Multi-site tumor sampling ( MSTS ) improves the performance of histological detection of intratumor heterogeneity in clear cell renal cell carcinoma ( CCRCC )

Current standard-of-care tumor sampling protocols for CCRCC (and other cancers) are not efficient at detecting intratumoural heterogeneity (ITH). We have demonstrated that an alternative protocol, multi-site tumor in silico sampling (MSTS) based upon the divide and conquer (DAC) algorithm, can significantly increase the efficiency of ITH detection without extra costs. Now we test this protocol on routine hematoxylin-eosin (HE) sections in a series of 38 CCRCC cases. MSTS was found to outperform traditional sampling when detecting either high grade (p=0.0136) or granular/eosinophilic cells (p=0.0114). We therefore propose that MSTS should be used in routine clinical practice. 1 2-4 3,5-7*

Clear cell renal cell carcinoma (CCRCC) is the most frequent form of renal cancer in Western Countries 1 and a paradigmatic example of intratumoural heterogeneity (ITH) 2-5 . ITH is a major factor in the unpredictable clinical behavior and treatment failure response that these tumors can display 5 and as a consequence, detection of ITH by pathologists is becoming an increasingly important metric of clinical practice.
We have recently demonstrated that a multi-site tumor sampling (MSTS) protocol following the divide-and-conquer (DAC) algorithm outperforms routine sampling protocols (RS) in detecting ITH when tested in silico 6 . DAC is a well-known strategy in computer science to solve many different practical problems and consists in recursively breaking down a given problem into simpler parts (divide) until they are simple enough to be efficiently solved (conquer). Then, partial solutions are combined to solve the original problem. Since such a strategy does not necessarily increase the cost of procedures and can be performed without significant changes in the pathologist's routine, we proposed its generalized implementation in pathology labs 6,7 . This study extends this hypothesis to a real life scenario by comparing the MSTS protocol with RS when detecting classic morphological ITH in a series of 38 CCRCC.

Material and methods
Thirty-eight CCRCC were prospectively collected from the Pathology Department of the Cruces University Hospital (Barakaldo, Spain). All patients were informed about the potential use for research of their surgically resected tissues, and accepted this eventuality by signing an information consent approved by the local Ethics Committee (CEIC). The two sampling protocols MSTS and RS were applied in each case. The RS 8 method consisted of selecting one tissue fragment per centimeter of tumor diameter plus an additional fragment of each suspicious area by the naked eye. Alternatively, the MSTS 6,7 method consisted of selecting a large number of small fragments including six to eight of them in the same cassette and fixing the number of cassettes to one per centimeter of tumor ( Figure 1). Thus, the two sampling protocols made use of the same number of cassettes and, consequently, the total surface of tumor tissue selected for analysis is equivalent (restricted by the cassette dimensions). Tissue samples were fixed in formalin and embedded in paraffin following routine methods. Four-micron-thick histological slides were processed in an automatized stainer (Symphony system, Ventana Medical Systems Inc., Tucson, USA).
The study was performed on hematoxylin-eosin (HE) stained histological slides exclusively. Two experienced pathologists (RG, JIL) reviewed all HE sections in a blind fashion. Fuhrman grade, cell type (clear vs. granular eosinophilic), and the presence of necrosis and/or sarcomatoid change were evaluated in all cases and in both sampling methods. Grade was grouped as low (G1/2) and high (G3/4) for higher consistency. The four histological

Amendments from Version 1
The authors thank all the comments and suggestions of the referees. A mention of what is really the divide and conquer strategy has been included in the Introduction. The Material and Methods section also includes additional information related to the amount of tissue evaluated in both protocols and the way we did it. Finally, a new paragraph (with an additional reference) has been included in the Discussion stressing the potential importance of this approach in detecting molecular intratumoural heterogeneity.

REVISED
parameters were considered if they were present in the sample, no matter they were focally or extensively found in RS or affecting one or more fragments in MSTS, since the final goal was "its detection".

Statistical analysis
Results of the two methods (RS and MSTS) were compared by applying a chi-squared test (χ 2 ), a test applied to sets of categorical data to evaluate the hypothesis of independence between the two groups. In particular, we made use of the script chi2test.m
Although MSTS also detected a higher number of tumors displaying sarcomatoid phenotype (12 vs. 6 cases, respectively) and a higher number of cases presenting tumor necrosis (10 vs. 7 cases, respectively), their figures did not reach significant levels (Table 1) probably because both were detected by the naked eye and were sampled in both protocols.

Discussion
The clinical importance of detecting ITH is becoming clearer as time passes and as a consequence represents one of the most challenging tasks facing pathologists today 5 . However, pathologists have not yet adapted the old sampling protocols and seem not aware of a concerning paradox: The success of sophisticated devices and expensive platforms in detecting key tumor mutations depends on the selection rightness of tumor pieces which are (very often) made by residents. The combination of lack of solid evidence for the necessity to change current practice and a reluctance to incur new costs and increased work load may be responsible of this attitude.
We present evidence that the MSTS protocol is much more effective than RS in detecting high grade areas and other histological parameters that determine tumor aggressiveness and prognosis in CCRCC. Importantly the MSTS protocol does not incur extra costs to pathology labs 6,7 . A similar approach (but for a different purpose) was already reported in 1990 by Battifora and Mehta to optimize the screening of new histologic reagents 9 .
A thorough histological analysis such as MSTS performs may also help the pathologists in detecting hidden or unexpected tumor histologies, i.e., hybrid tumors, collision neoplasms, histologically complex tumors, and minor but crucial components in a huge tumor, giving definite clues for a complete diagnosis.
However, the final objective of MSTS is to ensure the detection of the complete spectrum of molecular changes across each tumor as this achievement will open new possibilities for more efficient treatments, thus accomplishing oncologists expectancies 10 . Exhaustive molecular analyses of renal carcinomas are being performed nowadays 4,5 , but these approaches are difficult to be implemented in the routine practice for most of the hospitals due to their high costs. In this sense, MSTS could be regarded as an affordable alternative for a generalized application since it takes into account as a necessary premise the sustainability of health systems.