-treatment drug resistance often developed in melanoma patients. Non-invasive blood biomarker assays allow for regular dynamic monitoring of disease. "Liquid Biopsy" of blood, which exploits circulating tumor cells (CTCs), cell-free circulating tumor DNA (ctDNA) and cell-free circulating microRNA (cmiRNA), has been shown to detect prognostic factors for relapse in AJCC stage III and stage IV melanoma patients. Moreover, molecular characterization of CTC and analysis of various forms of ctDNA present promising potential in development of individualized therapy for melanoma patients. New approaches such as massive parallel sequencing (MPS) provide a comprehensive view of the disease progression, allowing for the selection of therapeutic options for individual patients. With advancements of improving molecular assays, liquid biopsy analysis as a powerful, routine clinical assay for melanoma patients, is highly promising prospective.

Circulating melanoma cells as a potential biomarker to detect metastasis and evaluate prognosis.  Hida, Yoneta, Wakamatsu, et al.  Australas J Dermatol. 2016 Mar 1.

TNM staging is mainly used to evaluate the prognosis of melanoma patients. Serum biomarkers such as 5-S-cysteinyldopa (5-S-CD) have occasionally been used but most do not respond until the tumour burden becomes high. Recently, circulating melanoma cells (CMC) have been reported as a possible new biomarker to detect metastasis, monitor treatment response and predict prognosis. The object of this exploratory study was to evaluate the efficacy of CMC to detect metastasis and predict prognosis by cross-sectional and prospective observational analyses, respectively. Altogether 15 patients with stages II-IV melanoma were enrolled and CMC were enumerated by CellSearch system with cut-off values of two cells/7.5 mL. Serum 5-S-CD and lactate dehydrogenase (LDH) were also measured. The sensitivity of CMC and 5-S-CD for the detection of metastasis was 33 and 50%, respectively. The combination of CMC and 5-S-CD showed a sensitivity of 67%, the best performance among CMC, 5-S-CD, LDH and any combination of two of the markers. Additionally, a 30-month prospective observation showed that CMC could segregate patients with poorer prognosis. The median survival time for the patients with less than 2 CMC and those with greater than or equal to 2 CMC was 19.5 and 4.5 months, respectively. The limitation of this study is the small sample size. These preliminary results indicate CMC may complement the efficacy of 5-S-CD to detect metastasis and can be a prognostic marker. Although there is still room for improvement to maximize the sensitivity, the CellSearch system is reproducible, standardized and suitable for multi-center studies.

Then, there's the examination of the properties of the tumor sample itself.  This article talks about looking at the tumor in regard to how well it is being recognized by the immune system....specifically t-cells:
 

Melanoma-specific MHC-II expression represents a tumour-autonomous phenotype and predicts response to anti-PD-1/PD-L1 therapy.  Johnson, Estrada, Salgado, Sosman, et al.  Nat Commun.  2016 Jan 29.

Anti-PD-1 therapy yields objective clinical responses in 30-40% of advanced melanoma patients. Since most patients do not respond, predictive biomarkers to guide treatment selection are needed. We hypothesize that MHC-I/II expression is required for tumour antigen presentation and may predict anti-PD-1 therapy response. In this study, across 60 melanoma cell lines, we find bimodal expression patterns of MHC-II, while MHC-I expression was ubiquitous. A unique subset of melanomas are capable of expressing MHC-II under basal or IFNγ-stimulated conditions. Using pathway analysis, we show that MHC-II(+) cell lines demonstrate signatures of 'PD-1 signalling', 'allograft rejection' and 'T-cell receptor signalling', among others. In two independent cohorts of anti-PD-1-treated melanoma patients, MHC-II positivity on tumour cells is associated with therapeutic response, progression-free and overall survival, as well as CD4(+) and CD8(+) tumour infiltrate. MHC-II(+) tumours can be identified by melanoma-specific immunohistochemistry using commercially available antibodies for HLA-DR to improve anti-PD-1 patient selection.

Here...the tumor is being characterized by surface molecules that are correlated with prognosis:

Molecular markers to complement sentinel node status in predicting survival in patients with high risk locally invasive melanoma.  Rowe, Tang, Hughes, et al.  Int J Cancer. 2016 Mar 14. 

Sentinel lymph node status is a major prognostic marker in locally invasive cutaneous melanoma. However this procedure is not always feasible, requires advanced logistics, and carries rare but significant morbidity. Previous studies have linked markers of tumour biology to patient survival. In this study we aimed to combine the predictive value of established biomarkers in addition to clinical parameters as indicators of survival in addition to or instead of sentinel node biopsy in a cohort of high risk melanoma patients. Patients with locally invasive melanomas undergoing sentinel lymph node biopsy were ascertained and prospectively followed. Information on mortality was validated through the National Death Index. Immunohistochemistry was used to analyse proteins previously reported to be associated with melanoma survival, namely Ki67, p16, and CD163. Evaluation and multivariate analyses according to REMARK criteria were used to generate models to predict disease-free and melanoma-specific survival. 189 patients with available archival material of their primary tumour were analysed. Our study sample was representative of the entire cohort (N=559). Average Breslow thickness was 2.5 mm. 32 (17%) patients in the study sample died from melanoma during the follow-up period. A prognostic score was developed and was strongly predictive of survival, independent of sentinel node status. The score allowed classification of risk of melanoma death in sentinel node negative patients. Combining clinicopathological factors and established biomarkers allows prediction of outcome in locally invasive melanoma and might be implemented in addition to or in cases when sentinel node biopsy cannot be performed.

Here the tumor is being analyzed for specific genetic variations:   

Genomic and Transcriptomic Features of Response to Anti-PD-1 Therapy in Metastatic Melanoma.  Hugo, Zaretsky, Sun, et al.  Cell. 2016 Mar 15. 

PD-1 immune checkpoint blockade provides significant clinical benefits for melanoma patients. We analyzed the somatic mutanomes and transcriptomes of pretreatment melanoma biopsies to identify factors that may influence innate sensitivity or resistance to anti-PD-1 therapy. We find that overall high mutational loads associate with improved survival, and tumors from responding patients are enriched for mutations in the DNA repair gene BRCA2. Innately resistant tumors display a transcriptional signature (referred to as the IPRES, or innate anti-PD-1 resistance), indicating concurrent up-expression of genes involved in the regulation of mesenchymal transition, cell adhesion, extracellular matrix remodeling, angiogenesis, and wound healing. Notably, mitogen-activated protein kinase (MAPK)-targeted therapy (MAPK inhibitor) induces similar signatures in melanoma, suggesting that a non-genomic form of MAPK inhibitor resistance mediates cross-resistance to anti-PD-1 therapy. Validation of the IPRES in other independent tumor cohorts defines a transcriptomic subset across distinct types of advanced cancer. These findings suggest that attenuating the biological processes that underlie IPRES may improve anti-PD-1 response in melanoma and other cancer types.

While none of these (other than those that are simple measures of recognized blood components) are validated and ready for daily screening of cancer patients across this country....every step is important in helping docs...eventually....determine the best treatment options for the individual and monitor their response to that treatment in ways that are specific, easily measured, and less invasive and damaging to that patient.  What a beautiful day that will be!!!  Minimized only by the day we figure out how to avoid cancer entirely!  Hey....might as well live large and dream big!!! - c

More blood tests to determine prognosis as well as response to therapy in melanoma patients!!!

It is becoming more and more clear (at least to me!!!!!!!!!) that there are all sorts of ways of looking at blood markers in order to diagnose, predict progression and response, as well as evaluate response to a particular treatment in melanoma patients.  Here is my latest post with a zillion links within:  Circulating tumor DNA to help differentiate true progression of melanoma vs pseudoprogression in patients treated with anti-PD-1  Now, there's this ~

Performance of a prognostic 31-gene expression profile in an independent cohort of 523 cutaneous melanoma patients. Zager, Gastman, Leachman, et al.  BMC Cancer. 2018 Feb 5.

The heterogeneous behavior of patients with melanoma makes prognostication challenging. To address this, a gene expression profile (GEP) test to predict metastatic risk was previously developed. This study evaluates the GEP's prognostic accuracy in an independent cohort of cutaneous melanoma patients.

This multi-center study analyzed primary melanoma tumors from 523 patients, using the GEP to classify patients as Class 1 (low risk) and Class 2 (high risk). Molecular classification was correlated to clinical outcome and assessed along with AJCC v7 staging criteria. Primary endpoints were recurrence-free (RFS) and distant metastasis-free (DMFS) survival.
The 5-year RFS rates for Class 1 and Class 2 were 88% and 52%, respectively, and DMFS rates were 93% versus 60%, respectively. The GEP was a significant predictor of RFS and DMFS in univariate analysis , along with Breslow thickness, ulceration, mitotic rate, and sentinel lymph node (SLN) status . GEP, tumor thickness and SLN status were significant predictors of RFS and DMFS in a multivariate model that also included ulceration and mitotic rate .

The GEP test is an objective predictor of metastatic risk and provides additional independent prognostic information to traditional staging to help estimate an individual's risk for recurrence. The assay identified 70% of stage I and II patients who ultimately developed distant metastasis. Its role in consideration of patients for adjuvant therapy should be examined prospectively.

Think how great this sort of information would be when trying to determine whether or not to utilize adjuvant therapy!!!

But, that's not all!  There's also this ~

Molecular signatures of circulating melanoma cells for monitoring early response to immune checkpoint therapy.  Hong, Sullivan, Kalinich, et al.  Proc Natl Acad Sci U S A. 2018 Feb 16.

A subset of patients with metastatic melanoma have sustained remissions following treatment with immune checkpoint inhibitors. However, analyses of pretreatment tumor biopsies for markers predictive of response, including PD-1 ligand (PD-L1) expression and mutational burden, are insufficiently precise to guide treatment selection, and clinical radiographic evidence of response on therapy may be delayed, leading to some patients receiving potentially ineffective but toxic therapy. Here, we developed a molecular signature of melanoma circulating tumor cells (CTCs) to quantify early tumor response using blood-based monitoring. A quantitative 19-gene digital RNA signature (CTC score) applied to microfluidically enriched CTCs robustly distinguishes melanoma cells, within a background of blood cells in reconstituted and in patient-derived (n = 42) blood specimens. In a prospective cohort of 49 patients treated with immune checkpoint inhibitors, a decrease in CTC score within 7 weeks of therapy correlates with marked improvement in progression-free survival and overall survival. Thus, digital quantitation of melanoma CTC-derived transcripts enables serial noninvasive monitoring of tumor burden, supporting the rational application of immune checkpoint inhibition therapies.

Wouldn't it be great to know where you stand 7 weeks into therapy?  You wouldn't waste time (and in melanoma world that "time" can make all the difference!!!) with a therapy that is not working for you!!!

Blood draws with more detailed info than repeated radiological studies!!!  C'mon man!!!  Let's get some consensus and make these tests available for patients in need! - c

Circulating DNA (ctDNA) Yes, again! A noninvasive method of diagnosing and monitoring melanoma patients

Yep.  Another thing I have been yelling about for years.  Circulating tumor DNA.  Something we can measure in the blood of melanoma patients.  Here are zillions of reports:  Important stuff floating in our blood - tumor DNA, micro RNA, cytokines - can determine tumor burden, predict response, and side effects for melanoma patients!!!  Now, there's this:

ctDNA as a noninvasive monitoring tool in metastatic melanoma.  2019 ASCO.  Varaljai, Wistuba-Hamprecht, Seremet, et al.  J Clin Oncology 37, 2019.

Background: The field of liquid biopsy provides a promising alternative to standard tissue biopsies. Previous work has shown that plasma circulating cell-free DNA (ctDNA) can reflect the heterogeneous spectrum of mutations in cancer including metastatic melanoma. Our project aimed to establish and statistically validate plasma-based assays for tumour load and therapy monitoring in melanoma. Methods: On a large cohort of stage III and stage IV melanoma patients (N = 96) who received signalling targeted or immune checkpoint inhibitors we showed that the most common oncogenic drivers of this disease such as the BRAFV600E, NRASQ61 and the TERTC250T and TERTC228T promoter mutations (termed TERTprom) can be analysed in ctDNA with highly sensitive droplet digital PCR technology (detection of mutant ctDNA down to 0.01% analytical sensitivity). Results: Our research has demonstrated that ctDNA (irrespective of the genotype) significantly correlates with tumour stage. Using receiver operating characteristics (ROC) analyses thresholds were established for risk stratification and response prediction. Elevated ctDNA at baseline was a significant predictor of disease progression compared to elevated LDH or S100 in multivariable cox proportional hazards model. During therapy, patients with low ctDNA load (below the ROC threshold) had significantly better radiological outcomes and prolonged progression free survival (PFS) compared to patients with high ctDNA load. Our findings were confirmed on an independent cohort of metastatic melanoma patients (N = 35) treated with immune checkpoint inhibitors, where also during therapy low ctDNA load correlated with prolonged PFS. An added benefit of ctDNA was demonstrated in about 80% of the patients, where ctDNA analyses preceded the radiological diagnosis of response or relapse. Progression was detected in plasma ctDNA in average 3.5 months earlier as compared to routine imaging techniques. Finally, we demonstrated that the occurrence of NRASQ61 mutation in BRAFV600-inhibitor treated patients at therapy baseline was associated with treatment failure. The sub-clonal NRASQ61 mutation at therapy baseline was an independent predictor of shorter PFS as compared to BRAFV600E patients without the NRASQ61 mutation at therapy baseline. Conclusions: In sum, our results support the value of ctDNA as a sensitive biomarker for real-time therapy monitoring and early detection of disease progression.

It works!  Doesn't require surgical biopsy.  Shows results months sooner than radiological imaging techniques and doesn't expose the patient to radiation.  What are we waiting on??????

There is also, this:

Circulating Cell-Free DNA-Diagnostic and Prognostic Applications in Personalized Cancer Therapy.  Oellerich, Schutz, Beck, Walson.  Ther Drug Monit. 2019 Apr.

Genomic analyses in oncologic care allow for the development of more precise clinical laboratory tests that will be critical for personalized pharmacotherapy. Traditional biopsy-based approaches are limited by the availability of sequential tissue specimens to detect resistance. Blood-based genomic profiling ("liquid biopsy") is useful for longitudinal monitoring of tumor genomes and can complement biopsies. Tumor-associated mutations can be identified in cell-free tumor DNA (ctDNA) from patient blood samples and used for monitoring disease activity. The US Food and Drug Administration approved a liquid biopsy test for EGFR-activating mutations in patients with non-small-cell lung cancer as a companion diagnostic for therapy selection. ctDNA also allows for the identification of mutations selected by treatment such as EGFR T790M in non-small-cell lung cancer. ctDNA can also detect mutations such as KRAS G12V in colorectal cancer and BRAF V600E/V600K in melanoma. Chromosomal aberration pattern analysis by low-coverage whole genome sequencing is a new, broader approach. Genomic imbalances detected in cell-free DNA (cfDNA) can be used to compute a copy number instability (CNI) score. In clinical studies, it was demonstrated that the change in CNI score can serve as an early predictor of therapeutic response to chemotherapy/immunotherapy of many cancer types. In multivariable models, it could be shown that the CNI score was superior to clinical parameters for prediction of overall survival in patients with head and neck cancer. There is emerging evidence for the clinical validity of ctDNA testing regarding identification of candidates for targeted therapies, prediction of therapeutic response, early detection of recurrence, resistance mutation detection, measuring genetic heterogeneity, tumor burden monitoring, and risk stratification. Improvement of sensitivity to detect tumors at very early stages is difficult due to insufficient mutant DNA fraction of less than/= to 0.01%. Further developments will include validation in prospective multicenter interventional outcome studies and the development of digital platforms to integrate diagnostic data.

The technology exists to make this simple test available - impacting melanoma patients in lots of ways!  Let's make it so!!!

And, yes.  There is still beauty -

 - despite the storm.  ~ les

PCR testing for melanoma

Polymerase chain reaction (PCR) is a technique that is used to amplify trace amounts of DNA (and in some instances, RNA) located in or on almost any liquid or surface where DNA strands may be deposited.  It is an exceedingly sensitive technique to detect molecules.  In everyday medicine it can be used to check for the presence of a wide variety of substances - pertussis from a nasal swab, chlamydia and gc in urine, viral load of HIV patients from their blood sample, fungus in skin scrapings, and even genetic mutations.  It would be super cool if we could utilize this technique to accurately identify the presence and characteristics of melanoma cells....both for diagnosis as well as monitoring effects of treatment.  Here are some peeps who are working on it:

SNPase-ARMS qPCR:  Ultrasensitive Mutation-Based Detection of Cell-Free Tumor DNA in Melanoma Patients.  PLoS One. 2015 Nov 12.  Stadler, Eder, Pratscher, et al.

"Cell-free circulating tumor DNA in the plasma of cancer patients has become a common point of interest as indicator of therapy options and treatment response in clinical cancer research. Especially patient- and tumor-specific single nucleotide variants that accurately distinguish tumor DNA from wild type DNA are promising targets. The reliable detection and quantification of these single-base DNA variants is technically challenging. Currently, a variety of techniques is applied, with no apparent "gold standard". Here we present a novel qPCR protocol that meets the conditions of extreme sensitivity and specificity that are required for detection and quantification of tumor DNA. By consecutive application of two polymerases, one of them designed for extreme base-specificity, the method reaches unprecedented sensitivity and specificity. Three qPCR assays were tested with spike-in experiments, specific for point mutations BRAF V600E, PTEN T167A and NRAS Q61L of melanoma cell lines. It was possible to detect down to one copy of tumor DNA per reaction , at a background of up to 200 000 wild type DNAs. To prove its clinical applicability, the method was successfully tested on a small cohort of BRAF V600E positive melanoma patients."

Still needs to be tested on a greater scale...but sounds like progress. - c

Simple blood tests that tell us how melanoma patients are responding to therapy and identify their disease in the first place....AGAIN!!!!

Yep.  It's me again, Margaret.  I'm still yelling about simple blood draws that can provide critical information related to the diagnosis of, feasibility of treatment for, response to treatment, and prognosis generally in melanoma patients!  Just last week I posted this:  More blood tests to determine prognosis as well as response to therapy in melanoma patients!!!

Now, there's this....

Clinical response to PD-1 blockade correlates with a sub-fraction of peripheral central memory CD4+ T cells in patients with malignant melanoma. Takeuchi, Tanemura, Tada, et al.  Int Immunol. 2017 Dec 23.

Cancer immunotherapy that blocks immune checkpoint molecules, such as PD-1/PD-L1, unleashes dysfunctional antitumor T-cell responses and has durable clinical benefits in various types of cancers. Yet its clinical efficacy is limited to a small proportion of patients, highlighting the need for identifying biomarkers that can predict the clinical response by exploring antitumor responses crucial for tumor regression. Here we explored T-cell responses associated with clinical benefits using peripheral blood mononuclear cells (PBMCs) from patients with malignant melanoma treated with anti-PD-1 monoclonal antibody (mAb). Pre- and post-treatment samples were collected from two different cohorts (discovery set and validation set) and subjected to mass cytometry assays that measured the expression levels of 35 proteins. Screening by high dimensional clustering in the discovery set identified increases in three micro-clusters of CD4+ T cells, a subset of central memory CD4+ T cells harboring CD27+FAS-CD45RA-CCR7+ phenotype, after treatment in long-term survivors, but not in non-responders. The same increase was also observed in clinical responders in the validation set. We propose that increases in this subset of central memory CD4+ T cells in peripheral blood can be potentially used as a predictor of clinical response to PD-1 blockade therapy in patients with malignant melanoma.

Here researchers looked at the blood of folks before and after patients with melanoma were treated with anti-PD-1.  They found that a particular type of CD4+ T cell increased in long term survivors and clinical responders...but NOT in non-responders.

That seems pretty straight forward to me!  But, there's more....

A high neutrophil to lymphocyte ratio prior to BRAF inhibitor treatment is a predictor of poor progression-free survival in patients with metastatic melanoma. Finon, Zaragoza, Maillard, et al. Eur J Dermatol. 2018 Jan 16.

Some studies have shown that a high neutrophil/lymphocyte ratio (NLR) greater than or = to 4 before initiating ipilimumab treatment is an independent prognostic indicator of poor survival in patients with metastatic melanoma (MM). To determine whether the NLR before starting BRAF inhibitor (BRAFi) treatment in patients with (MM) is associated with progression-free survival (PFS). This retrospective study included 49 patients consecutively receiving BRAFi for MM between July 2012 and December 2014. Cox proportional hazards regression was used to analyse the relationship between NLR and other factors, such as lactate dehydrogenase (LDH), performance status, BRAFi as first- or second-line therapy, and corticosteroid intake with PFS. The NLR before starting BRAFi was significantly associated with PFS based on univariate analysis and multivariate analysis adjusted for potential confounding factors, such as LDH activity, ulceration, performance status, first-line therapy, and corticosteroid intake. A high NLR (continuous variable) was associated with short PFS, and NLR greater than or = to 4 was associated with shorter PFS . Corticosteroid intake was not associated with short PFS based on multivariate analysis. An NLR greater than 4, before starting BRAFi treatment, is an independent prognostic indicator of poor progression-free survival.

SO.....again ~ folks with a high NLR (greater than or = to 4) did worse!!!  This is NOT news people.  I've been yelling about this since 2015!!!

That fact was valid when the NLR was tested in folks without metastatic disease ~  Neutrophil-to-lymphocyte ratios as a predictor of death from melanoma in patients with NONmetastatic disease
That fact was valid when the NLR was examined in regard to ipi (as noted in the above article) ~  Neutrophils as a prognostic predictor in patients trated with ipi/Yervoy
That fact was valid when first postulated in 2015 ~  Lab values that may predict response to Ipi/Yervoy????

Do we really need to examine that any further???  There is also this...

Development and validation of a plasma-based melanoma biomarker suitable for clinical use.  Van, Lincoln, Van Laar.  Br J Cancer. 2018 Jan 23.

In Australia, more money is spent on skin cancer than any other malignancy. Despite this, the mortality rate of melanoma, the deadliest form, has steadily increased over the past 50 years. Diagnostic imprecision and a lack of complimentary molecular biomarkers are partially responsible for this lack of progress.

Whole-microRNAome profiling was performed on plasma samples from 32 patients with histologically confirmed melanoma and 16 normal controls. A classification algorithm was trained on these data and independently validated on multiple previously published microRNA data sets, representing (i) melanoma patient- and normal-blood, (ii) melanoma and nevi biopsy tissue, and (iii) cell lines and purified exosomes.

38 circulating microRNAs had biologically and statistically significant differences between melanoma and normal plasma samples (MEL38). A support vector machine algorithm, trained on these markers, showed strong independent classification accuracy (AUC 0.79-0.94). A majority of MEL38 genes have been previously associated with melanoma and are known regulators of angiogenesis, metastasis, tumour suppression, and treatment resistance.

MEL38 exhibits disease state specificity and robustness to platform and specimen-type variation. It has potential to become an objective diagnostic biomarker and improve the precision and accuracy of melanoma detection and monitoring.

Here folks looked at blood samples for microRNA whose presence helped define the difference between melanoma and normal plasma.  

I have endless posts regarding the examination of tumor DNA in the blood of melanoma patients, allowing for the identification of disease, BRAF status, progression, response, etc.  Earlier this month, I posted this:  Circulating tumor DNA to help differentiate true progression of melanoma vs pseudoprogression in patients treated with anti-PD-1  Here are about a zillion additional posts:  Circulating melanoma DNA

Really people!  Can someone not do a definitive study on a larger scale...rather than piecemeal this and that....without sharing data...to come up with a valid and specific melanoma panel???  We have simple lipid panels that help guide heart health.  We have lab draws and tests that help diagnosis and monitor diabetes.  No set of labs is an absolute for ANYTHING.  BUT!!!!  They are incredibly important tools in diagnosing, treating and managing a wide variety of disease processes.  They can do that for melanoma, too!!!   We have learned that we can look at T cells, other basic blood cells, tumor DNA, RNA, all sorts of bits and bobs....floating in the blood of patients with melanoma, to provide meaningful information that could allow melanoma patients and their doctors a mechanism for making better choices in their treatment and care.

Radiologic scans expose us to radiation, "find" lots of red herrings, and are a huge financial burden.  They also have limits regarding what they can see.  Sub-centimeter tumors, not to mention little demon melanoma cells floating about, are not visualized.  Additionally, imaging studies are cumbersome and have a built in time lag that can delay needed therapy or a change in current therapy as patients wait for "their next scan" to be compared to a prior one in order to define disease status.  Determining pathology from tumor samples is often confusing.  I had to have my initial slides examined by 3 pathologists to gain consensus that I was in fact dealing with melanoma.  Folks need and deserve answers. A basic algorithim that incorporates all forms of investigation including blood markers, nuclear, radiographic, and tissue examination needs to be established for melanoma. NOW!!!

Seriously..NOW!!! - c