Potential Unblinding due to Imbalance in Adverse Event Profiles

In a previous post “Is blinded study really blinded? - assessment of blinding/unblinding in clinical trials”, we discussed the potential unblinding if the subjects on different treatment groups experienced a different type of adverse events. For the ethic reasons, the informed consent form needs to list all the potential side effects of the experimental drug. As required by GCP, the investigator’s brochure needs to discuss the details of the potential side effects caused by the experimental drug. If there is an obvious imbalance in adverse event profiles between the two treatment groups (experiment drug versus control), it is possible that the study blinding is not well maintained and the patient or the investigator can guess or predict based on the AE profile which treatment group the patient is on.

This potential unblinding issue can be even more evident if the crossover design is employed where the same subject will have an chance to experience two different drugs. If there are differences in side effects, the subjects will be likely to feel the difference.

In FDA’s recent guidance for industry “Hematologic Malignancy and Oncologic Disease: Considerations for Use of Placebos and Blinding in Randomized Controlled Clinical Trials for Drug Product Development”, the potential unblinding issue was brought up.

“In many cases, because of the toxicity profile of the active treatment, patients and investigators may infer which treatment patients are receiving and thus use of a placebo control may not, in fact, blind the treatment. …”

The guidance also cited the ethic issue for maintaining blinding when the unblinding is necessary to give the best care for the subjects (patients).

“Continued blinding of patients and investigators at the time of disease progression or occurrence of serious adverse events presents additional challenges. For example, in a blinded immunotherapy trial, a patient who develops adverse events on the control arm may receive unnecessary treatments (e.g., immunosuppressive drug products including a high dose of glucocorticoids, cyclophosphamide, interleukin-6 antagonist, or infliximab) for management of adverse events incorrectly attributed to the investigational drug product. Maintaining the blind after disease progression could also affect a patient’s subsequent therapy, potentially preventing a patient who had been on a placebo arm from receiving an approved therapy, or delaying or preventing the patient’s entry into other clinical trials (for those trials of similar drug products that may have specific exclusion criteria based on prior treatment with an active drug or class of drugs). Unblinding would allow informed decision-making with respect to additional treatment 61 options (see below). “

In an analysis by Shah et al Adverse events appear to unblind clinical trials in irritable bowel syndrome, the result “suggests that higher AE incidence on active therapy is associated with more beneficial patient‐reported outcomes in IBS clinical trials. This raises the issue of spontaneous unblinding”

Maintaining the blinding is one of the cornerstones of the RCT (randomized, controlled trial). As mentioned in an article by Stefan Dürr Avoid Unintentional Unblinding In Clinical Trials

“The knowledge of the patient’s treatment could potentially lead to conscious or unconscious bias in the way the site staff recruits the patients (selection bias), how they are treated (performance bias), the assessment of endpoints (detection bias), the handling of withdrawals, and how data is excluded from analysis. The patient attitude to the treatment if known can lead to a difference in reporting symptoms (response bias) or withdrawal from the study (attrition bias). To reduce the chance of bias to the data, clinical trials should be conducted in a double-blind design whenever possible.”

However, in some situation especially when there is an imbalance in the side effects of two treatment groups, blinding may either be difficult to maintain or not be ethical to implement, and alternative approaches are needed. As suggested in FDA's guidance "

“Hematologic Malignancy and Oncologic Disease: Considerations for Use of Placebos and Blinding in Randomized Controlled Clinical Trials for Drug Product Development”, an open-label study or an add-on trial design may be more appropriate. In an early post, I also discussed some blinding techniques. 

Latin Squares for Constructing "Williams Designs" - Balanced for First-order Carry-over (Residual) Effects

Courtesy from the website http://statpages.info/latinsq.html, the list below contains the Williams design with 2 to 26 treatment groups. It is unlikely to have a clinical trial with 26 treatment groups. The most number of treatment groups I see is a study with 6 treatment groups. 

In one of my previous posts, I described the Williams design and its usefulness in phase I or clinical pharmacology studies. In a SAS SUGI paper "Generating Randomization Schedules Using SAS Programming", I described how to use SAS program to construct the randomization schedule for a study with Williams design. 

With the limited sample size, the Williams design is the most efficient design and minimizes the potential impact of the carryover effect. In Williams design with an even number of n treatment groups, there will be n number of sequences or an "n by n" square. Each treatment only occurs one time in one sequence, in one period. Furthermore, each treatment only follows another treatment one time. 

If the number of treatments (n) is an odd number, there will be 2 x n number of sequences or two n by n squares. Each treatment only occurs once in one sequence and in two periods. Each treatment only follows another treatment twice. 

With the Latin Square listed below, we can easily construct the crossover design with treatments, periods, and sequences. For example, a study design with 3 treatment groups will have the following assignments: three treatment groups (A, B, C), three periods (period 1, period 2, and period 3), and six sequences (ABC, BCA, CAB, CBA, ACB, and BAC). 

Treatment Sequence	Period 1	Period 2	Period 3
1	A	B	C
2	B	C	A
3	C	A	B
4	C	B	A
5	A	C	B
6	B	A	C

• Since it is the crossover design, there should be a washout period between each period. It is very common that the washout period is 5 times the half-life. 
• Randomization is for the sequence, not the treatment group. 
• Once we know the number of subjects is needed for each sequence, we will know the total number of subjects for the entire study. 
• Usually, the mixed model is needed for statistical analysis. The statistical model will need to consider the treatment, period, and sequence. An example of the SAS codes for analysis was provided in one of the previous posts "Cookbook SAS Codes for Bioequivalence Test in 2x2x2 Crossover Design"

Williams Design by the Number of Treatment Groups:

Size = 2

A B 
B A 
 

Size = 3

A B C 
B C A 
C A B 
C B A 
A C B 
B A C 
 

Size = 4

A B D C 
B C A D 
C D B A 
D A C B 
 

Size = 5

A B E C D 
B C A D E 
C D B E A 
D E C A B 
E A D B C 
D C E B A 
E D A C B 
A E B D C 
B A C E D 
C B D A E 
 

Size = 6

A B F C E D 
B C A D F E 
C D B E A F 
D E C F B A 
E F D A C B 
F A E B D C 
 

Size = 7

A B G C F D E 
B C A D G E F 
C D B E A F G 
D E C F B G A 
E F D G C A B 
F G E A D B C 
G A F B E C D 
E D F C G B A 
F E G D A C B 
G F A E B D C 
A G B F C E D 
B A C G D F E 
C B D A E G F 
D C E B F A G 
 

Size = 8

A B H C G D F E 
B C A D H E G F 
C D B E A F H G 
D E C F B G A H 
E F D G C H B A 
F G E H D A C B 
G H F A E B D C 
H A G B F C E D 
 

Size = 9

A B I C H D G E F 
B C A D I E H F G 
C D B E A F I G H 
D E C F B G A H I 
E F D G C H B I A 
F G E H D I C A B 
G H F I E A D B C 
H I G A F B E C D 
I A H B G C F D E 
F E G D H C I B A 
G F H E I D A C B 
H G I F A E B D C 
I H A G B F C E D 
A I B H C G D F E 
B A C I D H E G F 
C B D A E I F H G 
D C E B F A G I H 
E D F C G B H A I 
 

Size = 10

A B J C I D H E G F 
B C A D J E I F H G 
C D B E A F J G I H 
D E C F B G A H J I 
E F D G C H B I A J 
F G E H D I C J B A 
G H F I E J D A C B 
H I G J F A E B D C 
I J H A G B F C E D 
J A I B H C G D F E 
 
 
Size = 11

A B K C J D I E H F G 
B C A D K E J F I G H 
C D B E A F K G J H I 
D E C F B G A H K I J 
E F D G C H B I A J K 
F G E H D I C J B K A 
G H F I E J D K C A B 
H I G J F K E A D B C 
I J H K G A F B E C D 
J K I A H B G C F D E 
K A J B I C H D G E F 
G F H E I D J C K B A 
H G I F J E K D A C B 
I H J G K F A E B D C 
J I K H A G B F C E D 
K J A I B H C G D F E 
A K B J C I D H E G F 
B A C K D J E I F H G 
C B D A E K F J G I H 
D C E B F A G K H J I 
E D F C G B H A I K J 
F E G D H C I B J A K 
 

Size = 12

A B L C K D J E I F H G 
B C A D L E K F J G I H 
C D B E A F L G K H J I 
D E C F B G A H L I K J 
E F D G C H B I A J L K 
F G E H D I C J B K A L 
G H F I E J D K C L B A 
H I G J F K E L D A C B 
I J H K G L F A E B D C 
J K I L H A G B F C E D 
K L J A I B H C G D F E 
L A K B J C I D H E G F 
 
 
Size = 13

A B M C L D K E J F I G H 
B C A D M E L F K G J H I 
C D B E A F M G L H K I J 
D E C F B G A H M I L J K 
E F D G C H B I A J M K L 
F G E H D I C J B K A L M 
G H F I E J D K C L B M A 
H I G J F K E L D M C A B 
I J H K G L F M E A D B C 
J K I L H M G A F B E C D 
K L J M I A H B G C F D E 
L M K A J B I C H D G E F 
M A L B K C J D I E H F G 
H G I F J E K D L C M B A 
I H J G K F L E M D A C B 
J I K H L G M F A E B D C 
K J L I M H A G B F C E D 
L K M J A I B H C G D F E 
M L A K B J C I D H E G F 
A M B L C K D J E I F H G 
B A C M D L E K F J G I H 
C B D A E M F L G K H J I 
D C E B F A G M H L I K J 
E D F C G B H A I M J L K 
F E G D H C I B J A K M L 
G F H E I D J C K B L A M 
 
 
Size = 14

A B N C M D L E K F J G I H 
B C A D N E M F L G K H J I 
C D B E A F N G M H L I K J 
D E C F B G A H N I M J L K 
E F D G C H B I A J N K M L 
F G E H D I C J B K A L N M 
G H F I E J D K C L B M A N 
H I G J F K E L D M C N B A 
I J H K G L F M E N D A C B 
J K I L H M G N F A E B D C 
K L J M I N H A G B F C E D 
L M K N J A I B H C G D F E 
M N L A K B J C I D H E G F 
N A M B L C K D J E I F H G 
 
 
Size = 15

A B O C N D M E L F K G J H I 
B C A D O E N F M G L H K I J 
C D B E A F O G N H M I L J K 
D E C F B G A H O I N J M K L 
E F D G C H B I A J O K N L M 
F G E H D I C J B K A L O M N 
G H F I E J D K C L B M A N O 
H I G J F K E L D M C N B O A 
I J H K G L F M E N D O C A B 
J K I L H M G N F O E A D B C 
K L J M I N H O G A F B E C D 
L M K N J O I A H B G C F D E 
M N L O K A J B I C H D G E F 
N O M A L B K C J D I E H F G 
O A N B M C L D K E J F I G H 
I H J G K F L E M D N C O B A 
J I K H L G M F N E O D A C B 
K J L I M H N G O F A E B D C 
L K M J N I O H A G B F C E D 
M L N K O J A I B H C G D F E 
N M O L A K B J C I D H E G F 
O N A M B L C K D J E I F H G 
A O B N C M D L E K F J G I H 
B A C O D N E M F L G K H J I 
C B D A E O F N G M H L I K J 
D C E B F A G O H N I M J L K 
E D F C G B H A I O J N K M L 
F E G D H C I B J A K O L N M 
G F H E I D J C K B L A M O N 
H G I F J E K D L C M B N A O 
 

Size = 16

A B P C O D N E M F L G K H J I 
B C A D P E O F N G M H L I K J 
C D B E A F P G O H N I M J L K 
D E C F B G A H P I O J N K M L 
E F D G C H B I A J P K O L N M 
F G E H D I C J B K A L P M O N 
G H F I E J D K C L B M A N P O 
H I G J F K E L D M C N B O A P 
I J H K G L F M E N D O C P B A 
J K I L H M G N F O E P D A C B 
K L J M I N H O G P F A E B D C 
L M K N J O I P H A G B F C E D 
M N L O K P J A I B H C G D F E 
N O M P L A K B J C I D H E G F 
O P N A M B L C K D J E I F H G 
P A O B N C M D L E K F J G I H 
 

Size = 17

A B Q C P D O E N F M G L H K I J 
B C A D Q E P F O G N H M I L J K 
C D B E A F Q G P H O I N J M K L 
D E C F B G A H Q I P J O K N L M 
E F D G C H B I A J Q K P L O M N 
F G E H D I C J B K A L Q M P N O 
G H F I E J D K C L B M A N Q O P 
H I G J F K E L D M C N B O A P Q 
I J H K G L F M E N D O C P B Q A 
J K I L H M G N F O E P D Q C A B 
K L J M I N H O G P F Q E A D B C 
L M K N J O I P H Q G A F B E C D 
M N L O K P J Q I A H B G C F D E 
N O M P L Q K A J B I C H D G E F 
O P N Q M A L B K C J D I E H F G 
P Q O A N B M C L D K E J F I G H 
Q A P B O C N D M E L F K G J H I 
J I K H L G M F N E O D P C Q B A 
K J L I M H N G O F P E Q D A C B 
L K M J N I O H P G Q F A E B D C 
M L N K O J P I Q H A G B F C E D 
N M O L P K Q J A I B H C G D F E 
O N P M Q L A K B J C I D H E G F 
P O Q N A M B L C K D J E I F H G 
Q P A O B N C M D L E K F J G I H 
A Q B P C O D N E M F L G K H J I 
B A C Q D P E O F N G M H L I K J 
C B D A E Q F P G O H N I M J L K 
D C E B F A G Q H P I O J N K M L 
E D F C G B H A I Q J P K O L N M 
F E G D H C I B J A K Q L P M O N 
G F H E I D J C K B L A M Q N P O 
H G I F J E K D L C M B N A O Q P 
I H J G K F L E M D N C O B P A Q 
 
 

Size = 18

A B R C Q D P E O F N G M H L I K J 
B C A D R E Q F P G O H N I M J L K 
C D B E A F R G Q H P I O J N K M L 
D E C F B G A H R I Q J P K O L N M 
E F D G C H B I A J R K Q L P M O N 
F G E H D I C J B K A L R M Q N P O 
G H F I E J D K C L B M A N R O Q P 
H I G J F K E L D M C N B O A P R Q 
I J H K G L F M E N D O C P B Q A R 
J K I L H M G N F O E P D Q C R B A 
K L J M I N H O G P F Q E R D A C B 
L M K N J O I P H Q G R F A E B D C 
M N L O K P J Q I R H A G B F C E D 
N O M P L Q K R J A I B H C G D F E 
O P N Q M R L A K B J C I D H E G F 
P Q O R N A M B L C K D J E I F H G 
Q R P A O B N C M D L E K F J G I H 
R A Q B P C O D N E M F L G K H J I 
 

Size = 19

A B S C R D Q E P F O G N H M I L J K 
B C A D S E R F Q G P H O I N J M K L 
C D B E A F S G R H Q I P J O K N L M 
D E C F B G A H S I R J Q K P L O M N 
E F D G C H B I A J S K R L Q M P N O 
F G E H D I C J B K A L S M R N Q O P 
G H F I E J D K C L B M A N S O R P Q 
H I G J F K E L D M C N B O A P S Q R 
I J H K G L F M E N D O C P B Q A R S 
J K I L H M G N F O E P D Q C R B S A 
K L J M I N H O G P F Q E R D S C A B 
L M K N J O I P H Q G R F S E A D B C 
M N L O K P J Q I R H S G A F B E C D 
N O M P L Q K R J S I A H B G C F D E 
O P N Q M R L S K A J B I C H D G E F 
P Q O R N S M A L B K C J D I E H F G 
Q R P S O A N B M C L D K E J F I G H 
R S Q A P B O C N D M E L F K G J H I 
S A R B Q C P D O E N F M G L H K I J 
K J L I M H N G O F P E Q D R C S B A 
L K M J N I O H P G Q F R E S D A C B 
M L N K O J P I Q H R G S F A E B D C 
N M O L P K Q J R I S H A G B F C E D 
O N P M Q L R K S J A I B H C G D F E 
P O Q N R M S L A K B J C I D H E G F 
Q P R O S N A M B L C K D J E I F H G 
R Q S P A O B N C M D L E K F J G I H 
S R A Q B P C O D N E M F L G K H J I 
A S B R C Q D P E O F N G M H L I K J 
B A C S D R E Q F P G O H N I M J L K 
C B D A E S F R G Q H P I O J N K M L 
D C E B F A G S H R I Q J P K O L N M 
E D F C G B H A I S J R K Q L P M O N 
F E G D H C I B J A K S L R M Q N P O 
G F H E I D J C K B L A M S N R O Q P 
H G I F J E K D L C M B N A O S P R Q 
I H J G K F L E M D N C O B P A Q S R 
J I K H L G M F N E O D P C Q B R A S 
 

Size = 20

A B T C S D R E Q F P G O H N I M J L K 
B C A D T E S F R G Q H P I O J N K M L 
C D B E A F T G S H R I Q J P K O L N M 
D E C F B G A H T I S J R K Q L P M O N 
E F D G C H B I A J T K S L R M Q N P O 
F G E H D I C J B K A L T M S N R O Q P 
G H F I E J D K C L B M A N T O S P R Q 
H I G J F K E L D M C N B O A P T Q S R 
I J H K G L F M E N D O C P B Q A R T S 
J K I L H M G N F O E P D Q C R B S A T 
K L J M I N H O G P F Q E R D S C T B A 
L M K N J O I P H Q G R F S E T D A C B 
M N L O K P J Q I R H S G T F A E B D C 
N O M P L Q K R J S I T H A G B F C E D 
O P N Q M R L S K T J A I B H C G D F E 
P Q O R N S M T L A K B J C I D H E G F 
Q R P S O T N A M B L C K D J E I F H G 
R S Q T P A O B N C M D L E K F J G I H 
S T R A Q B P C O D N E M F L G K H J I 
T A S B R C Q D P E O F N G M H L I K J 
 

Size = 21

A B U C T D S E R F Q G P H O I N J M K L 
B C A D U E T F S G R H Q I P J O K N L M 
C D B E A F U G T H S I R J Q K P L O M N 
D E C F B G A H U I T J S K R L Q M P N O 
E F D G C H B I A J U K T L S M R N Q O P 
F G E H D I C J B K A L U M T N S O R P Q 
G H F I E J D K C L B M A N U O T P S Q R 
H I G J F K E L D M C N B O A P U Q T R S 
I J H K G L F M E N D O C P B Q A R U S T 
J K I L H M G N F O E P D Q C R B S A T U 
K L J M I N H O G P F Q E R D S C T B U A 
L M K N J O I P H Q G R F S E T D U C A B 
M N L O K P J Q I R H S G T F U E A D B C 
N O M P L Q K R J S I T H U G A F B E C D 
O P N Q M R L S K T J U I A H B G C F D E 
P Q O R N S M T L U K A J B I C H D G E F 
Q R P S O T N U M A L B K C J D I E H F G 
R S Q T P U O A N B M C L D K E J F I G H 
S T R U Q A P B O C N D M E L F K G J H I 
T U S A R B Q C P D O E N F M G L H K I J 
U A T B S C R D Q E P F O G N H M I L J K 
L K M J N I O H P G Q F R E S D T C U B A 
M L N K O J P I Q H R G S F T E U D A C B 
N M O L P K Q J R I S H T G U F A E B D C 
O N P M Q L R K S J T I U H A G B F C E D 
P O Q N R M S L T K U J A I B H C G D F E 
Q P R O S N T M U L A K B J C I D H E G F 
R Q S P T O U N A M B L C K D J E I F H G 
S R T Q U P A O B N C M D L E K F J G I H 
T S U R A Q B P C O D N E M F L G K H J I 
U T A S B R C Q D P E O F N G M H L I K J 
A U B T C S D R E Q F P G O H N I M J L K 
B A C U D T E S F R G Q H P I O J N K M L 
C B D A E U F T G S H R I Q J P K O L N M 
D C E B F A G U H T I S J R K Q L P M O N 
E D F C G B H A I U J T K S L R M Q N P O 
F E G D H C I B J A K U L T M S N R O Q P 
G F H E I D J C K B L A M U N T O S P R Q 
H G I F J E K D L C M B N A O U P T Q S R 
I H J G K F L E M D N C O B P A Q U R T S 
J I K H L G M F N E O D P C Q B R A S U T 
K J L I M H N G O F P E Q D R C S B T A U 
 


Size = 22

A B V C U D T E S F R G Q H P I O J N K M L 
B C A D V E U F T G S H R I Q J P K O L N M 
C D B E A F V G U H T I S J R K Q L P M O N 
D E C F B G A H V I U J T K S L R M Q N P O 
E F D G C H B I A J V K U L T M S N R O Q P 
F G E H D I C J B K A L V M U N T O S P R Q 
G H F I E J D K C L B M A N V O U P T Q S R 
H I G J F K E L D M C N B O A P V Q U R T S 
I J H K G L F M E N D O C P B Q A R V S U T 
J K I L H M G N F O E P D Q C R B S A T V U 
K L J M I N H O G P F Q E R D S C T B U A V 
L M K N J O I P H Q G R F S E T D U C V B A 
M N L O K P J Q I R H S G T F U E V D A C B 
N O M P L Q K R J S I T H U G V F A E B D C 
O P N Q M R L S K T J U I V H A G B F C E D 
P Q O R N S M T L U K V J A I B H C G D F E 
Q R P S O T N U M V L A K B J C I D H E G F 
R S Q T P U O V N A M B L C K D J E I F H G 
S T R U Q V P A O B N C M D L E K F J G I H 
T U S V R A Q B P C O D N E M F L G K H J I 
U V T A S B R C Q D P E O F N G M H L I K J 
V A U B T C S D R E Q F P G O H N I M J L K 
 


Size = 23

A B W C V D U E T F S G R H Q I P J O K N L M 
B C A D W E V F U G T H S I R J Q K P L O M N 
C D B E A F W G V H U I T J S K R L Q M P N O 
D E C F B G A H W I V J U K T L S M R N Q O P 
E F D G C H B I A J W K V L U M T N S O R P Q 
F G E H D I C J B K A L W M V N U O T P S Q R 
G H F I E J D K C L B M A N W O V P U Q T R S 
H I G J F K E L D M C N B O A P W Q V R U S T 
I J H K G L F M E N D O C P B Q A R W S V T U 
J K I L H M G N F O E P D Q C R B S A T W U V 
K L J M I N H O G P F Q E R D S C T B U A V W 
L M K N J O I P H Q G R F S E T D U C V B W A 
M N L O K P J Q I R H S G T F U E V D W C A B 
N O M P L Q K R J S I T H U G V F W E A D B C 
O P N Q M R L S K T J U I V H W G A F B E C D 
P Q O R N S M T L U K V J W I A H B G C F D E 
Q R P S O T N U M V L W K A J B I C H D G E F 
R S Q T P U O V N W M A L B K C J D I E H F G 
S T R U Q V P W O A N B M C L D K E J F I G H 
T U S V R W Q A P B O C N D M E L F K G J H I 
U V T W S A R B Q C P D O E N F M G L H K I J 
V W U A T B S C R D Q E P F O G N H M I L J K 
W A V B U C T D S E R F Q G P H O I N J M K L 
M L N K O J P I Q H R G S F T E U D V C W B A 
N M O L P K Q J R I S H T G U F V E W D A C B 
O N P M Q L R K S J T I U H V G W F A E B D C 
P O Q N R M S L T K U J V I W H A G B F C E D 
Q P R O S N T M U L V K W J A I B H C G D F E 
R Q S P T O U N V M W L A K B J C I D H E G F 
S R T Q U P V O W N A M B L C K D J E I F H G 
T S U R V Q W P A O B N C M D L E K F J G I H 
U T V S W R A Q B P C O D N E M F L G K H J I 
V U W T A S B R C Q D P E O F N G M H L I K J 
W V A U B T C S D R E Q F P G O H N I M J L K 
A W B V C U D T E S F R G Q H P I O J N K M L 
B A C W D V E U F T G S H R I Q J P K O L N M 
C B D A E W F V G U H T I S J R K Q L P M O N 
D C E B F A G W H V I U J T K S L R M Q N P O 
E D F C G B H A I W J V K U L T M S N R O Q P 
F E G D H C I B J A K W L V M U N T O S P R Q 
G F H E I D J C K B L A M W N V O U P T Q S R 
H G I F J E K D L C M B N A O W P V Q U R T S 
I H J G K F L E M D N C O B P A Q W R V S U T 
J I K H L G M F N E O D P C Q B R A S W T V U 
K J L I M H N G O F P E Q D R C S B T A U W V 
L K M J N I O H P G Q F R E S D T C U B V A W 
 

Size = 24

A B X C W D V E U F T G S H R I Q J P K O L N M 
B C A D X E W F V G U H T I S J R K Q L P M O N 
C D B E A F X G W H V I U J T K S L R M Q N P O 
D E C F B G A H X I W J V K U L T M S N R O Q P 
E F D G C H B I A J X K W L V M U N T O S P R Q 
F G E H D I C J B K A L X M W N V O U P T Q S R 
G H F I E J D K C L B M A N X O W P V Q U R T S 
H I G J F K E L D M C N B O A P X Q W R V S U T 
I J H K G L F M E N D O C P B Q A R X S W T V U 
J K I L H M G N F O E P D Q C R B S A T X U W V 
K L J M I N H O G P F Q E R D S C T B U A V X W 
L M K N J O I P H Q G R F S E T D U C V B W A X 
M N L O K P J Q I R H S G T F U E V D W C X B A 
N O M P L Q K R J S I T H U G V F W E X D A C B 
O P N Q M R L S K T J U I V H W G X F A E B D C 
P Q O R N S M T L U K V J W I X H A G B F C E D 
Q R P S O T N U M V L W K X J A I B H C G D F E 
R S Q T P U O V N W M X L A K B J C I D H E G F 
S T R U Q V P W O X N A M B L C K D J E I F H G 
T U S V R W Q X P A O B N C M D L E K F J G I H 
U V T W S X R A Q B P C O D N E M F L G K H J I 
V W U X T A S B R C Q D P E O F N G M H L I K J 
W X V A U B T C S D R E Q F P G O H N I M J L K 
X A W B V C U D T E S F R G Q H P I O J N K M L 
 
 

Size = 25

A B Y C X D W E V F U G T H S I R J Q K P L O M N 
B C A D Y E X F W G V H U I T J S K R L Q M P N O 
C D B E A F Y G X H W I V J U K T L S M R N Q O P 
D E C F B G A H Y I X J W K V L U M T N S O R P Q 
E F D G C H B I A J Y K X L W M V N U O T P S Q R 
F G E H D I C J B K A L Y M X N W O V P U Q T R S 
G H F I E J D K C L B M A N Y O X P W Q V R U S T 
H I G J F K E L D M C N B O A P Y Q X R W S V T U 
I J H K G L F M E N D O C P B Q A R Y S X T W U V 
J K I L H M G N F O E P D Q C R B S A T Y U X V W 
K L J M I N H O G P F Q E R D S C T B U A V Y W X 
L M K N J O I P H Q G R F S E T D U C V B W A X Y 
M N L O K P J Q I R H S G T F U E V D W C X B Y A 
N O M P L Q K R J S I T H U G V F W E X D Y C A B 
O P N Q M R L S K T J U I V H W G X F Y E A D B C 
P Q O R N S M T L U K V J W I X H Y G A F B E C D 
Q R P S O T N U M V L W K X J Y I A H B G C F D E 
R S Q T P U O V N W M X L Y K A J B I C H D G E F 
S T R U Q V P W O X N Y M A L B K C J D I E H F G 
T U S V R W Q X P Y O A N B M C L D K E J F I G H 
U V T W S X R Y Q A P B O C N D M E L F K G J H I 
V W U X T Y S A R B Q C P D O E N F M G L H K I J 
W X V Y U A T B S C R D Q E P F O G N H M I L J K 
X Y W A V B U C T D S E R F Q G P H O I N J M K L 
Y A X B W C V D U E T F S G R H Q I P J O K N L M 
N M O L P K Q J R I S H T G U F V E W D X C Y B A 
O N P M Q L R K S J T I U H V G W F X E Y D A C B 
P O Q N R M S L T K U J V I W H X G Y F A E B D C 
Q P R O S N T M U L V K W J X I Y H A G B F C E D 
R Q S P T O U N V M W L X K Y J A I B H C G D F E 
S R T Q U P V O W N X M Y L A K B J C I D H E G F 
T S U R V Q W P X O Y N A M B L C K D J E I F H G 
U T V S W R X Q Y P A O B N C M D L E K F J G I H 
V U W T X S Y R A Q B P C O D N E M F L G K H J I 
W V X U Y T A S B R C Q D P E O F N G M H L I K J 
X W Y V A U B T C S D R E Q F P G O H N I M J L K 
Y X A W B V C U D T E S F R G Q H P I O J N K M L 
A Y B X C W D V E U F T G S H R I Q J P K O L N M 
B A C Y D X E W F V G U H T I S J R K Q L P M O N 
C B D A E Y F X G W H V I U J T K S L R M Q N P O 
D C E B F A G Y H X I W J V K U L T M S N R O Q P 
E D F C G B H A I Y J X K W L V M U N T O S P R Q 
F E G D H C I B J A K Y L X M W N V O U P T Q S R 
G F H E I D J C K B L A M Y N X O W P V Q U R T S 
H G I F J E K D L C M B N A O Y P X Q W R V S U T 
I H J G K F L E M D N C O B P A Q Y R X S W T V U 
J I K H L G M F N E O D P C Q B R A S Y T X U W V 
K J L I M H N G O F P E Q D R C S B T A U Y V X W 
L K M J N I O H P G Q F R E S D T C U B V A W Y X 
M L N K O J P I Q H R G S F T E U D V C W B X A Y 
 


Size = 26

A B Z C Y D X E W F V G U H T I S J R K Q L P M O N 
B C A D Z E Y F X G W H V I U J T K S L R M Q N P O 
C D B E A F Z G Y H X I W J V K U L T M S N R O Q P 
D E C F B G A H Z I Y J X K W L V M U N T O S P R Q 
E F D G C H B I A J Z K Y L X M W N V O U P T Q S R 
F G E H D I C J B K A L Z M Y N X O W P V Q U R T S 
G H F I E J D K C L B M A N Z O Y P X Q W R V S U T 
H I G J F K E L D M C N B O A P Z Q Y R X S W T V U 
I J H K G L F M E N D O C P B Q A R Z S Y T X U W V 
J K I L H M G N F O E P D Q C R B S A T Z U Y V X W 
K L J M I N H O G P F Q E R D S C T B U A V Z W Y X 
L M K N J O I P H Q G R F S E T D U C V B W A X Z Y 
M N L O K P J Q I R H S G T F U E V D W C X B Y A Z 
N O M P L Q K R J S I T H U G V F W E X D Y C Z B A 
O P N Q M R L S K T J U I V H W G X F Y E Z D A C B 
P Q O R N S M T L U K V J W I X H Y G Z F A E B D C 
Q R P S O T N U M V L W K X J Y I Z H A G B F C E D 
R S Q T P U O V N W M X L Y K Z J A I B H C G D F E 
S T R U Q V P W O X N Y M Z L A K B J C I D H E G F 
T U S V R W Q X P Y O Z N A M B L C K D J E I F H G 
U V T W S X R Y Q Z P A O B N C M D L E K F J G I H 
V W U X T Y S Z R A Q B P C O D N E M F L G K H J I 
W X V Y U Z T A S B R C Q D P E O F N G M H L I K J 
X Y W Z V A U B T C S D R E Q F P G O H N I M J L K 
Y Z X A W B V C U D T E S F R G Q H P I O J N K M L 
Z A Y B X C W D V E U F T G S H R I Q J P K O L N M