Wouldn’t you prefer that all patients with solid tumors undergoing a rule-out workup (for any kind of cancer) have the benefit of molecular testing or next generation sequencing (NGS) to characterize their cancer type?

You want to be aware of any cancer cell vulnerabilities that may qualify your patient for a precision medicine drug, even if still experimental, to avoid radiation and chemotherapy. If your hospital utilizes the Crow’s NestTM Biopsy Catchment System, every patient under your care who undergoes a standard core needle biopsy will have the benefit of NGS, with maximum diagnostic value because the tests will be run using fresh, cell culture-quality material undamaged by formalin.

As you know, the diagnosis of cancer has historically been made from the microscopic examination of a tissue biopsy. Cancer treatment modality selection has been based on a combination of the type of organ involved and its morphology.

Today, diagnostic techniques are rapidly expanding beyond microscopic examination to include molecular analysis of DNA and RNA in the tissue’s tumor cells. These increasing demands on the tissue biopsy have become problematic due to the markedly decreased amount of tissue available for testing (Figure 1). An approach capable of providing enough material to meet the demands of both the microscopic examination necessary to make a pathologic diagnosis and the molecular testing that will guide therapy is urgently needed. 

Trying to recover enough tissue from an already limited source often leads to a situation referred to in the literature as “tissue exhaustion”, wherein no more tissue is available for any kind of testing. Tissue exhaustion has direct implications on patient care and clinical medicine and may lead to the need for an additional biopsy procedure -- or in cases when this is not possible -- to patients being ineligible for cutting-edge treatments or enrollment in life-saving clinical trials. Additional biopsies involve additive risks and pain, delays in diagnosis and treatment, the potential for no reimbursement, and additional cost to the health care system of up to $5.1 billion per year.

Adding to this problem is that tissue specimens are processed by formalin-fixation, paraffin-embedding (FFPE), which has not changed in decades. While FFPE is ideally suited for microscopic examination, it is widely known to be suboptimal for the recovery and testing of that tissue’s nucleic acids (1).

Some approaches have sought to remediate the problems that arise from nucleic acid exposure to formalin, but all involve the introduction of additional chemicals, increased labor and time, and/or expensive instrumentation and have not been widely adopted. Additionally, these “back-ended” (e.g., figure 2C) approaches seek to remediate nucleic acids after they have been damaged rather than avoiding the damage in the first place.

The urgency to address this ongoing, unresolved clinical problem is underscored by the NIH, as well as the oncology and pathology communities, to think of innovative solutions that may be outside of the proverbial box.(2,3,4,5,6).

The Crow’s Nest™ Biopsy Catchment System addresses this problem by harvesting diagnostic material in a heretofore under-recognized space along the tissue biopsy specimen’s journey (Figure 2). This journey begins with the procurement of tissue by a proceduralist (usually an interventional radiologist) using a long thin needle known as a core needle biopsy (CNB) instrument, followed by fixation in formalin and laboratory processing to produce a section amenable for microscopic examination, and if clinically indicated, recovery of nucleic acids for molecular-based testing. 

The Crow’s Nest captures dissociated dislodged tumor cells (DDTCs) that have become separated from the parent tissue due to the microtrauma associated with the biopsy procedure but are routinely, unknowingly, discarded. These DDTCs are discarded because they are below the acuity of the human eye and are simply thrown away with the disposable needle. The Crow’s Nest separates the DDTCs from the parent tissue without exposing the former to fixatives and in the process creates two specimens from what was initially intended to be one (Figure 3).

This straightforward but never previously addressed concept requires no additional instrumentation to function, is both simple and rapid to perform, and again and most importantly, recovers nucleic acids without exposure to formalin fixation. It recovers cells like no other approach available today, because it does not expose nucleic acids to formalin, so it results in a far superior specimen for molecular testing than those recovered by back-ended, indirect method to extract nucleic acids from FFPE-treated material. 

Why are we confident this will work? Preliminary data using a precursor of the Crow’s Nest yielded promising findings on the quality and quantities of available DNA captured as DDTCs. Collection of these DDTCs was performed by Dr. Mojica in the interventional radiology (IR) suite during three scheduled procedures (7). The specimen was transported to the pathology laboratory where the DDTCs and tissue core were separated. The parent tissue core underwent routine FFPE. Once all three DDTC specimens were collected, they were transferred to a laboratory core facility where the DNA was extracted using a commercially-available kit. Quantitation was evaluated using a Quibit fluorometer and qualitative metrics assessed by molecular weight (MW) on an Agilent Tape Station. These three specimens were run through an illumina next-generation sequencer (NGS), and additional qualitative features (Phred score and Per Base N content [PBNC]) obtained.  

High MW DNA (between 15 to 18 kilobase-pairs) was recovered in each case (Table 1, Figure 4A). In contrast, traditionally-processed FFPE specimens have recovers DNA typically <500 base-pairs (bps) in size (8). Our results are 30-36x larger than expected in FFPE specimens and may be even higher because size measurement was limited by use of an extraction protocol not designed for ultra-high MW DNA recovery. The Genomic Quality Number (GQN), 

which is the calculated percentage of the total DNA in a sample that is above a set threshold (e.g., typically 500 bps for FFPE specimens) and reported in a range from 0 (worst) to 10 (best), was either 9.9 or 10 in the three samples. This compares favorably to the GQN range of 2.5 to 7 that has been reported in FFPE specimens (9). 

Sequencing metrics provided additional promising data. The Phred score, the numeric value reflective of the quality and confidence of the nucleic acids generated during NGS, were consistently ≥35, indicating a base calling accuracy of 99.97% (Figure 4B). Additionally, the Phred score was ≥35 throughout the entire sequence read, in contrast to the dip towards the tail end of the sequence normally observed. The PBNC metric that measures the ability of a sequencer to designate a base as either adenine, guanine, cytosine, or thymidine was consistently <<1% (Figure 4C). 

All together, these findings support the Crow’s Nest basic premise that recovering DNA before exposure to formalin yields higher-quality starting material for molecular studies. Confirmation that the recovered DDTCs are diagnostic and neoplastic in nature comes from the finding of Tier 1 (TSC2 gene mutations) and Tier 2 (p53 gene mutations) alterations in the DNA of these samples. This finding supports the hypothesis underlying use of the Crow’s Nest -- that microtrauma during tumor tissue biopsies has a built-in bias that more tumor cells than normal cells will become dislodged due to the former’s “friable” or invasive nature within the host tissue.

The benefits of next-generation diagnostics for oncology are many, but NGS carries preanalytical considerations, technical challenges, and inchoate available technologies that don’t address those challenges when the samples come from core needle biopsies (10).

If every patient who is biopsied has cells screened with NGS, more cancer will be caught – earlier. Some cancer patients may qualify for new precision medicine treatments that they otherwise wouldn’t know about. Because of this, the Crow’s Nest can be a life-saving device.