LRG Partners With Columbia University Medical Center, New York-Presbyterian on New Research Project

New York, NY & Wayne, NJ – Columbia University Medical Center, NewYork-Presbyterian and the Life Raft Group (LRG), a patient advocacy organization specializing in GIST cancer, have announced that they have entered into a collaborative research project to investigate the efficacy of a novel system biology approach for identifying the best treatment options for patients with advanced gastrointestinal stromal tumors (GIST).

The science behind the approach, developed in the Califano Lab at Columbia University, utilizes VIPER algorithm software (Virtual Inference of Protein activity by Enriched Regulon analysis) to investigate the molecular networks of GIST patients that have become resistant to approved tyrosine kinase inhibitors. Although oncogene targets are already established in GIST, this will identify the master regulators or “tumor-checkpoints” that represent the final on and off switches in the GIST cells. Personalized therapeutic agents can then be selected for patients currently lacking any effective therapeutic options.

Clinical and molecular data from the study participants will be stored in the Life Raft Group’s Patient Registry, a unique data management analytics tool developed by the LRG which tracks a patient’s clinical history and links it to a companion record of tissue and mutational data housed in the LRG’s Tissue Bank. The project will launch with the mapping of tissue samples donated by patients to the LRG.

Norman Scherzer, left, Executive Director of the
Life Raft Group, and Gary K. Schwartz, MD, Chief of the Division of Hematology and Oncology at New York Presbyterian/Columbia University Medical Center. The two groups have announced a collaborative effort to manage data on Gastrointestinal Stromal Tumors (GIST).

The LRG will also serve as the monitoring arm of the study and use their proprietary research collaboration platform, InterGR, to provide investigators a centralized repository for all data collected.

The collaboration is an example of the vital role patient advocacy groups play in bridging the gap between researchers and motivated patient populations who want to share their clinical histories and tissue to create new treatments and better outcomes.

According to Gary K. Schwartz, MD, Chief of NewYork-Presbyterian/Columbia University Medical Center’s Division of Hematology and Oncology who is spearheading the project, “I think this could be monumental. It shows how a major patient advocacy group and academic centers can work so closely together.“

Norman Scherzer, Executive Director of the Life Raft Group, said, “We agree that this is a historic occasion, and are both impressed and grateful for Columbia’s initiative in recognizing the importance of working with the patient community. We hope that this collaboration will serve as a model for other academic medical institutions. Following the inspiration of the Cancer Moonshot Initiative, this project illustrates high-level collaboration between a major academic medical research center and a patient advocacy group in action. This has incredible potential for GIST patients and presents a great opportunity to showcase the impact patient-powered science can make on cancer research.”

Collaboration begins with six other academic institutions including Fox Chase Cancer Center, Oregon Health & Science University, University of California San Diego, University of Miami, Washington University and Stanford University.  GSJ

 

 

 

The National Leiomyosarcoma Foundation, Empowering Patients, Encompassing Multiple Programs

The National Leiomyosarcoma Foundation was incorporated in 2001. The original patient/caregiver gatherings of support began in 1997 as a “HugFest” annual event. Today, the NLMSF is a robust patient/family/advocacy support organization that focuses on empowering patients diagnosed with Leiomyosarcoma within the cancer/sarcoma ecosystem.

The empowerment support offered by the Foundation encompasses multiple annual patient education programs throughout the United States and in 2018, Canada and the UK.  The Foundation collaborates with researchers and cancer research/treatment centers to ensure that LMS education opportunities are available to patients, their families and caregivers. Knowledge is power = Patient Power….to know more is to be able to ask the right questions and enhance one’s oncology appointment and strengthen the care team experience.

Funding the much needed LMS research is another important component of the mission and purpose of the NLMSF. Standing behind the researchers who give hope to extend survivorship through the much needed breakthrough research efforts – to accelerate advancement in treatment options is imperative. The average survival time is 5 years from diagnosis.

The National Leiomyosarcoma Foundation represents the LMS Community through its support programs as follows:

  • LMS Live Connect With Fellow Patients/Caregivers – monthly conference call in, building support, strength, courage, hope, and inspiration in sharing stories, treatment information and experiences.
  • Connect With  a Clinician Program – through the Foundation’s Facebook, patients have access to world renowned clinicians who provide general answers to the general questions posted by patients. This program does not take the place of a patient’s own oncologist or care team (disclaimer specifies this).
  • Annual Patient-Family/Caregiver LMS Education Program – throughout the year, across the US, several educational symposia are offered, to learn more about research updates, clinical trial updates, psychosocial as well as integrative medicine/nutrition guidance information is provided. The opportunity to connect with fellow patients, and experts in their field of medicine/oncology is the additional importance of such programs.
  • NLMSF E-Bulletins are posted  weekly for information updates on research, clinical trials, general medical summaries, as well as well being tips and information. Sometimes the E Bulletins are posted twice a week.The NLMSF Website:  www.nlmsf.org is the Patient Compass for a new diagnosis, navigating treatment, after treatment/survivorship. Guidance on what to ask, who to ask, when to ask for each step of the journey, plus a myriad of patient help resource information from financial assistance, to lodging for out of area appointments, meal preparation assistance, housecleaning assistance and more. Updates on research/medical information and clinical trials are continuously posted on the website. The Medical Advisory Committee of world renowned researchers  are supportive of the Foundation through their work in review of LMS specific research proposals received.
  • SideEQ – Patient Side Effects Information Program  -Collaboration and partnership with the The Life Raft Group, will facilitate the ability for patients to access information about possible side effects of drug therapy protocols and better prepare themselves for potential side effects as a result. This program became available to patients in April of this year.
  • Survivorship Program Information – The American Society of Clinical Oncology (ASCO) Survivorship Care Plan provides information and guidance for a smooth transition from end of treatment to survival mode. Visit www.nlmsf.org – your LMS Patient Compass for support and vital information.
  • Collaboration in Partnership – The NLMSF is in partnership with several organizations that offer additional support of enhancing quality of life/well-being of patients and their families.

 

The NLMSF is working with Pattern.org to empower patients to direct excess cancer tissue through the Rare Cancer Research Foundation’s Pilot Cell Line Project. The Foun- dation has done its due diligence, canvassing for input by researchers who are interested in the Foundation’s pursuit of this initiative in order to broaden their research capabilities for LMS Patients. The NLMSF was selected as the entity to represent the Leiomyosarcoma Community.

  • THE NLMSF was instrumental in collaborating for federal legislation to bring about Congressional approval for the  National Leiomyosarcoma Awareness Day, through the coordination efforts of NLMSF Legislative Liaison, Steve Baker, Mayor Pro Tem of Berkley, MI. Senator D. Stabenow, (D-MI) brought about the Congressional Resolution for the 2016 National Leiomyosarcoma Awareness Day. The Foundation is working once again for continuous annual recognition of this rare cancer/rare form of sarcoma as well as introduce a Resolution for a National Sarcoma Awareness Day or Week for hopeful Congressional support and 2017 approval.

Sarcoma is 1% of all cancers. There are approximately 50 – 100 sarcoma subtypes. Leiomyosarcoma is 13% – 17% of all sarcoma subtypes. There are approximately 5 Leiomyosarcoma subtypes, adding to the incredible complexity of this sarcoma subtype that creates the difficulty of its research to advance immunotherapy treatment and sustainability of treatment options.

Annie Achee
The National Leiomyosarcoma Foundation
http://www.nlmsf.org –
The Patient/Family/Care-partner Compass
Moving Forward Together –  Focusing on the Future –  in the Quest for a Cure!

.  .  .  .  .  .  .  .

Leiomyosarcoma – A Rare Disease With a Common Need
The NLMSF Patient/Family and Research Focus is a “Mission That Matters”

  • Patient/Family/Caregiver LMS Education Programs across the United States and abroad
  • Patient Resource Assistance and Advocacy
  • Foundation Website – THE PATIENT COMPASS
  • Leiomyosarcoma Research Funding

LMS Education Programs:  Provides current information on latest treatment strategies, clinical trial information, ongoing and future research efforts,  nutrition, physical and emotional wellbeing recommendations, as well as opportunity for connection opportunities with fellow patients as well as world renowned clinicians/experts in their medical field. Please visit www.nlmsf.org to access information about scheduled programs throughout the year.

Patient Advocacy – The Foundation represents the voices of patients and their families at annual oncology society conferences,  annual sarcoma conferences hosted by other cancer centers/institutes,  and patient advocacy summits / conferences. Additionally, the Foundation is in partnership with Cure Magazine, writing periodic articles that are posted online by CURE, and also sent to OncNursesNews.

The NLMSF coordinated efforts with the Senate starting in 2016 to successfully bring about Congressional approval for the first 2016 National Leiomyosarcoma Awareness Day.  The Foundation coordinates efforts annually to encourage ongoing Senate and Congressional support for subsequent yearly national awareness recoginition of this rare cancer/ sarcoma.

Contact Information:
Annie Achee, President, NLMSF  (303) 783-0924  or annieachee@aol.com
Together We Are Stronger –  Moving Forward . .  .
Focusing on the Future .  . ..Opening Doors .  . . .
Reaching for the Cure!

.  .  .  .  .  .  .  .

Foundation Mission and Focus:

  • Patient-Family/Caregiver Education And Support
  • Patient Advocacy Resource Program Collaboration
  • LMS Research Funding Support
  • Organizational Collaborative Partnerships – Bridging Patient
  • Resource Assistance

Connect With a Clinician Program – A Q & A opportunity for patients to post a general question to a world renowned clinician (one of 6 volunteers), and receive a general answer. This does not take the place of one’s own medical care team (disclaimer specification on the website). A highly successful support offered through the Foundation’s Facebook. The Facebook has three components: communication with Foundation followers;  the Q & A which can be public or private;  and the Blog.

The NLMSF / Rare Cancer Research Foundation Partnership
Collaboration with the Rare Cancer Research Foundation (RCRF) – a partnership to represent the LMS Community for the Pilot Cell Line Project – a program that promotes amplification of tumor tissue after surgery to amplify and accelerate research efforts. Researchers surveyed are in favor of this program, which has been connected to the NIH.

The NLMSF World Renowned Medical Advisory Committee
This committee of researchers/clinicians represent some of the best in the world. They are charged with vetting research project requests for funding by the Foundation. This is an important donation stewardship component of the Foundation, to ensure that all “investments in research” are used for the most promising LMS specific research projects.

NLMSF Weekly Bulletins
• Weekly information updates on research news, clinical trial listings, well-being articles, and more
• Communication of updates to the ACOR and Smart Patients Group, as well as community Patient Support Groups
• Article submissions by the Foundation to: Cure Magazine and Global Genes.

The NLMSF Website and Facebook Group –
All effective and helpful communication tools to convey the latest in information updates through the daily research efforts of volunteers.

Collaborative Partnerships With Other Sarcoma/Cancer Organizations

  • To enhance/expand patient resource assistance for patients and their families
  • Stronger together in mission and purpose on behalf of patients/families/caregivers

LMS Research Support
Through the Foundation’s Medical Advisory Committee, comprised of world renowned research clinicians, LMS specific research projects are reviewed and recommended for funding, once such projects are established as holding great promise for advancing treatment options in the future. Co-funding research projects are an ongoing goal of the Foundation. To date, several research projects have been co-funded, with more under current review. Additionally, the Foundation’s goal is to incentivize young resear-chers to continue their interest in LMS specific research endeavors, through monetary recognition of their published studies. GSJ

 

 

GSJ Interview
Liquid Biopsy May Help Guide Treatment Decisions for Advanced Solid Tumors

Blood Test Offers a Non-Invasive Alternative
to Tissue Biopsy

A study presented at one of the recent ASCO Scientific Symposia highlights the potential of liquid biopsy to help guide treatment decisions. The fact that genomic mutations vary not only from patient to patient, but also change over time has been a constant challenge in cancer treatment, especially in he precision medicine era, according to Sumanta Kumar Pal, MD, ASCO expert in developmental therapeutics. “Having a good, reliable option beyond a tumor biopsy could have a major impact on our ability to select the right therapy for the right patient. In this interview Philip Mack, PhD, reviews essential information on a blood test that could offer a noninvasive alternative to tissue biopsies. Dr. Mack is Professor and Director of Molecular Pharmacology at the University of California Davis Comprehensive Cancer Center, Sacramento, California. A large-scale genomic analysis has found that patterns of genetic changes detected in blood samples (liquid biopsy) closely mirror those identified in traditional tumor biopsy. With blood samples from more than 15,000 patients and 50 different tumor types, this is one of the largest cancer genomics studies ever conducted.

Q. How would you characterize the overall value and applicability of liquid biopsy?

Dr Mack: Findings suggest that analysis of shed tumor DNA in patient blood, also known as a liquid biopsy, can be a highly informative, minimally-invasive alternative when a tissue biopsy is insufficient for genotyping or cannot be obtained safely. Moreover, this test, known as Guardant360 – a digital next-generation sequencing panel, provides an unparalleled opportunity to monitor changes in the cancer as it evolves over time, which can be critical when patients and physicians are discussing treatment options for continued tumor control.

Q. What are the most widely used applications of liquid biopsy currently?

Dr Mack: The clinical use so far has mostly been in non-small cell lung cancer, but it also has had application in colorectal cancer, breast cancer and other tumor types. There have been additional applications using it to track biological markers.

Q. Do you see a potential application to soft-tissue sarcomas?

Dr Mack: Yes. Since there are molecularly targeted agents being used in this disease, it would have potential value in this setting. Perhaps most importantly, there is a good chance that liquid biopsy can be used to detect emergent resistance mechanisms, that could help assign effective therapies.

Q. What about speculation that liquid biopsy could ultimately replace tissue biopsy? Will tissue biopsy become obsolete?

Dr Mack:  ctDNA results will not necessarily replace tissue biopsy but will be an additional tool. I think there is always going to be a role for tissue-based histology. Tumor biopsy will remain the gold standard, as it yields important information about morphology, tumor type, possible site of origin, and somatic mutations. At least in the foreseeable future, that will always be required. Additionally, in about 15% of patients, no tumor DNA can be detected by the ctDNA test. There are simply tumors that do not shed DNA into the circulation at identifiable levels and tissue biopsy will be required to genotype those tumors. More likely, liquid biopsy will be used when tissue is of insufficient quality or quantity to allow a broader array of testing. Probably, the biggest benefit of plasma analysis is its convenient serial collection of samples over time. As the cancer continues to spread and the patients are progressing on therapies, their tumors are evolving, and we will need rapid, convenient ways to monitor genetic changes in those cancers.

Q. Can you further delineate the role of liquid biopsy in terms of identifying the genetic changes in a tumor as well as other related advantages that clinicians need to be aware of?

Dr Mack: A liquid biopsy can be used periodically to monitor disease progression, response to therapy, and development of treatment resistance. If a repeat test suggests that the cancer is progressing or becoming resistant to treatment, physicians can modify the treatment plan. Periodic liquid biopsy, which requires a simple blood draw, may be preferable to repeat tissue biopsy in terms of patient safety and convenience. In addition, because genetic changes in ctDNA often occur before signs of tumor growth are apparent on a scan, liquid biopsy can help doctors adjust treatment sooner.

Liquid biopsy has another important advantage over tissue biopsy. The genetic changes that drive tumor growth often differ in different parts of the tumor. Because tissue biopsy removes only small pieces of the tumor, key mutations can be missed, depending on what area of the tumor is sampled. Analysis of ctDNA provides information on all the different genetic changes that may be present in the tumor. Detection of tumor-specific mutations in a blood draw is an attractive alternative when tissue biopsies are not feasible. It is easy to do in any clinic and avoids biopsy-related complications. The ctDNA assay also facilitates disease monitoring and can potentially identify mutations in metastatic lesions and treatment-induced resistant mutations not observed in the original, archival tumor biopsy.

Q. Please discuss some of the findings from a large study in which liquid biopsy was used. You reported on this at last year’s sessions of the American Society of Clinical Oncology.

Dr Mack: This study included 15,191 patients with advanced lung cancer (37%), breast cancer (14%), colorectal cancer (10%), and other cancers (39%). Each patient provided one or more blood samples for analysis of ctDNA. This study assessed the accuracy of liquid biopsies, as compared to tumor samples, in two ways. First, it compared the patterns of genomic changes in ctDNA to those found in 398 patients with available results of genetic testing of the tumor tissue. When ctDNA was positive for key abnormalities in EGFR, BRAF, KRAS, ALK, RET, and ROS1 that drive tumor growth, the same mutations were reported in tissue 94-100% of the time.

Q. How do you know that liquid biopsy provides an accurate snapshot of the tumor?

Dr Mack: The study also assessed consistency in the distribution and frequencies of specific changes in ctDNA against previously published data from genomic analyses of tumor tissue, including data from The Cancer Genome Atlas. The studies showed highly significant concordance between the ctDNA and the published data. The findings suggest that liquid biopsy provides an accurate snapshot of the genomic landscape of the tumor.

Across multiple cancer genes and different classes of alterations, correlations typically ranged from 0.92-0.99. However, one general exception was found in which ctDNA findings were often not seen in tumor biopsies: detection of new genomic alterations associated with resistance to targeted cancer drugs, such as the EGFR T790M resistance mutations in patients on EGFR inhibitor therapy. The study hypothesized that these alterations would be absent in the tissue-based population data because those patients had yet to receive treatment.

In summary, the results of the assay were compared with publicly available, population-scale tumor-sequencing data, most notably from The Cancer Genome Atlas. We found that ctDNA mutation patterns were highly consistent with distribution in tumor tissue by the [The Cancer Genome Atlas]. This was seen with commonly altered tumor suppressors and oncogenes alike. In the cancer genes we evaluated, we saw the exact same hot-spot and activating mutations as observed in The Cancer Genome Atlas and other publications, at very similar frequencies. Furthermore, we identified additional mutations associated with emergent resistant mechanisms that are not normally present at the time of initial biopsy.

Most of the ctDNA alterations were found at very low levels, with half occurring at a frequency below 0.4% of the total DNA in the circulation. Anecdotally, alterations observed at ctDNA fractions as low as 0.06% responded to treatment, which highlights the importance of assay sensitivity.

Q. What are the next steps in determining the value of circulating DNA testing?

Dr Mack: The next step for researchers is to work to increase the sensitivity of the Guardant360 assay to detect mutations at an extremely low ctDNA level, which is necessary for some tumors and will also enable its use in earlier-stage cancers. In about 15% of patients, no tumor DNA could be detected by the ctDNA test. There are simply tumors that do not shed DNA into the circulation at detectable levels, so we are bound to miss them. More importantly, we need to perform rigorous clinical testing of digital next-generation sequencing approaches to prove they can predict patient benefit from targeted treatment as well as-or perhaps better than-repeat tissue biopsies. This class of technologies has earned the right to be validated in the clinical trial setting. GSJ

 

 

CASE REPORT
Liquid Biopsies Monitor Disease Progression in a Sarcoma Patient

[Editor’s note: This report is adapted from Namløs HM, Zaikova O,Bjerkehagen B. Use of liquid biopsies to monitor disease progression in a sarcoma patient: a case report. BMC Cancer. 2017;17:29. DOI: 10.1186/s12885-016-2992-8. Additional material is included from PubMed sources.]

Although circulating tumor cells can be detected from the peripheral blood of cancer patients—and their prognostic value has been well established for meta-static colorectal, breast, and prostate cancer—information on their presence in patients affected by sarcomas is scarce. The discovery of EpCAM mRNA expression in different sarcoma cell lines and in a small cohort of metastatic sarcoma patients supports further investigations on these rare tumors to deepen the importance of circulating tumor cell (CTC) isolation.1 Although it is not clear whether EpCAM expression might be originally present on tumor sarcoma cells or acquired during the mesenchymal-epithelial transition, the discovery of EpCAM on circulating sarcoma cells opens a new scenario in CTC detection in patients affected by a rare mesenchymal tumor.

Sarcomas are relatively rare but particularly lethal tumors, with over 5,800 deaths per year in the United States,2 accounting for about 1% of all adult cancers and approximately 20% of pediatric solid malignancies.3 More than 50 subtypes of soft tissue malignant tumors of mesenchymal origin have been documented.4,5 The diagnosis is performed by hematoxylin and eosin staining of sections and immunohistochemistry. To date, radiographic imaging and PET are performed to detect recurrence and metastasis.

Advances in different technologies are transforming this picture. There are now new techniques available for molecular characterization of such tumor types Analyses such as Polymerase Chain Reaction (PCR), fluorescent in situ hybridization (FISH), Real-Time PCR, and next-generation sequencing (NGS) or the presence of reciprocal chromosomal translocations and fusion genes may be useful for diagnosis and treatment of sarcoma patients.6 Since most soft tissue sarcomas use the hematogenous dissemination to metastasize to lungs, liver, bones, and subcutaneous tissue whereas a small percentage may spread to lymph nodes,7,8 it is possible to isolate and characterize circulating tumor cells (CTCs) from whole blood based on their biological and/or physical properties.9 Furthermore, CTCs provide through their molecular evaluation an example of “liquid biopsy” useful for cancer patient care.10

The circulating cell-free tumor DNA (ctDNA) has been shown to contain the various tumor-specific alterations seen in the primary and metastatic tumors, and may more accurately represent the genetic profile of the whole tumor mass compared to DNA from a single biopsy of a heterogeneous lesion.11 By repeated sampling of liquid biopsies, somatic mutations identified in cfDNA can be used as unique non-invasive tumor-specific biomarkers for monitoring tumor burden throughout the disease course. Similar procedures are now in use for screening of fetal genetic aberrations using the mother’s blood, and in several cases aberrations from malignant tumors have been detected presymtomatic in pregnant women.12

Several reports have demonstrated that high-throughput sequencing of cfDNA may be used for prognosis and molecular stratification, early detection of recurrence and metastasis, monitoring response to treatment and identification of resistance mechanisms.13 Sequencing of cfDNA has been performed for cancers like colorectal, ovarian and breast, showing that the level of tumor-specific mutations reflects the course of the disease and the treatment response.14-16

Sarcomas make up a heterogeneous group of malignant tumors of mainly mesenchymal origin. The overall five-year survival of all soft tissue sarcoma patients is approximately 70%,17,18 and about 75% of soft tissue sarcomas are highly malignant. Soft tissue sarcomas often recur locally and/or metastasize, and the median time to local recurrence is around 1-1½ year and to metastasis about 1 year,19,20 both decreasing long-term survival. From a molecular genetics perspective, sarcomas are genetic diverse and may have numerous somatic mutations.21 The use of high-throughput sequencing of cfDNAs longitudinally collected during disease progression, making simultaneous screening for multiple mutations during the disease course possible, has not yet been reported for sarcomas.

As part of an ongoing prospective study,1 the study collected primary tumor and plasma samples taken before and after surgery and at disease progression from a soft tissue sarcoma patient. Targeted resequencing was used to identify somatic mutations in the primary tumor and monitor the level of ctDNA from plasma samples during the course of the disease. Local recurrence or metastases after receiving potentially curative treatment is common, and early detec-tion of these events is important for disease control. Recent technological advances make it possible to use blood plasma containing circulating cell-free tumor DNA (ctDNA) as a liquid biopsy. The following case report suggests how serial liquid biopsies can be used to monitor disease course and detect disease recurrence in a sarcoma patient.

Case Presentation

  • A 55-year-old male presented with a rapidly growing, painful palpable mass in the left groin region, and a biopsy revealed a high-grade malignant spindle cell sarcoma. Magnetic resonance imaging performed 18 days before surgery revealed a 10.5 x 7.6 x 11.0 cm large intramuscular tumor. No metastases were detected on CT scans of the chest, abdomen and pelvic area performed 14 days before surgery.
  • Microscopic evaluation of a biopsy revealed a high-grade malignant spindle cell sarcoma. Due to extensive locoregional growth into the skeleton and intractable pain, a hemipelvectomy was performed. Small focus with metastatic disease was detected in two lymph nodes removed during the surgery.
  • Macroscopic examination showed a well demarcated nodular tumor with white and fleshy cutting surface with small necrotic areas and bleeding. Immunohistochemical analysis showed positive finding for CD99 and AE1/AE3, and negative staining for S-100, SMA, EMA and CD31. Cytogenetic analysis showed massive clonal chromosomal rearrangements, and PCR and FISH were negative for fusion genes normally seen in synovial sarcoma.
  • The differential diagnoses were synovial sarcoma and malignant peripheral nerve sheath tumor. Lymph node metastasis is more commonly seen in synovial sarcoma and the immunohistochemical finding is also in favor of a synovial sarcoma, but the genetic findings did not support that diagnosis. According to the WHO classi-fication,22 the tumor was classified as an undifferentiated spindle cell sarcoma.

Determining Somatic Mutations
Targeted resequencing of the tumor and normal genomic DNA was performed. The sequencing revealed eight somatic mutations in the primary tumor. Among these, seven point mutations were identified in the genes COL2A1 (intronic), NF1 (p.K354R), PTGS2 (intronic), LRP2 (p.Q4132E), KRAS (p.G12V), PRRC2C (p.R1257G) and GATA6 (p.A29A), as well as a frameshift deletion in PRG4 (p.R791fs). Copy number analysis revealed a homozygote deletion of TP53. Targeted resequencing using a smaller ThunderBolts Cancer panel (Raindance Technologies, Billerica, Massachusetts, US) confirmed the identified KRAS mutation in the primary tumor at an allele frequency of 66%, similar to the 60% frequency found using the 900 gene panel.

Treatment With Surgery and Postoperative Findings
The patient was scheduled for adjuvant chemotherapy, but repeated radiologic imaging six weeks postoperatively showed widespread macroscopic metastatic disease in the lungs and skeleton, as well as numerous soft tissue metastases in the pelvic region. Targeted resequencing of the plasma samples, using the NCGC 900 cancer gene panel, confirmed the presence of six of the eight above mutations in all three plasma samples with allele frequencies ranging from 2.1-75%. The level of total cfDNA was monitored during disease progression. High quantity of cfDNA was detected one day before surgery (110 ng/ml plasma), and a decrease was seen three days after surgery (76 ng/ml plasma). Six weeks after surgery, the quantity of cfDNA had increased to more than twice the initial level present before the surgery (316 ng/ml plasma).

The ctDNA level was estimated from the somatic allele frequency of the recurrent mutations in the genes COL2A1, NF1, PTGS2, LRP2, KRAS and PRRC2C. The ctDNA level in plasma collected one day before the surgery (Plasma1) was high, and comparable to the level in primary tumour. Three days after surgery, the ctDNA level had dropped, but was still detectable in plasma (Plasma2). In the sample collected six weeks after surgery (Plasma3), there was again an increase in ctDNA level similar to the levels before surgery. When also taking into account the amount of cfDNA released, the number of mutated genomes per ml of plasma were three times higher at this time point than before surgery. This reflected the disease progression of the patient and correlated with the tumor burden, as multiple distant metastases were detected at this time. The patient’s general condition was considered too poor for administering chemotherapy, and he succumbed to the disease 13 weeks after surgery.

Analyzing the Significance and Implications of ctDNA Findings
In this study, we prospectively collected primary tumor and normal sample material at surgery and several plasma samples during the disease course of a high-grade soft tissue sarcoma patient. Targeted resequencing of the primary tumor and the normal sample identified eight somatic mutations of which six were also present in the plasma samples. Among the mutations, KRAS (p.G12V) and NF1 (p.K354R) were predicted by dbNSFP to have a deleterious effect on the protein function. It has been reported that simultaneous inactivation of TP53 and activation of KRAS induced quick formation of spindle-cell sarcoma in soft tissues in double transgenic mice. The homozygous deletion of TP53 found in the primary tumor strengthens the histology observed in the primary tumor.

The patient in our study had an unusually aggressive spindle-cell sarcoma, supporting KRAS not only as biomarker, but as a driving gene of the disease progression. NF1, a tumor suppressor that functions as a negative regulator of the Ras pathway, is among the most frequently mutated genes in several subtypes of sarcomas. Germline and somatic loss of NF1 in neurofibromatosis patients cause malignant peripheral nerve sheath tumors and GISTs. In addition, somatic NF1 mutations, including deletions, have been reported in a wide variety of pediatric and adult soft-tissue sarcomas with complex karyotypes . Although no therapeutics that target KRAS or NF1 are available, our study shows that repeated sampling using liquid biopsies opens new possibilities to identify and monitor biomarkers that can be used in targeted therapies.

The allele frequencies of the six mutated genes in the cfDNA represent the ctDNA level during disease progression. Three days after surgery, ctDNA was still detectable in the liquid biopsy. cfDNA has a rapid clearance, with reported half-life from 15 min to 13 h for fetal cfDNA in plasma. Although not detectable by CT before surgery, metastatic disease was detected in two lymph nodes removed during the hemipelvectomy, and a small amount of ctDNA detected was likely released from additional undiscovered local metastases that could not be detected by conventional diagnostic modalities. The patient had a very aggressive course of the disease, and metastases were detected both in soft tissue, skeleton and lungs only a few weeks after surgery. The plasma collected six weeks after surgery showed an increase in ctDNA relative to the levels before surgery, reflecting the presence of a tumor and rapid disease progression.

The cfDNA can originate from both normal and tumour cells. Based on the high mutated allele frequencies determined in plasma, the initial level of cfDNA is dominated by DNA from the tumor. Most of the cfDNA present three days after surgery is believed to originate from tissue injury and inflammation of normal cells as a consequence of the extensive surgery, which would explain the apparently higher normal contribution to the cfDNA at this time point. After six weeks, there was a large increase in cfDNA accompanied with an increase of the mutated allele frequencies. Thus, the quantities of cfDNA present in the plasma reflected the clinical status of the patient due to the fact that most of the cfDNA released during disease progression was tumour derived.

Conclusion
This study is the first report of using targeted resequencing of cfDNA from serial plasma samples to monitor disease progression in a soft tissue sarcoma patient. The findings show that the level of tumour-specific mutations in liquid biopsies is correlated to disease course in sarcomas, including clinical manifestation of metastatic disease. The longitudinally collected ctDNA allow for near real-time monitoring of the tumor genome during disease progression, and the ctDNA gives a good representation of the genomic profile of the tumor supporting the use of ctDNA from plasma as a liquid biopsy.

References
1. Namløs HM, Zaikova O,Bjerkehagen B. Use of liquid biopsies to monitor disease progression in a sarcoma patient: a case report. BMC Cancer. 201717:29. DOI: 10.1186/s12885-016-2992-8.
2. Siegel R, D. Naishadham D, Jemal A. Cancer statistics, CA—A Cancer Journal for Clinicians, 2012;62:10–29.
3. Amankwah EK, Conley AP, Reed DR, Epidemiology and therapies for metastatic sarcoma,” Clinical Epidemiology. 2013;5(1):147–162.
4. Fletcher CD, Hogendoorn P, Mertens F, et al. WHO Classification of Tumours of Soft Tissue and Bone, IARC Press, Lyon, France, 4th edition, 2013.
5. Doyle LA, Sarcoma classification: an update based on the 2013 world health organization classification of tumors of soft tissue and bone. Cancer. 2014; 120:12:1763–1774.
6. Smith SM, Coleman J, Bridge JA, et al. Molecular diagnostics in soft tissue sarcomas and gastrointestinal stromal tumors. J Surg Oncol. 2015; 111:5:520–531.
7. Pennacchioli E, Tosti, Barberis GM, et al. Sarcoma spreads primarily through the vascular system: are there biomarkers associated with vascular spread? Clinical and Experimental Metastasis. 2012; 29:7:757–773.
8. Gronchi A, Lo Vullo S, Colombo C, et al., Extremity soft tissue sarcoma in a series of patients treated at a single institution: local control directly impacts survival. Annals of Surgery. 2010; 251:3:506–511.
9. Vincenzi B, Rossi E, Zoccoli A, et al. Circulating tumor cell in soft tissue sarcomas patients. Ann of Oncol. 2012;23:489.
10. Pantel K, Alix-Panabières C. Real-time liquid biopsy in cancer patients: fact or fiction? Cancer Research. 2013;73:21:6384–6388.
11. De Mattos-Arruda L, Weigelt B, Cortes J, et al. Capturing intra-tumor genetic heterogeneity by de novo mutation profiling of circulating cell-free tumor DNA: a proof-of-principle. Ann Oncol. 2014;25(9).
12. Amant F, Verheecke M, Wlodarska I, et al. Presymptomatic identification of cancers in pregnant women during noninvasive prenatal testing. JAMA Oncol. 2015, doi:10.1001/jamaoncol.2015.1883.
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