Solving Riddles Through Sequencing

Study Overview
=================
Brief Summary
-----------------
During the last decades hematologists have excelled at improving and refining the classification, diagnosis, and thus ultimately the therapeutic decision-making process for their patients. This continuous evolution proceeded in parallel to seminal discoveries in basic science such as FISH, PCR and NGS. So far, the current WHO classification serves as reference to diagnostic decision making and is largely based on 5 diagnostic pillars: cytomorphology of peripheral blood and/or bone marrow smears, histology and immunohistochemistry of bone marrow trephine biopsies or lymph nodes, immunophenotyping, chromosome banding analysis supplemented by FISH analysis, molecular genetics including PCR and targeted panel sequencing via NGS. This leads to a swift diagnosis in 90 % of all cases. The leftover 10 % remain a challenge for hematopathologists and clinicians alike and are resolved through interdisciplinary teams in the context of specialized boards. With the advent of high throughput sequencing (mainly WGS and WTS) the possibility of a comprehensive and detailed portrait of the genetic alterations - specifically in challenging cases - has become a realistic alternative to classical methods. In SIRIUS the investigators will prospectively challenge this hypothesis to address the question of how often a better or final diagnosis can be delivered by WGS and/or WTS and if unclear cases can be efficiently resolved.

Detailed Description
-----------------
Background and Rationale Treatment of hematological diseases relies on a single cardinal prerequisite: correct classification within the broad specter of malignant diseases arising from the hematopoietic system. With the ever-expanding availability of distinct yet complementary diagnostic tools, our understanding of the landscape of hematological diseases steadily increases. As such, the current consensus classification as summarized through the WHO classification (2017) represents a compass guiding diagnostic algorithms to the correct diagnosis. Today, the gold standard of routine diagnostic process relies on five methodological pillars: cytomorphology, histology, chromosomal cytogenetics, immunophenotyping, and molecular genetic testing. This leads to a treatment enabling diagnosis in the vast majority of cases. However, approximately 10 % of cases remain unresolved from a diagnostic point of view and hence do not lead to a satisfactory diagnosis according to current WHO standards (2017). The investigators intend to solve this issue to provide illuminating diagnostic guidance for the best possible patient´s care. Objectives To address this problem, the investigators hypothesize that novel high throughput sequencing methods, e.g., whole genome and/or whole transcriptome sequencing are able - by virtue of painting a more delicate genetic portrait of a tumor sample - to provide a more accurate diagnosis. To this end the investigators generated a reference collection of 5,500 samples with the full spectrum of hematological malignancies, for which the investigators performed whole-genome sequencing as well as whole-transcriptome sequencing. Moreover, gold standard diagnoses according to WHO classification with all needed techniques, all performed in MLL, clinical data and therapy response data are fully available for these cases. The main advantage of this reference collection consists of the unambiguity of each diagnosis, providing a reference framework for any further classification and diagnosis especially in difficult cases. Therefore, SIRIUS will compare the diagnostic superiority of WGS or WTS to the combined approach with gold standard results and by matching the obtained results to the nearest digital sibling within our reference cohort of more than 5,500 WGS and WTS (both in 93% of cases). To this end, the investigators will use an inhouse developed matching algorithm, which is able to match genomic or transcriptomic profiles to a group of similar cases and gold standard results from timepoint of this study. Current workflows intended to generate WTS/WGS data from patient samples - all while fulfilling state of the art accreditation (ISO 15189) - require up to 5 - 7 days. This is largely comparable to classical methods but holds the promise to replace error prone and arduous iterations in the methodological work up. The objective is to test whether WTS and/or WGS based approaches can surpass classical methods regarding diagnostic precision and routine reliability. Here the investigators will test this hypothesis in a prospective real-world setting under diagnostically difficult circumstances.

Official Title
-----------------
Testing the Diagnostic Supremacy of Sequencing-only Approaches in Hematologic Malignancies: an Observational Trial

Conditions
-----------------
Leukemia, Hematologic Malignancy, Rare Diseases, Refractory Leukemia, Refractory Lymphoma, Unknown Primary Tumors

Intervention / Treatment
-----------------
* Diagnostic Test: Next Generation Sequencing


Participation Criteria
=================
Eligibility Criteria
-----------------
Inclusion Criteria: Patients having been investigated with a suspected hematological disorder and: Having unclear diagnosis after internal routine diagnosis Unusual clinical course Unusual r/r status or non-responder Multiple parallel hematological conditions Difficult/rare therapy associated/secondary neoplasms Current diagnostic workup is not satisfactory in terms of (1) accuracy (2) clinical behavior Only samples of patients min. 18 years of age will be used Material with a minimum of 20% tumor content in bone marrow or peripheral blood sample Patient´s informed consent Exclusion Criteria: Sample is not fit for state-of-the-art diagnosis, fails initial quality control. For quality insurance we will exclude samples with wrong anticoagulant sent. Samples with damage due to meteorological reasons (freeze-thaw damage or elevated temperature) will be excluded. Samples with to scarce material jeopardizing routine gold-standard diagnosis will be excluded (tumor content < 20 %).

Ages Eligible for Study
-----------------
Minimum Age: 18 Years
Maximum Age: 99 Years

Sexes Eligible for Study
-----------------
All

Accepts Healthy Volunteers
-----------------
No

Study Plan
=================
How is the study designed?
-----------------

Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Unclear diagnosis via conventional methods<br>The study population consists of carefully chosen patients with potential hematological malignancy, for which current diagnostic methods were not sufficient to provide clear-cut diagnosis and definitive clinical guidance. SIRIUS will be conducted for a total number of 110 patients with inconclusive diagnosis by gold standard techniques for a total of up to nine months after the first enrollment. | Diagnostic Test: Next Generation Sequencing<br>* NON-Interventional Observation only study comparing sequencing-only approaches to classical diagnostic methods<br>* Other names: NGS, Whole genome Sequencing;|

What is the study measuring?
-----------------
Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| sequencing only versus gold standard diagnosis | The primary endpoint will be assessed as follows: unclear cases will be subjected to three diagnostic algorithms: Inhouse at referring site by histopathological diagnosis in the context of a hematological tumor board according to current standards Current gold-standard diagnostic workup as performed routinely by the MLL WGS and WTS sequencing plus matching to nearest digital sibling in 5,5k cohort We will compare the accuracy for approach #3 for each patient/case by comparing the sequencing results with the therapy guiding approach in domo and Current gold-standard diagnostic workup as performed routinely by the MLL. Accuracy and overlap or discordance will be measured in percentage (%) of total cohort. Time Frame of Outcome: At diagnosis for each case/patient throughout the complete duration of study for approximately 1 year. | Time Frame of Outcome: At diagnosis for each case/patient throughout the complete duration of study for approximately 1 year. | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| turnaround time | Turnaround time until potential therapy guiding diagnosis will be measure and compared in days. | The time frame will be consisting of an assessment at diagnosis for next 14 days per case/patient. | 
| actionable targets | The number of potential actionable targets will be determined for each case. We will assess this number in absolute numbers for each patient and for which a therapy has been approved but identification was missed in during classical gold standard diagnosis. | This outcome will be measured one year after diagnosis. (1 year after diagnosis) | 
| disease stage | We will Identify the putative disease stage based on sequencing only approaches in comparison to normal gold standard diagnosis as well as clinical history. Results will be denoted as percentage (%) of correctly classified cases. | This outcome will be measured one year after diagnosis. (1 year after diagnosis) | 
| Micro-cost analysis | We will measure all costs for respective assays and personnel to finally compare workflows 2 and 3 (see primary outcome 1.). | The estimated timeframe for this outcome will be the timepoint of diagnosis for each patient (approximately 5 days) |